Transgenic plants expressing photorhabdus toxin

Abstract

Novel polynucleotide sequences that encode insect toxins TcdA and TcbA have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots. Transgenic plants with a genome comprising a nucleic acid of SEQ ID NO: 3 or SEQ ID NO:4 are insect resistant.

Description

BACKGROUND OF THE INVENTION

As reported in WO98/08932, protein toxins from the genus Photorhabdus have been shown to have oral toxicity against insects. The toxin complex produced by Photorhabdus luminescens (W-14), for example, has been shown to contain ten to fourteen proteins, and it is known that these are produced by expression of genes from four distinct genomic regions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotide sequences for the native toxin genes.

Of the separate toxins isolated from Photorhabdus luminescens (W-14), those designated Toxin A and Toxin B are especially potent against target insect species of interest, for example corn rootworm. Toxin A is comprised of two different subunits. The native gene tcdA (SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is an approximately 282.9 kDA protein (2516 aa) that is processed to provide TcdAii, an approximately 208.2 kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1, and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded by nucleotides 5812-7551 of SEQ ID NO:1.

Toxin B is similarly comprised of two different subunits. The native gene tcbA (SEQ ID NO:2) encodes protoxin TcbA (see SEQ ID NO:2). As determined by mass spectrometry, TcbA is processed by one or more proteases to provide Toxin B. More specifically, TcbA is an approximately 280.6 kDA (2504 aa) protein that is processed to provide TcbAii, an approximately 207.7 kDA (1844 aa) protein encoded by nucleotides 262-5793 of SEQ ID NO:2 and TcbAiii, an approximately 62.9 kDA (573 aa) protein encoded by nucleotides 5794-7512 of SEQ ID NO:2.

The native tcdA and tcbA genes are not well suited for high level expression in plants. They encode multiple destabilization sequences, mRNA splice sites, polyA addition sites and other possibly detrimental sequence motifs. In addition, the codon compositions are not like those of plant genes. WO98/08932 gives general guidance on how the toxin genes could be reengineered to more efficiently expressed in the cytoplasm of plants, and describes how plants can be transformed to incorporate the Photorhabdus toxin genes into their genomes.

SUMMARY OF THE INVENTION

In a preferred embodiment, the invention provides novel polynucleotide sequences that encode TcdA and TcbA. The novel sequences have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots, and this feature is designated by referring to the sequences as the “hemicot” criteria, which is set forth in detail hereinafter. Other important features of the sequences are that potentially deleterious sequences have been eliminated, and unique restriction sites have been built in to enable adding or changing expression elements, organellar targeting signals, engineered protease sites and the like, if desired.

In a particularly preferred embodiment, the invention provides polynucleotide sequences that satisfy hemicot criteria and that comprise a sequence encoding an endoplasmic reticulum signal or similar targeting sequence for a cellular organelle in combination with a sequence encoding TcdA or TdbA.

More broadly, the invention provides engineered nucleic acids encoding functional Photorhabdus toxins wherein the sequences satisfy hemicot criteria.

The invention also provides transgenic plants with genomes comprising a novel sequence of the invention that imparts functional activity against insects.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is the native tcdA DNA sequence together with the corresponding encoded amino acid sequence for TcdA.

SEQ ID NO:2 is the native tcbA DNA sequence together with the corresponding encoded amino acid sequence for TcbA.

SEQ ID NO:3 is an artificial sequence encoding TcdA that is suitable for expression in monocot and dicot plants.

SEQ ID NO:4 is an artificial sequence encoding TdbA that is suitable for expression in monocot and dicot plants.

SEQ ID NO:5 is an artificial hemicot sequence that encodes the 21 amino acid ER signal peptide of 15 kDa zein from Black Mexican Sweet maize.

SEQ ID NO:6 is an artificial hemicot sequence that encodes for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).

DETAILED DESCRIPTION

The native Photorhabdus toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus. Of particular interest are the proteins produced by the species Photorhabdus luminescens . The protein complexes have a molecular size of approximately 1,000 kDa and can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon ingestion by a number of insects.

A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabditis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (Rockville, Md.) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce insecticidal toxins. Such sources in the environment could be either terrestrial or aquatic based.

The genus Photorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits atypical of this family. For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and bioluminescent. This latter trait is otherwise unknown within the Enterobacteriaceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on DNA-DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus) or absence (Xenorhabdus) of catalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Photorhabdus; Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Photorhabdus from Xenorhabdus.

Currently, the bacterial genus Photorhabdus is comprised of a single defined species, Photorhabdus luminescens (ATCC Type strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A variety of related strains have been described in the literature (e.g., Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56, 181-186).

The following toxin producing Photorhabdus strains have been deposited:

	strain	accession number	date of deposit
	W-14	ATCC 55397	Mar. 5, 1993
	WX1	NRRL B-21710	Apr. 29, 1997
	WX2	NRRL B-21711	Apr. 29, 1997
	WX3	NRRL B-21712	Apr. 29, 1997
	WX4	NRRL B-21713	Apr. 29, 1997
	WX5	NRRL B-21714	Apr. 29, 1997
	WX6	NRRL B-21715	Apr. 29, 1997
	WX7	NRRL B-21716	Apr. 29, 1997
	WX8	NRRL B-21717	Apr. 29, 1997
	WX9	NRRL B-21718	Apr. 29, 1997
	WX10	NRRL B-21719	Apr. 29, 1997
	WX11	NRRL B-21720	Apr. 29, 1997
	WX12	NRRL B-21721	Apr. 29, 1997
	WX14	NRRL B-21722	Apr. 29, 1997
	WX15	NRRL B-21723	Apr. 29, 1997
	H9	NRRL B-21727	Apr. 29, 1997
	Hb	NRRL B-21726	Apr. 29, 1997
	Hm	NRRL B-21725	Apr. 29, 1997
	HP88	NRRL B-21724	Apr. 29, 1997
	NC-1	NRRL B-21728	Apr. 29, 1997
	W30	NRRL B-21729	Apr. 29, 1997
	WIR	NRRL B-21730	Apr. 29, 1997
	B2	NRRL B-21731	Apr. 29, 1997
	ATCC 43948	ATCC 55878	Nov. 5, 1996
	ATCC 43949	ATCC 55879	Nov. 5, 1996
	ATCC 43950	ATCC 55880	Nov. 5, 1996
	ATCC 53951	ATCC 55881	Nov. 5, 1996
	ATCC 43952	ATCC 55882	Nov. 5, 1996
	DEPI	NRRL B-21707	Apr. 29, 1997
	DEP2	NRRL B-21708	Apr. 29, 1997
	DEP3	NRRL B-21709	Apr. 29, 1997
	P. zealandrica	NRRL B-21683	Apr. 29, 1997
	P. hepialus	NRRL B-21684	Apr. 29, 1997
	HB-Arg	NRRL B-21685	Apr. 29, 1997
	HB Oswego	NRRL B-21686	Apr. 29, 1997
	Hb Lewiston	NRRL B-21687	Apr. 29, 1997
	K-122	NRRL B-21688	Apr. 29, 1997
	HMGD	NRRL B-21689	Apr. 29, 1997
	Indicus	NRRL B-21690	Apr. 29, 1997
	GD	NRRL B-21691	Apr. 29, 1997
	PWH-5	NRRL B-21692	Apr. 29, 1997
	Megidis	NRRL B-21693	Apr. 29, 1997
	HF-85	NRRL B-21694	Apr. 29, 1997
	A. Cows	NRRL B-21695	Apr. 29, 1997
	MP1	NRRL B-21696	Apr. 29, 1997
	MP2	NRRL B-21697	Apr. 29, 1997
	MP3	NRRL B-21698	Apr. 29, 1997
	MP4	NRRL B-21699	Apr. 29, 1997
	MP5	NRRL B-21700	Apr. 29, 1997
	GL98	NRRL B-21701	Apr. 29, 1997
	Gl101	NRRL B-21702	Apr. 29, 1997
	GL138	NRRL B-21703	Apr. 29, 1997
	GL155	NRRL B-21704	Apr. 29, 1997
	GL217	NRRL B-21705	Apr. 29, 1997
	GL257	NRRL B-21706	Apr. 29, 1997

All strains were deposited in accordance with the terms of the Budapest Treaty. Strains having accession numbers prefaced by “ATTC” were deposited on the indicated date in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA. Strains prefaced by “NRRL” were deposited on the indicated date in the Agricultural Research Service Patent Culture Collection (NRRL), National Center for Agricultural Utilization Research, ARS-USDA, 1815 North University St., Peoria Ill. 61604 USA.

The present invention provides hemicot nucleic acid sequences encoding toxins from any Photorhabdus species or strain that produces a toxin having functional activity. Hemicot nucleic acid sequences encoding proteins homologous to such toxins are also encompassed by the invention.

Several terms that are used herein have a particular meaning and are defined as follows:

By “functional activity” it is meant herein that the protein toxins) function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect. When an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression, formulated protein compositions), sprayable protein compositions), a bait matrix or other delivery system, the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.

By “homolog” it is meant an amino acid sequence that is identified as possessing homology to a reference Photorhabdus toxin polypeptide amino acid sequence.

By “homology” it is meant an amino acid sequence that has a similarity index of at least 33% and/or an identity index of at least 26% to a reference Photorhabdus toxin polypeptide amino acid sequence, as scored by the GAP algorithm using the B10sum 62 protein scoring matrix Wisconsin Package Version 9.0, Genetics Computer Group GCG), Madison, Wis.).

By “identity” is meant an amino acid sequence that contains an identical residue at a given position, following alignment with a reference Photrhabdus toxin polypeptide amino acid sequence by the GAP algorithm.

By the use of the term “Photorhabdus toxin” it is meant any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via baculovirus, or delivered by any other applicable host or delivery system.

By the use of the term “toxic” or “toxicity” as used herein it is meant that the toxins produced by Photorhabdus have “functional activity” as defined herein.

By “substantial sequence homology” is meant either: a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to produce a protein having similar biochemical properties; or a polypeptide having an amino acid sequence sufficiently similar to another polypeptide to exhibit similar biochemical properties.

As with other bacterial toxins, the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist. Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein. Preferably, the toxins are active against Lepidoptera, Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina. The inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative strains thereof, as well as any protein toxins produced by Photorhabdus. These homologous proteins may differ in sequence, but do not differ in function from those toxins described herein. Homologous toxins are meant to include protein complexes of between 300 kDa to 2,000 kDa and are comprised of at least two 2) subunits, where a subunit is a peptide which may or may not be the same as the other subunit. Various protein subunits have been identified and are taught in the Examples herein. Typically, the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.

As discussed above, some Photorhabdus strains can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the phylum Nematoda, have evolved an ability to exploit insect larvae as a favored growth environment. The insect larvae provide a source of food for growing nematodes and an environment in which to reproduce. One dramatic effect that follows invasion of larvae by certain nematodes is larval death. Larval death results from the presence of, in certain nematodes, bacteria that produce an insecticidal toxin which arrests larval growth and inhibits feeding activity.

Interestingly, it appears that each genus of insect parasitic nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode. In the interim since this research was initiated, the name of the bacterial genus Xenorhabdus was reclassified into the Xenorhabdus and the Photorhabdus. Bacteria of the genus Photorhabdus are characterized as being symbionts of Heterorhabditus nematodes while Xenorhabdus species are symbionts of the Steinernema species. This change in nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the inventions described herein.

The peptides and genes that are disclosed herein are named according to the guidelines recently published in the Journal of Bacteriology “Instructions to Authors” p. i-xii January 1996), which is incorporated herein by reference.

Transformation methods useful in carrying out the invention are well known, and are described, for example, in WO98/08932.

Hemicot tcdA and tcbA

SEQ ID NO: 3 is the nucleotide sequence for an engineered tcdA gene in accordance with the invention. SEQ ID NO: 4 is the nucleotide sequence for an engineered tcbA gene in accordance with the invention.

The following Tables 1 and 2 identify significant features of the engineered tcdA and tcbA genes.

TABLE 1
tcdA
Feature           nucleotides of SEQ ID NO: 3
NcoI              1-6
HindIII           48-53
KpnI              246-254
sequence encoding 267-5798
TcbAii
NheI              333-338
BglII             1215-1220
ClaI              2604-2609
PstI              4015-4020
AgeI              5088-5093
MunI              5598-5603
XbaI              5778-5783
sequence encoding 5799-7517
TcbAiii
AflII             5853-5858
SphI              6439-6444
SfuI              7392-7397
SacI              7519-7524
XhoI              7522-7527
StuI              7528-7533
NotI              7533-7538

TABLE 2
tcbA
Feature           nucleotides of SEQ ID NO: 5
NcoI              1-6
HindIII           48-53
KpnI              246-251
sequence encoding 267-5798
TcbAii
NheI              333-338
BglII             1215-1220
ClaI              2604-2609
PstI              4015-4020
AgeI              5088-5093
MunI              5598-5603
XbaI              5778-5783
sequence          5799-7517
encoding TcbAiii
AflII             5853-5858
SphI              6439-6444
SfuI              7392-7397
SacI              7519-7524
SfuI              7392-7397
SacI              7519-7524
XhoI              7522-7527
StuI              7528-7533
NotI              7535-7540

It should be noted that the proteins encoded by the plant-optimized tcdA (SEQ ID NO:3) and tcbA (SEQ ID NO:5) differ from the native proteins by the addition of an Ala residue at position #2. This modification was made to accommodate the NcoI site which spans the ATG start codon.

The following Table 3 compares the codon composition of the engineered tcdA gene of SEQ ID NO:3 and engineered tcbA gene of SEQ ID NO:5 with the codon compositions of the native genes, the typical dicot genes, and maize genes.

TABLE 3
		% in		% in
		SEQ		SEQ
amino		ID	% in	ID	% in	% in	% in
acid	codon	NO:3	tcdA	NO:5	tcbA	dicot	maize
Ala	GCT	62	21	69	41	42	24
	GCC	26	32	27	17	27	34
	GCA	11	25	4	22	25	18
	GCG	0	21	0	21	6	24
Arg	AGG	48	0	60	2	25	26
	CGC	22	36	18	16	11	24
	AGA	20	11	15	6	30	15
	CGT	11	39	7	57	21	11
	CGG	0	7	0	13	4	15
	CGA	0	8	0	6	8	9
Asn	AAC	100	32	100	33	55	68
	AAT	0	68	0	67	45	32
Asp	GAC	67	22	70	25	42	63
	GAT	33	78	30	75	58	37
Cys	TGC	100	30	100	19	56	68
	TGT	0	70	0	81	44	32
End	TGA	100	0	100	0	33	59
	TAG	0	0	0	0	19	21
	TAA	0	100	0	100	48	20
Gln	CAA	65	61	74	53	59	38
	CAG	35	39	26	47	41	62
Glu	GAG	100	24	98	36	51	71
	GAA	0	76	2	64	49	29
Gly	GGT	67	37	64	44	33	20
	GGC	32	36	36	22	16	42
	GGA	1	20	0	19	38	19
	GGG	0	8	0	16	12	20
His	CAC	62	40	72	31	46	62
	CAT	38	60	28	69	54	38
Ile	ATC	73	34	65	24	37	58
	ATT	27	51	35	59	45	28
	ATA	0	15	0	17	18	14
Leu	CTC	54	11	59	7	28	26
	TTG	29	17	25	32	26	15
	CTT	16	9	15	7	19	17
	TTA	0	18	0	19	10	5
	CTG	0	32	0	29	9	29
	CTA	0	13	0	7	8	8
Lys	AAG	99	79	99	75	61	78
	AAA	1	21	1	25	39	22
Met	ATG	100	100	100	100	100	100
Phe	TTC	100	42	100	41	55	71
	TTT	0	58	0	59	45	29
Pro	CCA	74	30	91	26	42	26
	CCT	22	28	7	20	32	22
	CCC	4	14	3	7	17	24
	CCG	0	27	0	47	9	28
Ser	TCC	47	19	55	11	18	23
	TCT	35	15	30	15	25	15
	AGC	18	22	15	18	18	23
	AGT	0	20	0	31	14	9
	TCG	0	7	0	8	6	14
	TCA	0	17	0	17	19	16
Thr	ACC	60	41	64	31	30	37
	ACT	28	25	32	34	35	20
	ACA	12	21	4	18	27	21
	ACG	0	13	0	18	8	22
Trp	TGG	100	100	100	100	100	100
Tyr	TAC	100	24	100	19	57	73
	TAT	0	76	0	81	43	27
Val	GTC	69	27	73	11	20	31
	GTG	21	17	22	27	29	39
	GTT	10	34	3	48	39	21
	GTA	0	22	2	14	12	8

EXAMPLE 1

Design of Plant Codon-Biased Genes Encoding W-14 Peptides TcbA and TcdA

A. Gene Design

The coding strands of the native DNA sequences of the Photorhabdus W-14 genes encoding peptides TcbA and TcdA were scanned for the presence of deleterious sequences such as the Shaw/Kamen RNA destabilizing motif ATTTA, intron splice recognition sites, and poly A addition motifs. This was done using the MacVector Sequence Analysis Software (Oxford Molecular Biology Group, Symantec Corp.), using a custom Nucleic Acid Subsequence File. The native sequence was also searched for runs of 4 or more of the same base.

Motif searching of the native W-14 tcbA and tcdA genes revealed the presence of many potentially deleterious sequences in the protein coding strands, as summarized in Table 4. Not shown, but also present, were many runs of four or more single residues (e.g. the native tcbA gene has 81 runs of four A's).

TABLE 4
Native				Poly A	RNAP II
Gene	ATTTA	5′ splice	3′ Splice	Addition*	term.
tcbA	18	7	17	46	0
tcdA	18	7	13	77	1
*Totals of 16 different motifs.

Analyses of eukaryotic genes and plant genes in particular have shown that CG & TA doublets are underrepresented, while the genes are enriched in CT & TG doublets. The sequences of the hemicot biased genes have accordingly been adjusted to encompass these base compositions and to have G+C compositions of about 53%, similar to many plant genes. When compared to the native W-14 tcbA and tcdA genes, the plant-biased genes have a much more uniform G+C distribution.

Nucleotide changes to remove potentially deleterious sequences were chosen to simultaneously adjust the codon composition of the coding region to more closely reflect that of plant genes. A framework for these changes was provided by the codon bias tables prepared for maize and dicot genes shown in Table 3.

Comparison of codon compositions of the native W-14 genes to maize and dicot genes revealed that the W-14 genes contain a very different preference set of the degenerate codons for the 18 amino acids for which there is a choice (Table 3). For each of 8 amino acids (Phe, Tyr, Cys, Arg, Asn, Lys, Glu, and Gly) in both W-14 genes, the most abundant codon is different from the preferred codons found in either maize or dicot genes. One might expect that translational difficulties would be encountered in efforts to produce in plants proteins (such as TcbA and TcdA) having high relative amounts of these amino acids from mRNAs having large numbers of nonpreferred codons. There is a marked difference in distribution of the codon compositions specifying the other 10 amino acids. For His, Gln, Ile, Val, and Asp, the dicot-preferred codons are found as the most abundant ones in both W-14 genes. For Leu, Thr, Ser, and Ala, the maize preferred codons are the most abundant codon choices found in the tcdA gene. In contrast, the tcbA gene contains only the CCG (Pro) maize-preferred codon as the highest abundance choice.

In making the codon choices, doublet contents were considered, so that adjacent codons preferably did not form CG or TA doublets (which are underrepresented in eukaryotic genes; 1, 4), while CT or TG doublets (which are enriched in eukaryotic genes ibid.) were created when possible.

Choices were also made to utilize a diversity of codons for Met, Trp, Asn, Asp, Cys, Glu, His, Ile, Lys, Phe, Thr, and Tyr.

The sequences were also designed to encode unique 6-bp recognition sites for restriction enzymes, spaced about every 1200 bp. Finally, an additional codon (GCT; Ala) was inserted at the second position to encode an Nco I recognition site encompassing the ATG (Met) start codon. Additional recognition sites were included after the stop codon to facilitate subsequent cloning steps into expression vectors. These features are set forth above in Tables 1 and 2.

The new tcdA and tcbA genes of SEQ ID NO:3 and SEQ ID NO:4 share 73.5%, and 72.6%% identity, respectively, to their native W-14 counterparts (Wisconsin Genetics Computer Group, GAP algorithm).

B. Gene Synthesis

The complete synthesis of the plant codon-biased tcbA and tcdA genes was performed under contract by Operon Technologies, Inc. (OPTI, Alameda, Calif.). Basically, chemically synthesized oligonucleotides of appropriate sequence were assembled into DNA pieces about 500 bases long. These were joined together end-to-end (presumably by means of appropriately placed restriction enzyme sites) into four larger pieces of roughly 2 kilobase pairs (kbp) each; therefore each comprised about ¼ of the entire coding region of the particular gene. DNA sequence of the pieces was confirmed at this step. If mistakes in sequence were present, the appropriate oligonucleotides were re-synthesized, and the assembly process was repeated. Once gene fractional parts were sequence verified, they were assembled in pairs to make the gene halves, and again sequence verified. Finally, the two halves were joined, and the sequences of the junctions between the halves was verified. Therefore, each part of the new gene was sequence verified at least twice.

It should be noted that attempts to express the native tcbA or tcdA genes in standard Escherichia coli cloning strains suggests that production of these proteins is lethal. Lethality problems may be encountered if standard cloning vectors having leaky expression from inherent lacZ promoters are used to assemble these genes.

C. Addition of Endoplasmic Reticulum Targeting Peptide to Tcda Coding Region

It is known to those in the field of plant gene expression that proteins are specifically directed into the endoplasmic reticulum (ER) by means of a short signal peptide which is removed during or after the transport process through the ER membrane. The mature (processed) protein is incorporated into the ER endomembrane or is released into the ER lumen where the transported protein may be uniquely folded (aided by chaperoning), modified by glycosylation, accumulated in the vacuole, or additionally translocated (by secretion). These processes are reviewed by Gomord and Faye [V. Gomord and L. Faye, (1996) Signals and mechanisms involved in intracellular transport of secreted proteins in plants . Plant Physiol. Biochem. 34:165-181] and by Bar-Peled et al. [M. Bar-Peled, D. C. Bassham, and N. V. Raikhel, (1996) Transport of proteins in eukaryotic cells: more questions ahead . Plant Molec. Biology 32:223-249]. It is also known that the subcellular recognition mechanisms for an ER signal peptide are evolutionarily somewhat conserved, since the ER signal for a protein normally produced in monocot (maize) cells is recognized and processed normally by dicot (tobacco) cells. This is exemplified by the maize 15 kDa zein ER signal peptide [L. M. Hoffman, D. D. Donaldson, R. Bookland, K. Rashka, and E. M. Herman, (1987) Synthesis and protein body deposition of maize 15- kd zein in transgenic tobacco seeds . EMBO J. 6:3213-3221, and U.S. Pat. No. 5,589,616]. Further, it is known that the ER signal peptide derived from one protein can direct the translocation of a different protein if it is appropriately attached to the second protein by genetic engineering methods [D. C. Hunt and M. J. Chrispeels, (1991) The signal peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein . Plant Physiol. 96:18-25, and Denecke, J., J. Botterman, and R. Deblaere (1990) Protein secretion in plants can occur via a default pathway . Plant Cell 2:51-59]. Therefore, one may expose a protein in vivo to different biochemical environments by directing its accumulation in the cytosol (by not providing a signal peptide sequence), or in the ER/vacuole (by provision of an appropriate signal peptide.)

The ER signal peptide of maize 15 kDa zein proteins is known to comprise the first 20 amino acids encoded by the zein coding region. Two examples of such signal peptides the ER signal peptide of 15 kDa zein from A5707 maize, NCBI Accession #M72708, and the ER signal peptide of 15 kDa zein from Black Mexican Sweet maize, NCBI Accession #M13507. There is only a single amino acid difference (Ser vs Cys at residue 17) between these signal peptides.

SEQ ID NO:5 is a modified sequence coding the ER signal peptide of 15 kDa zein from Black Mexican Sweet maize. The modifications embodied in this sequence were made to accommodate the different monocot/dicot codon usages and other sequence motif considerations discussed above in the design of the plant-optimized tcdA coding region. The sequence includes an additional Ala residue at position #2 to accommodate the NcoI site which spans the ATG start codon.

SEQ ID NO:6 gives a sequence coding for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).

EXAMPLE 2

Transformation of Tobacco with Agrobacterium Carrying Plasmid pDAB2041 Encoding Photorhabdus Toxins

A. Plasmid pDAB2041

Preparation of tobacco transformation vectors was accomplished in three steps. First, a modified plant-optimized tcdA coding region was ligated into a tobacco plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in tobacco plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. Two plasmids designed to function in this role are pDAB1507 and pDAB2006. In the second step, the complete gene comprised of the promoter, coding region, and terminator region was ligated between the T-DNA borders of an Agrobacterium binary vector, pDAB1542. Also positioned between the T-DNA borders was a plant selectable marker gene to allow selection of transformed tobacco plant cells. In the third step, the engineered binary vector plasmid was conjugated from its E. coli host strain into a disabled Agrobacterium tumefaciens strain capable of transforming tobacco plant cells that regenerate into fertile transgenic plants.

It is a feature of plasmid pDAB1507 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB1507, is placed under the transcriptional control of an enhanced version of the CaMV 35S promoter. It is also a feature of pDAB1507 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a modified version of the 5′ UTR of the MSV coat protein gene, into which has been cloned an internally deleted version of the maize Adh1S intron 1. Additionally it is a feature of pDAB1507 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB1507 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of plasmid pDAB2006 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB2006, is placed under the transcriptional control of the CaMV 35S promoter. It is also a feature of pDAB2006 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a polylinker. Additionally it is a feature of pDAB2006 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB2006 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of pDAB1542 that any DNA fragment flanked by NotI sites can be cloned into the unique NotI site of pDAB1542, thus placing the introduced fragment between the T-DNA borders, and adjacent to the neomycin phosphotransferase II (kanamycin resistance) gene.

To prepare a plant-expressible gene to produce the non-targeted TcdA protein in tobacco plant cells, DNA of a plasmid (pAOH — 4-OPTI) containing the plant-optimized tcdA coding region, (SEQ ID No:3) was cleaved with restriction enzymes NcoI and SacI, and the large 7550 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1507 to produce plasmid pDAB2040. DNA of pDAB2040 was then digested with NotI, and the 8884 bp fragment was ligated to NotI digested DNA of pDAB1542 to produce plasmid pDAB2041. This plasmid was then conjugated by triparental mating [Firoozabady, E., D. L. DeBoer, D. J. Merlo, E. L. Halk, L. N. Amerson, K. E. Rashka, and E. E. Murray (1987) Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants . Plant Molec. Biol. 10:105-116] from the host Escherichia coli strain (XL1-Blue, Stratagene, La Jolla, Calif.), into the nontumorigenic Agrobacterium tumefaciens strain EHA101S, which is a spontaneous streptomycin-resistant mutant of strain EHA101 (Hood, E. E., G. L. Helmer, R. T. Fraley, and M. D. Chilton (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo 542 outside of T-DNA . J. Bacteriol. 168:1291-1301). Strain EHA101S(pDAB2041) was then used to produce transgenic tobacco plants that expressed the TcdA protein.

B. Plasmid pRK2013

To prepare a plant-expressible gene to produce the endoplasmic reticulum-targeted TcdA protein in tobacco plant cells, DNA of a plasmid (pAOH — 4-ER) containing the plant-optimized, ER-targeted tcdA coding region, (SEQ ID No:6) was cleaved with restriction enzymes NcoI and SacI, and the large 7610 bp fragment was ligated to similarly-cut DNA of plasmid pDAB2006 to produce plasmid pDAB1833. DNA of pDAB1833 was then digested with NotI, and the 8822 bp fragment was ligated to NdtI digested DNA of pDAB1542 to produce plasmid pDAB2052. This plasmid was then conjugated by triparental mating from the host Escherichia coli strain (XL1-Blue), into the nontumorigenic Agrobacterium tumefaciens strain EHA101S. Strain EHA101S (pDAB2052) was then used to produce transgenic tobacco plants that expressed the TcdA protein containing an amino terminus endoplasmic reticulum targeting peptide.

C. Transfer of Plasmid pDAB2041 into Agrobacterium Strain EHA101S

Cultures of E. coli carrying the engineered Ti plasmid pDAB2041 (plasmid containing the rebuilt Toxin A gene, tcdA), E. coli carrying the plasmid pRK2013, and Agrobacterium strain EHA101S were grown overnight, then mixed 1:1:1 on plain LB medium solidified with agar and cultured in the dark at 28° C. Two days later, the lawn of bacteria was scraped up with a loop, suspended in plain LB medium, vortexed, and then diluted 1:10 4 , 1:10 5 , and 1:10 6 fold in plain LB liquid medium. Aliquots of these dilutions were spread on selective plates containing medium YEP plus erythromycin (100 mg/L) and streptomycin (250 mg/L) and grown at 28° C. Two days later, single colonies were picked and streaked onto the same medium, then spread to give single colonies. Single colonies were picked again and streaked, then spread for single colonies. Single colonies were picked a third time, grown as streaks, then subjected to a quality analysis involving growth on lactose medium and chromogenic assay with Benedict's reagent. Of ten strains developed in this way, the fastest coloring colony was chosen for further work.

D. Transformation of Tobacco with Agrobacterium Carrying Plasmid pDAB2041

Tobacco transformation with Agrobacterium tumefaciens was carried out by a method similar, but not identical, to published methods (R Horsch et al, 1988. Plant Molecular Biology Manual, S. Gelvin et al, eds., Kluwer Academic Publishers, Boston). To provide source tissue for the transformation, tobacco seed ( Nicotiana tabacum cv. Kentucky 160) were surface sterilized and planted on the surface of TOB−, which is a hormone-free Murashige and Skoog medium (T. Murashige and F. Skoog, 1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol. 75: 473-497) solidified with agar. Plants were grown for 6-8 weeks in a lighted incubator room at 28-30° C. and leaves were collected sterilely for use in the transformation protocol. Approximately one cm 2 pieces were sterilely cut from these leaves, excluding the midrib. Cultures of the Agrobacterium strains (EHA101S containing pDAB2041), which had been grown overnight on a rotor at 28° C., were pelleted in a centrifuge and resuspended in sterile Murashige & Skoog salts, adjusted to a final optical density of 0.7 at 600 nm. Leaf pieces were dipped in this bacterial suspension for approximately 30 seconds, then blotted dry on sterile paper towels and placed right side up on medium TOB+ (Murashige and Skoog medium containing 1 mg/L indole acetic acid and 2.5 mg/L benzyladenine) and incubated in the dark at 28° C. Two days later the leaf pieces were moved to medium TOB+ containing 250 mg/L cefotaxime (Agri-Bio, North Miami, Fla.) and 100 mg/L kanamycin sulfate (AgriBio) and incubated at 28-30° C. in the light. Leaf pieces were moved to fresh TOB+ with cefotaxime and kanamycin twice per week for the first two weeks and once per week thereafter. Leaf pieces which showed regrowth of the Agrobacterium strain were moved to medium TOB+ with cefotaxime and kanamycin, plus 100 mg/l carbenicillin (Sigma). Four to six weeks after the leaf pieces were treated with the bacteria, small plants arising from transformed foci were removed from this tissue preparation and planted into medium TOB− containing 250 mg/L cefotaxime and 100 mg/L kanamycin in Magenta GA7 boxes (Magenta Corp., Chicago). These plantlets were grown in a lighted incubator room. After 3-4 weeks the primary transgenic plants had rooted and grown to a size sufficient that leaf samples could be analyzed for expression of protein from the transgene. Twenty-five independent transgenic events were recovered as single plants from the pDAB2041 transformation.

Eight independent lines expressing various levels of transgenic protein from the T-DNA of pDAB2041 were propagated in vitro from leaf pieces as follows. Twelve to sixteen approximately one cm 2 pieces were sterilely cut from leaves of each primary transgenic plant, excluding the midrib and all naturally occurring edges. These leaf pieces were placed on medium TOB+ containing 250 mg/L cefotaxime and 100 mg/L kanamycin, and cultured in the lighted incubator at 28-30° C. for 3-4 weeks, at which time small plants could be cut from the proliferating tissue mass. Several small plantlets from each transgenic line were moved into Magenta boxes containing medium TOB− plus cefotaxime and kanamycin and allowed to root and grow. The proliferating tissue mass was further cultured on medium TOB+ with cefotaxime and kanamycin, and additional plants could be cut out and grown up as needed.

Plants were moved into the greenhouse by washing the agar from the roots, transplanting into soil in 5{fraction ( 1 / 2 )}″ square pots, placing the pot into a Ziploc bag (DowBrands), placing plain water into the bottom of the bag, and placing in indirect light in a 30° C. greenhouse for one week. After one week the bag could be opened; the plants were fertilized and allowed to grow further, until the plants were acclimated and the bag was removed. Plants were grown under ordinary warm greenhouse conditions (30° C., 16 H light). Plants were suitable for sampling four weeks post transplant.

EXAMPLE 3

Chacterization of Transgenic Tobacco Plants Expressing Photorhabdus Toxin that Confer Insect Control

A. Polyclonal Antibody Production

The E. coli produced recombinant TcdA protein was purified by a series of column purification. The protein was sent to Berkley Antibody Company (Richmond, Calif.) for the production of antiserum in a rabbit. Inoculations with the antigen were initiated with 0.5 mg of protein followed by four boosting injections of 0.25 mg each at about three week intervals. The rabbit serum was tested by the standard Western analysis using the recombinant TcdA protein as the antigen and enhanced chemi-luminescens, ECL method (Amersham, Arlington Heights, Ill.). The antibodies (PAb-EA 0 ) were purified using a PURE I antibody purification kit (Sigma, St. Luis, Mo.). PAb-EA 0 antibodies recognize the full-length TcdA and its processed components.

B. Expression of TcdA Protein in Tobacco

Protein was extracted from the leaf tissue of transformed and non-transformed tobacco plants following the procedure described immediately below.

Two leaf disks of 1.4 cm in diameter were harvested from the middle portion of a fully expanded leaf. The disks were placed on a 1.6×4 cm piece of 3M Whatman paper. The paper was folded lengthwise and inserted in a flexible straw. Four hundred micro liters of the extraction buffer (9.5 ml of 0.2 M NaH 2 PO 4 , 15.5 ml of 0.2 M Na 2 HPO 4 , 2 ml of 0.5 M Na 2 EDTA, 100 ml of Triton X100, 1 ml of 10% Sarkosyl, 78 ml of beta-mercaptoethanol, H 2 O to bring total volume to 100 ml) was pipetted on to the paper. The straw containing the sample was then passed through a rolling device used for squeezing out the extract 1.5 mL micro centrifuge tube was placed at the other end of the straw to collect the extract. The extract was centrifuged for 10 minutes at 14,000 rpm in an Eppendorf regrigerated microcentrifuge. The supernatant was transferred into a new tube. Protein quantitation analysis was performed using the standard Bio-Rad Protein Analysis protocol (Bio-Rad Laboratories, Hercules, Calif.). The extract was diluted to 2 mg/ml of total protein using the extraction buffer.

For the detection of transgenic protein, Western blot analysis was performed. Following a standard procedure for protein separation (Laemmli, 1970), 40 μg of protein was loaded in each well of 4-20% gradient polyacrylamide gel (Owl Scientific Co., Mass.) for electrophoresis. Subsequently, the protein was transferred onto a nitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKB Biotechnology, Piscataway, N.J.). The membrane was incubated for one hour in Blotto (5% milk in TBST solution; 25 mM Tris HCL pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, Blotto was replaced by the primary antibody solution (in Blotto). After one hour in the primary antibody, the membrane was washed with TBST for five minutes three times. Then the secondary antibody in Blotto (1:2000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase; Bio-Rad Laboratories). was added to the membrane. After one hour of incubation, the membrane was washed with an excess amount of TBST for 10 minutes four times. The protein was visualized by using the enhanced chemi-luminescens, ECL method (Amersham, nArlington Heights, Ill.). The differential intensity of the protein bands were measured using densitometer (Molecular Dynamics Inc., Sunnyvale, Calif.).

To determine the expression of TcdA protein in tobacco transformed with pDAB2041, PAb-EA 0 antibodies were used as the primary antibodies. The expression levels of TcdA protein varied among independent transformation events. The primary plant generated from the event #2041-13 showed the highest level of pre-pro TcdA expression of extractable protein. When the leaf pieces from this plant (#2041-13) were used in in vitro propagation, several plants were obtained. Seven of these plants were analyzed for the expression of the TcdA protein. All but one plant produced the full-length TcdA protein as well as some processed peptide components. Using the antibodies specific to Neomycin phospho-transferase, NPT (5 prime-3 prime, Boulder, Colo.), the expression the selectable marker gene (npt II) was detected. Similar results were obtained for #2041-29.

TABLE 5
Western analysis of plants derived from event #2041-13.
			NPT
	Plant #	TcdA	(selectable marker)
	2041-13A	+	not done
	2041-13B	+	not done
	2041-13-1	−	+
	2041-13-2	+	+
	2041-13-3	+	+
	2041-13-4	+	+
	2041-13-5	+	+

C. Nucleic Acid Analysis of Transgenic Tobacco Lines

Genomic DNA was prepared from a group of 2041 transgenic events. The lines included Magenta box stage 2041-13, and greenhouse stage plants 2041-13-1, 2041-13-2, 2041-13-5, 2041-9, 2041-20A and 2041-20B. A transgenic GUS line (2023) was included as a negative control. Southern analysis of these lines was performed. The genomic tobacco DNA was restricted with the enzyme SstI which should result in a 8.9 kb hybridization product when hybridized to a tcdA gene specific probe. The 8.9 kb hybridization product should consist of the 35T promoter and the tcdA coding region. All 2041 plants contained a band of the expected size. Events 2041-9 and -20 appear to be the same line with 5 identical hybridizing bands. Event 2041-13 produced 6 hybridization fragments with the tcdA coding region probe. Magenta box and various greenhouse plants of 2041-13 all produced the same hybridization profile. This hybridization pattern was different from that of events 2041-9 and -20.

RNA analysis, using the tcdA coding region probe, was performed on the same group of greenhouse 2041 plants. Immunoblot analysis had revealed that plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1 produced no detectable TcdA protein; while 2041-13-2 and 2041-13-5 produced substantial amounts of full-length TcdA. Northern analysis was in agreement with the immunoblot result. A faint RNA signal was detected for plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1. Only faintly visible was a band corresponding to full-length tcdA transcript in plant 2041-13.1. In contrast, for plants 2041-13-2 and 2041-13-5 a strong RNA signal was detected, with a substantial amount of full-length size (˜8.0 kb) tcdA transcript. These data support the observed bioassay activity for this group of plants.

Genomic DNA was prepared from a second functionally active 2041 transgenic event, 2041-29. Southern analysis of this line was performed. A transgenic GUS line (2023) was included as a negative control, DNA of line 2041-9 was included as a positive control.

The genomic tobacco DNAs were restricted with the enzyme SstI which should result in a 8.9 kb hybridization product when hybridized to a tcdA gene specific probe. The 8.9 kb hybridization product should consist of the 35T promoter and the tcdA coding region. For plant 2041-29-5, three hybridization products larger than 8.9 kb the were detected with the tcdA gene specific probe. Immunoblot analysis has demonstrated pre-pro TcdA protein is made by this plant, it is therefore likely that a restriction site was lost during transformation or regeneration, or the 2041-29 genomic DNA was not thoroughly digested.

D. Tobacco Leaf-Disk Tests with Tobacco Hornworm Exhibiting Insect Control

Leaves were sampled from tobacco plants, Nicotiana tabaco , previously transplanted into the greenhouse. A single leaf was sampled from each plant.on each test date. Leaves were selected from the zone where younger elongate leaves transition into older ovate leaves. Excised leaves were placed into 12 oz. cups with the petiole submerged in water to maintain turgor, and transported to the laboratory.

Eight, 1.4 cm disks were cut from the center portion of one side of each leaf (right adaxial side up, with distal portion facing away from the observer). Each disk was placed individually into a well of a C-D International 128 well tray (Pitman, N.J.) into which 0.5 ml of a 1.6% aqueous agar solution had been previously pippetted. The solidified agar prevented the leaf disks from drying out. The adaxial surface of the disk was always oriented up.

A single neonate tobacco hornworm, Manduca sexta , was placed on each disk and the wells were sealed with vented plastic lids. The assay was held at 27° C. and 40% RH. Larval mortality and live-weight data were collected after 3 days. Data were subjected to analysis of variance and Duncan's multiple range test (α=0.05) (Proc GLM, SAS Institute Inc., Cary, N.C.). Data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

TABLE 6
Results of leaf-disk assays from greenhouse grown tobacco
plants with event 2041-13.
	Weight of Surviving Larvae (mg) & Duncan's Group 1
		Plant		Test	Test	Test	3 Test
TRT	Plant	Age	Pretest	1	2	3	Sum.
13	non-transformed - 2	young	—	—	—	18.8 a*	—
14	non-transformed - 3	young	—	—	—	17.0 ab
16	non-transformed - 5	young	—	—	—	16.4 ab
3	2041-13-1 (western −)	young	—	17.6 a	18.2 a	16.1 ab	17.3 a
9	Gus Control	old	19.3 a	14.6 a	16.3 a	14.5 ab	15.1 a
10	non-transformed - 1	young	—	8.3 b	16.8 a	13.9 b	13.0 b
11	2041-20B (western −)	old	—	10.0 b*	13.7 ab	14.6 ab	12.9 b
15	non-transformed - 4	young	—	—	—	13.0 bc	—
8	2041-20A (western −)	old	15.7 a	8.3 b	11.3 bc	9.2 cd	9.6 c
12	2041-9 (western −)	old	19.5 a	—	—	7.9 d	—
7	2041-13-5 (western +)	young	—	6.3 bc	9.6 cd	7.2 de	7.7 d
5	2041-13-3 (western +)	young	—	6.4 bc****	6.2 e	6.8 de**	6.4 de
1	2041-13A (western +)	old	7.2 b	6.8 bc*	7.0 de*	5.4 e	6.4 de
6	2041-13-4 (western +)	young	—	4.9 c****	5.8 e	7.6 d	6.4 de
4	2041-13-2 (western +)	young	—	5.7 bc	5.7 e**	7.5 d	6.3 de
2	2041-13B (western +)	old	—	4.7 c**	5.6 e	7.2 de	5.9 e
*Number of stars corresponds to the number of dead larvae per 8 tested.

1. Data transformed (logarithm) for analysis.

Means followed by the same letter are not significantly different (alpha=0.05).

TABLE 7
Results Of Leaf-Disk Assays From Greenhouse Grown Tobacco
Plants
With Event 2041-29.
	MEAN WGT (MG)/Duncan's Group
	Test	Test	Test	Test	Four Test
Plant	1	2	3	4	Summary
2014-6	15.8 a	16.6 a	**5.5 bc	*12.9 ab	13.2 a
GUS 1
2014-6	14.4 a	*6.6 bc	*13.4 a	15.2 a	12.6 a
GUS 2
KY-160	13.4 a	6.7 bc	7.9 b	8.5 bc	9.1 b
NTC
2041-29	*4.9 b	*7.3 b	****6.9 b	********	6.3 c
4P
2041-29 7	*5.9 b	5.1 bc	***6.7 b	***7.2 c	6.1 c
2041-29	*5.6 b	**7.9 b	*****6.5 b	***3.6 d	5.9 c
3P
2041-29	6.3 b	****4.7 c	******4.1 c	******4.6 d	5.4 c
2P
*Number of stars corresponds to the number of dead larvae per 8 tested.

1. Data transformed (logarithm) for analysis.

Means followed by the same letter are not significantly different (alpha=0.05).

All event 2041-29 plants significantly depressed THW larval weight gain compared to control plants. Average weight depression was 49%. Statistically significant mortality occurred in THW larvae exposed to foliage from 2041-29 plants. Mortality averaged 37.5% compared to 5.2% in controls.

E. Isolation and Characterization of Functional Photorhabdus Toxin Protein From Transgenic Plants

Seven grams of transgenic tobacco plants (2041-13) expressing TcdA (Toxin A) gene were homogenized with 10 ml 50 mM Potassium Phosphate buffer, pH 7.0 using a bead beater (Biospec Products, Bartlesville, Okla.) according to manufacturer's instructions. The homogenate was filtered through four layers of cheese cloth and then centrifuged at 35,000 g for 15 min. The supernant was collected and filtered through 0.22 μm Millipore Express™ membrane. It was then applied to a Superdex 200 cloumn (2.6×40 cm) which had been equilibrated with 20 mM Tris buffer, pH 8.0 (Buffer A). The protein was eluted in Buffer A at a flow rate of 3 ml/min. Fractions with 3 ml each were collected and subjected to southern corn rootworm (SCR) bioassay. It was found that fractions corresponding to a native molecular weight around 860 kDa had the highest insecticidal activity. Western analysis of the active fraction using a polyclonal antibody specific to Toxin A indicated the presence of full-length TcdA peptide. The active fractions were further combined and applied to a Mono Q 10/10 column which had been equilibrated with Buffer A. Proteins bound to the column were then eluted by a linear gradient of 0 to 1 M NaCl in Buffer A. Fractions with 2 ml each were collected and analyzed by both SCR bioassay and Western using antibody specific to Toxin A. The results again demonstrated the correlation between insecticidal activity and presence of full-length TcdA peptide.

F. Characterization of Progeny Transgenic Plants

The inheritability of the genetically engineering plants containing the Photorhabdus toxin gene was evaluated by generating F1 progeny. Progeny was generated from 2041-13 event by selfing expression positive plants. The 2041-13 plants in the greenhouse were allowed to self-pollinate. Seed capsules were collected when mature and were allowed to dry and after-ripen on the laboratory bench for two weeks. Seed from plant designated 2041-13A was surface-sterilized and distributed on the surface of medium TOB− without selection, to allow recovery of nonexpressing or nontransgenic progeny as well as expressing and segregating transgenic siblings. Seed was germinated in a C lighted incubator room (16 H light, 28 C). After 1 month, fifty-one seedlings, designated 2041-13A-S1 through S51, were distributed into Magenta boxes containing medium TOB− to grow further. Three weeks later, leaf samples from these Magenta-box grown seedlings were submitted for evaluation of the level of expression of TcdA toxin.

Leaf samples were tested for kanamycin response by placing sterile leaf segments on medium TOB+ containing 100 mg/L kanamycin in the light and scoring for tissue growth and color after two weeks. All leaf pieces showed some positive response, indicating complex segregation.

This group of in vitro grown event 2041-13 progeny seedlings were all transplanted into the greenhouse approximately two months after seeding onto medium, using the following method. After washing the agar from the roots, _plants were transplanted into 5½ inch square pots in a soil mix containing 75% MetroMix and 25% mineral soil. They were enclosed in a zip-lock bag and plain water added to leave 1-2 inches of water in the bottom of the bag after soil absorption. These bags were closed and placed under a cart in the greenhouse to protect them from direct sunlight. The bags were opened after 5-6 days, and removed after 7 days, when the plants were adapted to soil and were moved to the top of the cart for normal greenhouse culture. Plants were ready to test in insect bioassays at four weeks post transplant.

F1 progeny were evaluated for expression of protein toxin by immunological screen and for biological activity by plant bioassays, as described previously, using tobacco hornworm. There existed a positive correlation between levels of expression protein toxin and degree of growth inhibition and at higher expression levels mortality was observed. The biological activity was observed to be statistical significance with high cofidence levels between populations of non-transformed and transformed expressing protein toxin.

The following table summarizes the results of insect (tobacco hornworm) bioassays conducted with F1 progeny of self-fertilized 2041-13 plants genetically engineered to produce the “204” A toxin. The tests included 6 non-expressing progeny (protein-negative controls), 45 toxin A expressors, and 4 non-transformed controls (KY-160). Results are from three leaf-disk assays (method previously outlined) where eight disks were used per test. The data were analyzed using analysis of variance and were blocked by test.

The treatment effect for each of these analyses indicated the Pr>F was less than 0.0001. The Toxin A expressors produced significant control of tobacco hornworm compared to each of the control groups based on each of the three measures of efficacy. The two control groups behaved similarly. Statistical analysis using ANOVA and an LSD test with alpha equal to 0.01 (or 1%) showed differences between the 3 groups. The LSD test indicated that the non-expressors and the non-transformed plants were similar in larvae weights but the expressors gave weights significantly lower than either of the other two groups of plants. These data demonstrated that the genetic basis for insect control was inheritable and corresponded to the presence of expressed toxin gene.

TABLE 8
Tobacco hornworm results from F1 progeny of
self-fertilized 2041-13 tobacco plants.
	Mean Value and Duncan's Grouping d
		Total	Survivor	Leaf
	Treatment	Weight	Weight	Area
	Group	(mg) a	(mg) b	(cm 2 ) c
	Non-transformed	15.8 a	15.8 a	1.2 a
	Control
	Protein-negative	16.4 a	16.5 a	1.2 a
	Control
	Toxin A	8.1 b	9.2 b	4.9 b
	Expressor
	a Average insect weight with dead insects considered to weigh nothing.
	b Average insect weight with dead insects excluded from analysis.
	c Total leaf area remaining per eight leaf disks. Initial area was approximately 12 cm 2 .
	d Means followed by the same letter are not significantly different (alpha = 0.05).

EXAMPLE 4

Transformation of Maize with a Vector Carrying Plasmid pDAB1834 Encoding Photorhabdus Toxins

A. Preparation of Maize Transformation Vectors Containing Modified Plant-Optimized Tcda Coding Regions: Plasmid Pdab1834

Preparation of maize transformation vectors was accomplished in two steps. First, a modified plant-optimized tcdA coding region was ligated into a plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in maize plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. One plasmid designed to function in this role is pDAB1538. In the second step, the complete gene comprised of the promoter, coding region, and 3′ UTR terminator region was ligated to a plant transformation vector that contained a plant expressible selectable marker gene which allowed the selection of transformed maize plant cells amongst a background of nontransformed cells. An example of such a vector is pDAB367.

It is a feature of plasmid pDAB1538 that any coding region having an NcoI site at its 5′ end and a SacI site 3′ to the coding region, when cloned into the unique NcoI and SacI sites of pDAB1538, is placed under the transcriptional control of the maize ubiquitin1 (ubi1) promoter. It is also a feature of pDAB1538 that the 5′ untranslated leader (UTR) sequence preceding the NcoI site comprises a polylinker. Additionally it is a feature of pDAB1538 that transcription termination and polyadenylation of the mRNA containing the introduced coding region are mediated by termination/Poly A addition sequences derived from the nopaline synthase (Nos) gene. Finally, it is a feature of pDAB1538 that the entire assembly of promoter/coding region/3′UTR can be obtained as a single DNA fragment by cleavage at the flanking NotI sites.

It is a feature of pDAB367 that the phosphinothricin acetyl transferase protein, which has as its substrate phosphinothricin and related compounds, is produced in plant cells through transcription of its coding region mediated by the Cauliflower Mosaic Virus 35S promoter and that termination of transcription plus polyadenylation are mediated by the nopaline synthase terminator region. It is further a feature of pDAB367 that any DNA fragment containing flanking NotI sites can be cloned into the unique NotI site of pDAB367, thus physically linking the introduced DNA fragment to the aforementioned selectable marker gene.

To prepare a maize plant-expressible gene to produce the endoplasmic reticulum-targeted TcdA protein in plant cells, DNA of a plasmid (pAOH — 4-ER) containing the plant-optimized, ER-targeted tcdA coding region, (SEQ ID No:6) was cleaved with restriction enzymes NcoI and SacI, and the large 7610 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1538 to produce plasmid pDAB1832. DNA of pDAB1832 was then digested with NotI, and the 9984 bp NotI fragment was ligated into the unique NotI site of pDAB367 to produce plasmid pDAB1834.

It is a feature of plasmids pDAB1834 that the ubi1 and 35S promoters are encoded on the same DNA strand.

B. Transformation and Regeneration of Transgenic Maize Isolates

Type II callus cultures were initiated from immature zygotic embryos of the genotype “Hi-II.” (Armstrong et al, (1991) Maize Genet. Coop. Newslett., 65: 92-93). Embryos were isolated from greenhouse-grown ears from crosses between Hi-II parent A and Hi-II parent B or F 2 embryos derived from a self- or sib-pollination of a Hi-II plant. Immature embryos (1.5 to 3.5 mm) were cultured on initiation medium consisting of N6 salts and vitamins (Chu et al, (1978) The N6 medium and its application to anther culture of cereal crops. Proc. Symp. Plant Tissue Culture, Peking Press, 43-56), 1.0 mg/L 2,4-D, 25 mM L-proline, 100 mg/L casein hydrolysate, 10 mg/L AgNO 3 , 2.5 g/L GELRITE (Schweizerhall, South Plainfield, N.J.), and 20 g/L sucrose, with a pH of 5.8. After four to six weeks callus was subcultured onto maintenance medium (initiation medium in which AgNO 3 was omitted and L-proline was reduced to 6 mM). Selection for Type II callus took place for ca. 12-16 weeks.

Plasmid pDAB1834 was transformed into embryogenic callus. For blasting, 140 μg of plasmid DNA was precipitated onto 60 mg of alcohol-rinsed, spherical gold particles (1.5-3.0 μm diameter, Aldrich Chemical Co., Inc., Milwaukee, Wis.) by adding 74 μL of 2.5M CaCl 2 H 2 O and 30 μL of 0.1M spermidine (free base) to 300 μL of plasmid DNA and H 2 O. The solution was immediately vortexed and the DNA-coated gold particles were allowed to settle. The resulting clear supernatant was removed and the gold particles were resuspended in 1 ml of absolute ethanol. This suspension was diluted with absolute ethanol to obtain 15 mg DNA-coated gold/mL.

Approximately 600 mg of embryogenic callus tissue was spread over the surface of Type II callus maintenance medium as described herein lacking casein hydrolysate and L-proline, but supplemented with 0.2 M sorbitol and 0.2 M mannitol as an osmoticum. Following a 4 h pre-treatment, tissue was transferred to culture dishes containing blasting medium (osmotic media solidified with 20 g/L TC agar (PhytoTechnology Laboratories, LLC, Shawnee Mission, Kans.) instead of 7 g/L GELRITE. Helium blasting accelerated suspended DNA-coated gold particles towards and into the prepared tissue targets. The device used was an earlier prototype of that described in U.S. Pat. No. 5,141,131 which is incorporated herein by reference. Tissues were covered with a stainless steel screen (104 μm openings) and placed under a partial vacuum of 25 inches of Hg in the device chamber. The DNA-coated gold particles were further diluted 1:1 with absolute ethanol prior to blasting and were accelerated at the callus targets four times using a helium pressure of 1500 psi, with each blast delivering 20 μL of the DNA/gold suspension. Immediately post-blasting, the tissue was transferred to osmotic media for a 16-24 h recovery period. Afterwards, the tissue was divided into small pieces and transferred to selection medium (maintenance medium lacking casein hydrolysate and L-proline but containing 30 mg/L BASTA® (AgrEvo, Berlin, Germany)). Every four weeks for 3 months, tissue pieces were non-selectively transferred to fresh selection medium. After 7 weeks and up to 22 weeks, callus sectors found proliferating against a background of growth-inhibited tissue were removed and isolated. The resulting BASTA®-resistant tissue was subcultured biweekly onto fresh selection medium. Following western analysis, positive transgenic lines were identified and transferred to regeneration media. Western-negative lines underwent subsequent RNA spot blot analysis to identify negative controls for regeneration.

Regeneration was initiated by transferring callus tissue to cytokinin-based induction medium, which consisted of Murashige and Skoog salts, hereinafter MS salts, and vitamins (Murashige and Skoog, (1962) Physiol. Plant. 15: 473-497) 30 g/L sucrose, 100 mg/L myo-inositol, 30 g/L mannitol, 5 mg/L 6-benzylaminopurine, hereinafter BAP, 0.025 mg/L 2,4-D, 30 mg/L BASTA®, and 2.5 g/L GELRITE at pH 5.7. The cultures were placed in low light (125 ft-candles) for one week followed by one week in high light (325 ft-candles). Following a two week induction period, tissue was non-selectively transferred to hormone-free regeneration medium, which was identical to the induction medium except that it lacked 2,4-D and BAP, and was kept in high light. Small (1.5-3 cm) plantlets were removed and placed in 150×25 mm culture tubes containing SH medium (SH salts and vitamins (Schenk and Hildebrandt, (1972) Can. J. Bot. 50:199-204), 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL/L FeEDTA, and 2.5 g/L GELRITE, pH 5.8). Plantlets were transferred to 12 cm pots containing approximately 0.25 kg of METRO-MIX 360 (The Scotts Co. Marysville, Ohio) in the greenhouse as soon as they exhibited growth and developed a sufficient root system. They were grown with a 16 h photoperiod supplemented by a combination of high pressure sodium and metal halide lamps, and were watered as needed with a combination of three independent Peters Excel fertilizer formulations (Grace-Sierra Horticultural Products Company, Milpitas, Calif.). At the 6-8 leaf stage, plants were transplanted to five gallon pots containing approximately 4 kg METRO-MIX 360, and grown to maturity.

EXAMPLE 5

Characterization of Transgenic Maize Plants Expressing Photorhabdus Toxin that Confer Insect Control

A. Insect Bioassays

A single leaf was sampled from each plant in each test. Eight, 1.4 cm disks were cut from the outer portion of each leaf (approximately 30 cm long) avoiding the center vein. Each disk was placed individually into a well of a C-D International 128 well tray (Pitman, N.J.) into which 0.5 ml of a 1.6% aqueous agar solution had been previously pipetted. The solidified agar prevented the leaf disks from drying out. The adaxial surface of the disk was always oriented up.

Five neonate southern corn rootworms, Diabrotica undecimpunctata howardi, were placed on each disk and the wells were sealed with vented plastic lids. The assay was held at 27° C. and 40% RH. Larval mortality and live-weight data were collected after 3 days. Data were subjected to analysis of variance and Duncan's multiple range test (α=0.05)(Proc GLM, SAS Institute Inc., Cary, N.C.). Weight data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

TABLE 9
Results of Maize Leaf-disk Test vs SCR
			Mean Survival
		Mean % Kill	Weight (mg)
	Treatment	(Duncan's)	(Duncan's)
	1834 - 11	68 A	0.064 A
	1834 - 17	44 B	0.098 B
	1834 - 15	26 BC	0.127 C
	HiII control	13 C	0.161 C
	Note:
	Means followed by the same letter are not significantly different based on Duncan's multiple range test (alpha = 0.05). Insect groups weighing less than 0.1 mg were set to 0.03 mg instead of zero to conduct a more conservative analysis. Mortality (arcsin(sqrty)) and weight(log10) data were transformed for analyses.

The results shown in Table 9 demonstrated that two events expressing TcdA protein were statistically distinct from control lines bioassayed using SCR neonates by mortality and survival weight criteria. These results demonstrated that southern corn rootworm were functionally effected by feeding on maize plants containing and expressing the tcdA gene. Those plants from 1834-11 were used to generate progeny for testing of inheritability of transgene.

B. Production and Progeny Test of tcdA Transgenic Maize

Origin and growth of progeny plants: Sibling plants 1834-11-07 and 1834-11-08, clonally derived by regeneration from the callus of transgenic maize event 1834-11, were transplanted to the greenhouse and pollinated with inbred OQ414. Seeds obtained from these crosses, comprising seed lots 1834-11-07A and 1834-11-08A, were planted in Rootrainers (1 M inch×2 inch×8 inch deep, product #647, C. Hummert Intl., Earth City, Mo.) filled with Metro-Mix 360 soilless mix (Scotts Terra-Lite, available from Hummert Intl.) and top irrigated with Hoagland's nutrient solution. (Hoagland's solution contains 229 ppm nitrogen as nitrate, 24.6 ppm nitrogen as ammonium, 26 15 ppm P, 157 ppm K, 187 ppm Ca, 49 ppm Mg. and 30 ppm Na.)

Greenhouse conditions for this trial were: 16 hour days, daylight supplemented by metal halide lamps as needed to achieve a minimum of 600 ?Einsteins/cm 2 PAR, and ambient temperature 30 C days, 22 C nights.

Leaves were sampled for protein determination approximately one week after planting. Leaf bioassays were conducted 2-3 weeks after planting; root bioassays were initiated approximately 3 weeks post planting.

Protein analysis of progeny plants: Protein was extracted from leaf and root samples harvested from transgenic plants, line 1834-11 progenies, and non-transformed plants. Each sample was placed on a 1.6×4 cm piece of 3M Whatman™ paper. The paper was folded lengthwise and inserted in a flexible straw. A volume of 350 μl of an extraction buffer (9.5 ml of 0.2 M NaH 2 PO 4 , 15.5 ml of 0.2 M Na 2 HPO 4 , 2 ml of 0.5 M Na 2 EDTA, 100 ml of Triton X-100, 1 ml of 10% Sarkosyl, 78 ml of beta-mercaptoethanol, H 2 O to bring total volume to 100 ml, 50 μg/ml Antipain, 50 μg/ml Leupeptin, 0.1 mM Chymostatin, 5 μg/ml Pepstatin) was pipetted on to the paper. The straw containing the sample was then passed through a rolling device used for squeezing the extract into a 1.5 ml microcentrifuge tube.

The extract was centrifuged for 10 minutes at 14,000 rpm in an Eppendorf refrigerated micro-centrifuge. The supernatant was transferred into a new tube. The amount of the total extractable protein was determined using a standard BioRad Protein Analysis protocol (BioRad Laboratories, Hercules, Calif.).

The presence of the TcdA protein was visualized by Western blot analysis following a standard procedure for protein separation (Laemmli, 1970). A volume of twenty μl of extract was loaded in each well of 4-20% gradient polyacrylamide gel (Owl Scientific Co., Mass.) for electrophoresis. Subsequently, the protein was transferred onto a nitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKB Biotechnology, Piscataway, N.J.). The membrane was incubated for one hour in TBST-M solution (10% milk in TBST solution; 25 mM Tris HCL pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, the primary antibody (Anti-TcdA in TBST-M) was added. After one hour, the membrane was washed with TBST for five minutes, three times. Then the secondary antibody solution (goat anti-rabbit IgG conjugated to horseradish peroxidase; Bio-Rad Laboratories, in TBST-M) was added to the membrane. After one hour of incubation, the membrane was washed with an excess amount of TBST for 10 minutes, four times. The protein was visualized using the Super Signal® West Pico chemiluminescence method (Pierce Chemical Co., Rockford, Ill.). The protein blot was exposed on a Hyper-film (Amersham, Arlington Heights, Ill.) and was developed within 3 minutes. The intensity of the protein band was measured using a densitometer (Molecular Dynamics Inc., Sunnyvale, Calif.) and compared to standards.

Three of six plants from seed lot 1834-11-07A and three of six plants from seed lot 1834-11-08A produced detectable levels of TcdA protein (Table 1). Approximately 3.8 to 13.3 ppm of TcdA were detected in the leaf blades and 4.1 to 8.4 ppm were detected in the leaf tips of the protein-positive plants. The amounts of TcdA protein detected in the roots were slightly lower than those found in the leaves.

Insect bioassays with progeny plants: Plants were selected for bioassay based on results from Western blot analysis. Twelve (12), 6.4 mm diameter leaf discs were cut from the youngest leaf of each 2 week old seedling. Each disc was placed in a well of a 128-well tray (CD International) containing approximately 0.5 mL of a solidified 2% agar in water solution. Two neonate southern corn rootworm, Diabrotica undecimpunctata howardi (Barber)(SCR), were placed in each well with a leaf disc. Trays were covered with perforated lids and maintained under a controlled environment for 3 days (28 C; 16 hours light:8 hours dark; approx. 60% relative humidity). Living larvae from 4 leaf discs were pooled and weighed producing 3 weight determinations per plant. Average weights were calculated by dividing the pooled weight by the number of survivors. Differences in average weights of SCR fed leaf discs from protein positive and protein negative plants were assessed using analysis of variance on the natural log-transformed average weights (Minitab, v. 12.2, Minitab Inc., State College, Pa.).

Root bioassays were initiated approximately 1 week after the initiation of the leaf disc bioassays.

Approximately 24 h prior to eclosion, SCR eggs were suspended in a 0.15% solution of agar in water to a concentration of 100 eggs/ml. Plants were inoculated with SCR eggs by pipetting 2.0 ml of the egg suspension (ie., approximately 200 eggs) just below the soil surface at the base of each plant. Two weeks after inoculation, plants were removed from their Rootrainer pots, their roots washed free of potting mix, and scored for rootworm damage based on a 1 (resistant) to 9 (susceptible) rating system (Welch, 1977). The results of the root ratings were examined using non-parametric tests to determine if the distribution of root ratings from the protein positive plants was the same as the distribution of the ratings from the protein negative plants. Testing was done at the 5% significance level. (StatXact v.3, CYTEL Software Corporation, Cambridge Mass.)

Results from leaf and root bioassays of tcdA protein positive and protein negative progeny plants are summarized in Table 10. The average weights of SCR larvae fed leaf discs from protein positive plants were significantly lower than those of larvae fed leaf discs from protein negative plants (F=4.6; d.f.=1, 34; P≦0.001. The Kolmogorov-Smirnov 2 sample test (p=0.04) and the Wald Wolfowitz runs test (p=0.001) indicated that the protein positive and protein negative root rating distributions were not similar. The Wilcoxon-Mann-Whitney test (p=0.0206) and the Normal Scores test (p=0.206) indicated that the average score for the protein positive plants was lower than the average root rating from the protein negative plants.

TABLE 10
Protein analysis and insect bioassay results
with progeny of TcdA transgenic maize.
			Leaf Disc	Root Bioassay
	Plant	TcdA	Bioassay	Root Rating
	Number	Protein	Avg. Wt. (mg)	(1-9)
	1834-11-07A-30	PRO−	0.190	8
	1834-11-08A-21	PRO−	0.196	9
	1834-11-08A-16	PRO−	0.195	9
	1834-11-08A-14	PRO−	0.137	9
	1834-11-07A-22	PRO−	0.208	9
	1834-11-07A-20	PRO−	0.175	9
	1834-11-07A-26	PRO+	0.118	9
	1834-11-08A-17	PRO+	0.132	8
	1834-11-07A-14	PRO+	0.110	2
	1834-11-07A-11	PRO+	0.106	4
	1834-11-08A-28	PRO+	0.129	8
	1834-11-08A-27	PRO+	0.108	4

DNA analysis of progeny plants: Leaf samples from 1834-11.7A and 1834-11.8A progeny plants were in conical 50 ml polypropylene tubes and dried in a Labconco Freeze Dry Lyophilizer (Kansas City, Mo.) for 1-2 days. Lyophilized leaves were then ground in a Tecator Cyclotec 1093 Sample mill grinder (Hoganas, Sweden) and stored at −20C. Genomic DNA was extracted by the following procedure: (1) to a 25 ml Conical tube containing 300-500 mg of ground tissue, 9 ml of CTAB (cetyl trimethylammonium bromide solution) was added, and incubated at 65° C. for 1 hour; (2) 4.5 ml of chloroform:octanol (24:1) was added and mixed gently for 5 minutes; (3) samples were centrifuged at 2000 rpm and DNA was precipitated from the supernatant with an equal volume of isopropanol; (4) DNA was collected on a glass hook, washed in ethanol, and dissolved in TE (10 mM Tris.HCl, 0.5 mM EDTA, pH8.0).

Genomic DNA was digested at 37° C. for 2 hours in an Eppendorf tube containing the following mixture: 8 μl of 800 ug/ml DNA, 2 μl 1 mg/ml BSA (Bovine serum albumin),2 μl 10×buffer, 1 μl SacI, 1 μl EcoRI, and 6 μl H2O. Digested DNA samples were electrophoresed overnight at 40 mA in a 0.85% SeaKem LE agarose gel (FMC, Rockland, Me.). The gel was blotted onto Millipore Immobilon-Ny+ (Bedford, Mass.) membrane overnight in 20×SSC (NaCl 175.2 g/l, Na citrate 88 g/l). The probe DNA was cut with BamHI/SacI (NEB, Beverly, Mass.) from pDAB1551 plasmid, which released a 7356 bp fragment containing the open reading frame of the rebuilt tcdA gene. This 7356 bp fragment was labeled with P32 using a Stratagene Prime-it RmT dCTP-Labeling Reactions kit (La Jolla, Calif.) and used for Southern hybridization. Hybridization was conducted in hybridization buffer (10% polyethylene glycol, 7% SDS [Sodium dodecyl sulfate], 0.6×SSC, 10 mM NaPO 4 , 5 mM EDTA, 10 μg/ml denatured salmon sperm) at 60° C. overnight. After hybridization, the membrane was washed with 10×SSC plus 0.1% SDS at 60° C. for 30 min and exposed to X ray film (Hyperfilm® MP, Amershan Life Sciences, Piscataway, N.J.) for 1-2 days.

Results summarized indicate that a pattern of 8 hybridizing bands (the size of the expected fragment and larger) cosegregated with protein expression in 50% of all progeny assayed. These results are characteristic of a complex insertion at a single site. All seedlings containing the insert also expressed toxin protein.

EXAMPLE 6

Transformation of Rice with a Vector Carrying Plasmid pDAB1553 Encoding Photorhabdus Toxins

A. Plasmid pDAB1553

Plasmid pDAB1553 containing tcdA driven by the maize ubiquitinl promoter and hpt (hygromycin phosphotransferase providing resistance to the antibiotic hygromycin) under the control of 35T (a modified 35S promoter), was used for transformation.

Preparation of rice transformation vectors was accomplished in two steps. First, a modified plant-optimized tcdA coding region was ligated into a rice plant expression cassette plasmid. In this step, the coding region was placed under the transcriptional control of a promoter functional in plant cells. RNA transcription termination and polyadenylation were mediated by a downstream copy of the terminator region from the Agrobacterium nopaline synthase gene. One plasmid designed to function in this role is plasmid pDAB1538 (described in the section on maize transformation vectors). In the second step, the complete gene comprised of the promoter, coding region, and terminator region was ligated to a rice plant transformation vector that contained a plant expressible selectable marker gene which allowed the selection of transformed rice plant cells amongst a background of nontransformed cells. An example of such a vector is pDAB354-Not1.

It is a feature of pDAB354-Not1 that the hygromycin phosphotransferase protein, which has as its substrate hygromycin B and related compounds, is produced in plant cells through transcription of its coding region mediated by the Cauliflower Mosaic Virus 35S promoter and that termination of transcription plus polyadenylation are mediated by the nopaline synthase terminator region. It is further a feature of pDAB354-Not1 that any DNA fragment containing flanking NotI sites can be cloned into the unique NotI site of pDAB354-Not1, thus physically linking the introduced DNA fragment to the aforementioned selectable marker gene.

To prepare a planti-expressible gene to produce the non-targeted TcdA protein in rice plant cells, DNA of a plasmid (pAOH — 4-OPTI) containing the planti-optimized tcdA coding region, (SEQ ID No:3) was cleaved with restriction enzymes NcoI and SacI, and the large 7550 bp fragment was ligated to similarly-cut DNA of plasmid pDAB1538 to produce plasmid pDAB1551. DNA of pDAB1551 was then digested with NotI, and the large 9933 bp fragment was ligated to NotI digested DNA of pDAB354-Not1 to produce plasmid pDAB1553.

It is a feature of plasmid pDAB1553 that the ubi1 and 35S promoters are encoded on the same DNA strand.

B. Production of Rice Transgenics

For initiation of embryogenic callus, mature seeds of a Japonica cultivar, Taipei 309 were dehusked and surface-sterilized in 70% ethanol for 2-5 min. followed by a 30-45 min soak in 50% commercial bleach (2.6% sodium hypochlorite) with a few drops of ‘Liquinox’ soap. The seeds were then rinsed 3 times in sterile distilled water and placed on filter paper before transferring to ‘callus induction’ medium (i.e., NB). The NB medium consisted of N6 macro elements (Chu, 1978, The N6 medium and its application to anther culture of cereal crops. Proc. Symp. Plant Tissue Culture, Peking Press, p43-56), B5 micro elements and vitamins (Gamborg et al., 1968, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158), 300 mg/L casein hydrolysate, 500 mg/L L-proline, 500 mg/L L-glutamine, 30 g/L sucrose, 2 mg/L 2,4-dichloro-phenoxyacetic acid (2,4-D), and 2.5 g/L gelrite (Schweizerhall, N.J.) with the pH adjusted to 5.8. The mature seed cultured on ‘induction’ media were incubated in the dark at 28° C. After 3 weeks of culture, the emerging primary callus induced from the scutellar region of mature embryo was transferred to fresh NB medium for further maintenance.

About 140 μg of plasmid pDAB1553 DNA was precipitated onto 60 mg of 1.0 micron (Bio-Rad) gold particles as described herein.

For helium blasting, actively growing embryogenic callus cultures, 2-4 mm in size, were subjected to a high osmoticum treatment. This treatment included placing of callus on NB medium with 0.2 M mannitol and 0.2 M sorbitol (Vain et al., 1993, Osmoticum treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12: 84-88) for 4 h before helium blasting. Following osmoticum treatment, callus cultures were transferred to ‘blasting’ medium (NB+2% agar) and covered with a stainless steel screen (230 micron). The callus cultures were blasted at 2,000 psi helium pressures twice per target. After blasting, callus was transferred back to the media with high osmoticum overnight before placing on selection medium, which consisted NB medium with 30 mg/L hygromycin. After 2 weeks, the cultures were transferred to fresh selection medium with a higher concentration of selection agent, i.e., NB+50 mg/L hygromycin (Li et al., 1993, An improved rice transformation system using the biolistic method. Plant Cell Rep. 12: 250-255).

Compact, white-yellow, embryogenic callus cultures, recovered on NB+50 mg/L hygromycin, were regenerated by transferring to ‘pre-regeneration’ (PR) medium+50 mg/L hygromycin. The PR medium consisted of NB medium with 2 mg/L benzyl aminopurine (BAP), 1 mg/L naphthalene acetic acid (NAA), and 5 mg/L abscisic acid (ABA). After 2 weeks of culture in the dark, they were transferred to ‘regeneration’ (RN) medium. The composition of RN medium is NB medium with 3 mg/L BAP, and 0.5 mg/L NAA. The cultures on RN medium were incubated for 2 weeks at 28° C. under high fluorescent light (325-ft-candles). The plantlets with 2 cm shoot were transferred to ½ MS medium (Murashige and Skoog, 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant.15:473-497) with ½ B5 vitamins, 10 g/L sucrose, 0.05 mg/L NAA, 50 mg/L hygromycin and 2.5 g/L gelrite adjusted to pH 5.8 in magenta boxes. When plantlets were established with well-developed root systems, they were transferred to soil (1 metromix: 1 top soil) and raised in the greenhouse (29/24° C. day/night cycle, 50-60% humidity, 12 h photoperiod) until maturity.

EXAMPLE 7

Chacterization of Transgenic Rice Plants Expressing Photorhabdus Toxin that Confer Insect Control

A. Insect Bioassays

Insect bioassays were performed using leaf discs and shown to be highly effective in controlling Southern corn rootworm. Diabrotica undecimpunctata howardi eggs are obtained from French Ag Research and hatched in petri dishes held at 28.5° C. and 40% RH. The aerial parts are sampled from the transgenic plants and placed, singly into inverted petri dishes (100×15 mm) containing 15 ml of 1.6% aqueous agar in the bottom to provide humidity and filter paper in the top to absorb condensation. These preparations are infested with five neonate larvae per dish and held at 28.5° C. and 40% RH for 3 days. Mortality and larval weights are recorded. Weight data were transformed using a logarithmic function to correct a correlation between the magnitude of the mean and variance.

	TABLE 11
		Average Survivor	Presence TcdA
		Weight in mg 1	greenhouse-grown
		(Duncan's	plants (number of +/
	Treatment	Grouping)	number of plants tested)
	GUS	0.390 A	−
	Control
	1553-33	0.170 BCD	++
	1553-44	0.167 BCD	+++
	1553-62	0.125 CD	+++
	1553-41	0.100 D	+++
	Note:
	1 Means followed by the same letter are not significantly different based on Duncan's multiple range test (alpha = 0.05).

Insect groups weighing less than 0.1 mg were set to 0.03 mg instead of zero to conduct a more conservative analysis. Weight data were transformed (Log10) for analyses. A single replicate was used on each of three test dates. Plants were sampled from magenta boxes.

The results demonstrate that in leaf disc bioassays, several rice events derived by transformation with tcdA gene were demonstrated to statistically have a functional affect on corn rootworm neonate.

6 1 7551 DNA Photorhabdus luminescens CDS (1)..(7548) 1atg aac gag tct gta aaa gag ata cct gat gta tta aaa agc cag tgt 48Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys 1 5 10 15ggt ttt aat tgt ctg aca gat att agc cac agc tct ttt aat gaa ttt 96Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe 20 25 30cgc cag caa gta tct gag cac ctc tcc tgg tcc gaa aca cac gac tta 144Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu 35 40 45tat cat gat gca caa cag gca caa aag gat aat cgc ctg tat gaa gcg 192Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala 50 55 60cgt att ctc aaa cgc gcc aat ccc caa tta caa aat gcg gtg cat ctt 240Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu 65 70 75 80gcc att ctc gct ccc aat gct gaa ctg ata ggc tat aac aat caa ttt 288Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe 85 90 95agc ggt aga gcc agt caa tat gtt gcg ccg ggt acc gtt tct tcc atg 336Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met 100 105 110ttc tcc ccc gcc gct tat ttg act gaa ctt tat cgt gaa gca cgc aat 384Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn 115 120 125tta cac gca agt gac tcc gtt tat tat ctg gat acc cgc cgc cca gat 432Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp 130 135 140ctc aaa tca atg gcg ctc agt cag caa aat atg gat ata gaa tta tcc 480Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser145 150 155 160aca ctc tct ttg tcc aat gag ctg tta ttg gaa agc att aaa act gaa 528Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu 165 170 175tct aaa ctg gaa aac tat act aaa gtg atg gaa atg ctc tcc act ttc 576Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe 180 185 190cgt cct tcc ggc gca acg cct tat cat gat gct tat gaa aat gtg cgt 624Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg 195 200 205gaa gtt atc cag cta caa gat cct gga ctt gag caa ctc aat gca tca 672Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser 210 215 220ccg gca att gcc ggg ttg atg cat caa gcc tcc cta ttg ggt att aac 720Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn225 230 235 240gct tca atc tcg cct gag cta ttt aat att ctg acg gag gag att acc 768Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr 245 250 255gaa ggt aat gct gag gaa ctt tat aag aaa aat ttt ggt aat atc gaa 816Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu 260 265 270ccg gcc tca ttg gct atg ccg gaa tac ctt aaa cgt tat tat aat tta 864Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu 275 280 285agc gat gaa gaa ctt agt cag ttt att ggt aaa gcc agc aat ttt ggt 912Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly 290 295 300caa cag gaa tat agt aat aac caa ctt att act ccg gta gtc aac agc 960Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser305 310 315 320agt gat ggc acg gtt aag gta tat cgg atc acc cgc gaa tat aca acc 1008Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr 325 330 335aat gct tat caa atg gat gtg gag cta ttt ccc ttc ggt ggt gag aat 1056Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn 340 345 350tat cgg tta gat tat aaa ttc aaa aat ttt tat aat gcc tct tat tta 1104Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu 355 360 365tcc atc aag tta aat gat aaa aga gaa ctt gtt cga act gaa ggc gct 1152Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala 370 375 380cct caa gtc aat ata gaa tac tcc gca aat atc aca tta aat acc gct 1200Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala385 390 395 400gat atc agt caa cct ttt gaa att ggc ctg aca cga gta ctt cct tcc 1248Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser 405 410 415ggt tct tgg gca tat gcc gcc gca aaa ttt acc gtt gaa gag tat aac 1296Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn 420 425 430caa tac tct ttt ctg cta aaa ctt aac aag gct att cgt cta tca cgt 1344Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg 435 440 445gcg aca gaa ttg tca ccc acg att ctg gaa ggc att gtg cgc agt gtt 1392Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val 450 455 460aat cta caa ctg gat atc aac aca gac gta tta ggt aaa gtt ttt ctg 1440Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu465 470 475 480act aaa tat tat atg cag cgt tat gct att cat gct gaa act gcc ctg 1488Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu 485 490 495ata cta tgc aac gcg cct att tca caa cgt tca tat gat aat caa cct 1536Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro 500 505 510agc caa ttt gat cgc ctg ttt aat acg cca tta ctg aac gga caa tat 1584Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr 515 520 525ttt tct acc ggc gat gag gag att gat tta aat tca ggt agc acc ggc 1632Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly 530 535 540gat tgg cga aaa acc ata ctt aag cgt gca ttt aat att gat gat gtc 1680Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val545 550 555 560tcg ctc ttc cgc ctg ctt aaa att acc gac cat gat aat aaa gat gga 1728Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly 565 570 575aaa att aaa aat aac cta aag aat ctt tcc aat tta tat att gga aaa 1776Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys 580 585 590tta ctg gca gat att cat caa tta acc att gat gaa ctg gat tta tta 1824Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu 595 600 605ctg att gcc gta ggt gaa gga aaa act aat tta tcc gct atc agt gat 1872Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp 610 615 620aag caa ttg gct acc ctg atc aga aaa ctc aat act att acc agc tgg 1920Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp625 630 635 640cta cat aca cag aag tgg agt gta ttc cag cta ttt atc atg acc tcc 1968Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser 645 650 655acc agc tat aac aaa acg cta acg cct gaa att aag aat ttg ctg gat 2016Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp 660 665 670acc gtc tac cac ggt tta caa ggt ttt gat aaa gac aaa gca gat ttg 2064Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu 675 680 685cta cat gtc atg gcg ccc tat att gcg gcc acc ttg caa tta tca tcg 2112Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser 690 695 700gaa aat gtc gcc cac tcg gta ctc ctt tgg gca gat aag tta cag ccc 2160Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro705 710 715 720ggc gac ggc gca atg aca gca gaa aaa ttc tgg gac tgg ttg aat act 2208Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr 725 730 735aag tat acg ccg ggt tca tcg gaa gcc gta gaa acg cag gaa cat atc 2256Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile 740 745 750gtt cag tat tgt cag gct ctg gca caa ttg gaa atg gtt tac cat tcc 2304Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser 755 760 765acc ggc atc aac gaa aac gcc ttc cgt cta ttt gtg aca aaa cca gag 2352Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu 770 775 780atg ttt ggc gct gca act gga gca gcg ccc gcg cat gat gcc ctt tca 2400Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser785 790 795 800ctg att atg ctg aca cgt ttt gcg gat tgg gtg aac gca cta ggc gaa 2448Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu 805 810 815aaa gcg tcc tcg gtg cta gcg gca ttt gaa gct aac tcg tta acg gca 2496Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala 820 825 830gaa caa ctg gct gat gcc atg aat ctt gat gct aat ttg ctg ttg caa 2544Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln 835 840 845gcc agt att caa gca caa aat cat caa cat ctt ccc cca gta act cca 2592Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro 850 855 860gaa aat gcg ttc tcc tgt tgg aca tct atc aat act atc ctg caa tgg 2640Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp865 870 875 880gtt aat gtc gca caa caa ttg aat gtc gcc cca cag ggc gtt tcc gct 2688Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala 885 890 895ttg gtc ggg ctg gat tat att caa tca atg aaa gag aca ccg acc tat 2736Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr 900 905 910gcc cag tgg gaa aac gcg gca ggc gta tta acc gcc ggg ttg aat tca 2784Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser 915 920 925caa cag gct aat aca tta cac gct ttt ctg gat gaa tct cgc agt gcc 2832Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala 930 935 940gca tta agc acc tac tat atc cgt caa gtc gcc aag gca gcg gcg gct 2880Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala945 950 955 960att aaa agc cgt gat gac ttg tat caa tac tta ctg att gat aat cag 2928Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln 965 970 975gtt tct gcg gca ata aaa acc acc cgg atc gcc gaa gcc att gcc agt 2976Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser 980 985 990att caa ctg tac gtc aac cgg gca ttg gaa aat gtg gaa gaa aat gcc 3024Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala 995 1000 1005aat tcg ggg gtt atc agc cgc caa ttc ttt atc gac tgg gac aaa tac 3072Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr 1010 1015 1020aat aaa cgc tac agc act tgg gcg ggt gtt tct caa tta gtt tac tac 3120Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr1025 1030 1035 1040ccg gaa aac tat att gat ccg acc atg cgt atc gga caa acc aaa atg 3168Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met 1045 1050 1055atg gac gca tta ctg caa tcc gtc agc caa agc caa tta aac gcc gat 3216Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp 1060 1065 1070acc gtc gaa gat gcc ttt atg tct tat ctg aca tcg ttt gaa caa gtg 3264Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val 1075 1080 1085gct aat ctt aaa gtt att agc gca tat cac gat aat att aat aac gat 3312Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp 1090 1095 1100caa ggg ctg acc tat ttt atc gga ctc agt gaa act gat gcc ggt gaa 3360Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu1105 1110 1115 1120tat tat tgg cgc agt gtc gat cac agt aaa ttc aac gac ggt aaa ttc 3408Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe 1125 1130 1135gcg gct aat gcc tgg agt gaa tgg cat aaa att gat tgt cca att aac 3456Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn 1140 1145 1150cct tat aaa agc act atc cgt cca gtg ata tat aaa tcc cgc ctg tat 3504Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr 1155 1160 1165ctg ctc tgg ttg gaa caa aag gag atc acc aaa cag aca gga aat agt 3552Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser 1170 1175 1180aaa gat ggc tat caa act gaa acg gat tat cgt tat gaa cta aaa ttg 3600Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu1185 1190 1195 1200gcg cat atc cgc tat gat ggc act tgg aat acg cca atc acc ttt gat 3648Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp 1205 1210 1215gtc aat aaa aaa ata tcc gag cta aaa ctg gaa aaa aat aga gcg ccc 3696Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro 1220 1225 1230gga ctc tat tgt gcc ggt tat caa ggt gaa gat acg ttg ctg gtg atg 3744Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met 1235 1240 1245ttt tat aac caa caa gac aca cta gat agt tat aaa aac gct tca atg 3792Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met 1250 1255 1260caa gga cta tat atc ttt gct gat atg gca tcc aaa gat atg acc cca 3840Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro1265 1270 1275 1280gaa cag agc aat gtt tat cgg gat aat agc tat caa caa ttt gat acc 3888Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr 1285 1290 1295aat aat gtc aga aga gtg aat aac cgc tat gca gag gat tat gag att 3936Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile 1300 1305 1310cct tcc tcg gta agt agc cgt aaa gac tat ggt tgg gga gat tat tac 3984Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr 1315 1320 1325ctc agc atg gta tat aac gga gat att cca act atc aat tac aaa gcc 4032Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala 1330 1335 1340gca tca agt gat tta aaa atc tat atc tca cca aaa tta aga att att 4080Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile1345 1350 1355 1360cat aat gga tat gaa gga cag aag cgc aat caa tgc aat ctg atg aat 4128His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn 1365 1370 1375aaa tat ggc aaa cta ggt gat aaa ttt att gtt tat act agc ttg ggg 4176Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly 1380 1385 1390gtc aat cca aat aac tcg tca aat aag ctc atg ttt tac ccc gtc tat 4224Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr 1395 1400 1405caa tat agc gga aac acc agt gga ctc aat caa ggg aga cta cta ttc 4272Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe 1410 1415 1420cac cgt gac acc act tat cca tct aaa gta gaa gct tgg att cct gga 4320His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly1425 1430 1435 1440gca aaa cgt tct cta acc aac caa aat gcc gcc att ggt gat gat tat 4368Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr 1445 1450 1455gct aca gac tct ctg aat aaa ccg gat gat ctt aag caa tat atc ttt 4416Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe 1460 1465 1470atg act gac agt aaa ggg act gct act gat gtc tca ggc cca gta gag 4464Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu 1475 1480 1485att aat act gca att tct cca gca aaa gtt cag ata ata gtc aaa gcg 4512Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala 1490 1495 1500ggt ggc aag gag caa act ttt acc gca gat aaa gat gtc tcc att cag 4560Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln1505 1510 1515 1520cca tca cct agc ttt gat gaa atg aat tat caa ttt aat gcc ctt gaa 4608Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu 1525 1530 1535ata gac ggt tct ggt ctg aat ttt att aac aac tca gcc agt att gat 4656Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp 1540 1545 1550gtt act ttt acc gca ttt gcg gag gat ggc cgc aaa ctg ggt tat gaa 4704Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu 1555 1560 1565agt ttc agt att cct gtt acc ctc aag gta agt acc gat aat gcc ctg 4752Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu 1570 1575 1580acc ctg cac cat aat gaa aat ggt gcg caa tat atg caa tgg caa tcc 4800Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser1585 1590 1595 1600tat cgt acc cgc ctg aat act cta ttt gcc cgc cag ttg gtt gca cgc 4848Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg 1605 1610 1615gcc acc acc gga atc gat aca att ctg agt atg gaa act cag aat att 4896Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile 1620 1625 1630cag gaa ccg cag tta ggc aaa ggt ttc tat gct acg ttc gtg ata cct 4944Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro 1635 1640 1645ccc tat aac cta tca act cat ggt gat gaa cgt tgg ttt aag ctt tat 4992Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr 1650 1655 1660atc aaa cat gtt gtt gat aat aat tca cat att atc tat tca ggc cag 5040Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln1665 1670 1675 1680cta aca gat aca aat ata aac atc aca tta ttt att cct ctt gat gat 5088Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp 1685 1690 1695gtc cca ttg aat caa gat tat cac gcc aag gtt tat atg acc ttc aag 5136Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys 1700 1705 1710aaa tca cca tca gat ggt acc tgg tgg ggc cct cac ttt gtt aga gat 5184Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp 1715 1720 1725gat aaa gga ata gta aca ata aac cct aaa tcc att ttg acc cat ttt 5232Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe 1730 1735 1740gag agc gtc aat gtc ctg aat aat att agt agc gaa cca atg gat ttc 5280Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe1745 1750 1755 1760agc ggc gct aac agc ctc tat ttc tgg gaa ctg ttc tac tat acc ccg 5328Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro 1765 1770 1775atg ctg gtt gct caa cgt ttg ctg cat gaa cag aac ttc gat gaa gcc 5376Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala 1780 1785 1790aac cgt tgg ctg aaa tat gtc tgg agt cca tcc ggt tat att gtc cac 5424Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His 1795 1800 1805ggc cag att cag aac tac cag tgg aac gtc cgc ccg tta ctg gaa gac 5472Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp 1810 1815 1820acc agt tgg aac agt gat cct ttg gat tcc gtc gat cct gac gcg gta 5520Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val1825 1830 1835 1840gca cag cac gat cca atg cac tac aaa gtt tca act ttt atg cgt acc 5568Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr 1845 1850 1855ttg gat cta ttg ata gca cgc ggc gac cat gct tat cgc caa ctg gaa 5616Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu 1860 1865 1870cga gat aca ctc aac gaa gcg aag atg tgg tat atg caa gcg ctg cat 5664Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His 1875 1880 1885cta tta ggt gac aaa cct tat cta ccg ctg agt acg aca tgg agt gat 5712Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp 1890 1895 1900cca cga cta gac aga gcc gcg gat atc act acc caa aat gct cac gac 5760Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp1905 1910 1915 1920agc gca ata gtc gct ctg cgg cag aat ata cct aca ccg gca cct tta 5808Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu 1925 1930 1935tca ttg cgc agc gct aat acc ctg act gat ctc ttc ctg ccg caa atc 5856Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile 1940 1945 1950aat gaa gtg atg atg aat tac tgg cag aca tta gct cag aga gta tac 5904Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr 1955 1960 1965aat ctg cgt cat aac ctc tct atc gac ggc cag ccg tta tat ctg cca 5952Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro 1970 1975 1980atc tat gcc aca ccg gcc gat ccg aaa gcg tta ctc agc gcc gcc gtt 6000Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val1985 1990 1995 2000gcc act tct caa ggt gga ggc aag cta ccg gaa tca ttt atg tcc ctg 6048Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu 2005 2010 2015tgg cgt ttc ccg cac atg ctg gaa aat gcg cgc ggc atg gtt agc cag 6096Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln 2020 2025 2030ctc acc cag ttc ggc tcc acg tta caa aat att atc gaa cgt cag gac 6144Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp 2035 2040 2045gcg gaa gcg ctc aat gcg tta tta caa aat cag gcc gcc gag ctg ata 6192Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile 2050 2055 2060ttg act aac ctg agc att cag gac aaa acc att gaa gaa ttg gat gcc 6240Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala2065 2070 2075 2080gag aaa acg gtg ttg gaa aaa tcc aaa gcg gga gca caa tcg cgc ttt 6288Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe 2085 2090 2095gat agc tac ggc aaa ctg tac gat gag aat atc aac gcc ggt gaa aac 6336Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn 2100 2105 2110caa gcc atg acg cta cga gcg tcc gcc gcc ggg ctt acc acg gca gtt 6384Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val 2115 2120 2125cag gca tcc cgt ctg gcc ggt gcg gcg gct gat ctg gtg cct aac atc 6432Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile 2130 2135 2140ttc ggc ttt gcc ggt ggc ggc agc cgt tgg ggg gct atc gct gag gcg 6480Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala2145 2150 2155 2160aca ggt tat gtg atg gaa ttc tcc gcg aat gtt atg aac acc gaa gcg 6528Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala 2165 2170 2175gat aaa att agc caa tct gaa acc tac cgt cgt cgc cgt cag gag tgg 6576Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp 2180 2185 2190gag atc cag cgg aat aat gcc gaa gcg gaa ttg aag caa atc gat gct 6624Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala 2195 2200 2205cag ctc aaa tca ctc gct gta cgc cgc gaa gcc gcc gta ttg cag aaa 6672Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys 2210 2215 2220acc agt ctg aaa acc caa caa gaa cag acc caa tct caa ttg gcc ttc 6720Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe2225 2230 2235 2240ctg caa cgt aag ttc agc aat cag gcg tta tac aac tgg ctg cgt ggt 6768Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly 2245 2250 2255cga ctg gcg gcg att tac ttc cag ttc tac gat ttg gcc gtc gcg cgt 6816Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg 2260 2265 2270tgc ctg atg gca gaa caa gct tac cgt tgg gaa ctc aat gat gac tct 6864Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser 2275 2280 2285gcc cgc ttc att aaa ccg ggc gcc tgg cag gga acc tat gcc ggt ctg 6912Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu 2290 2295 2300ctt gca ggt gaa acc ttg atg ctg agt ctg gca caa atg gaa gac gct 6960Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala2305 2310 2315 2320cat ctg aaa cgc gat aaa cgc gca tta gag gtt gaa cgc aca gta tcg 7008His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser 2325 2330 2335ctg gcc gaa gtt tat gca gga tta cca aaa gat aac ggt cca ttt tcc 7056Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser 2340 2345 2350ctg gct cag gaa att gac aag ctg gtg agt caa ggt tca ggc agt gcc 7104Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala 2355 2360 2365ggc agt ggt aat aat aat ttg gcg ttc ggc gcc ggc acg gac act aaa 7152Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys 2370 2375 2380acc tct ttg cag gca tca gtt tca ttc gct gat ttg aaa att cgt gaa 7200Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu2385 2390 2395 2400gat tac ccg gca tcg ctt ggc aaa att cga cgt atc aaa cag atc agc 7248Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser 2405 2410 2415gtc act ttg ccc gcg cta ctg gga ccg tat cag gat gta cag gca ata 7296Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile 2420 2425 2430ttg tct tac ggc gat aaa gcc gga tta gct aac ggc tgt gaa gcg ctg 7344Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu 2435 2440 2445gca gtt tct cac ggt atg aat gac agc ggc caa ttc cag ctc gat ttc 7392Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe 2450 2455 2460aac gat ggc aaa ttc ctg cca ttc gaa ggc atc gcc att gat caa ggc 7440Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly2465 2470 2475 2480acg ctg aca ctg agc ttc cca aat gca tct atg ccg gag aaa ggt aaa 7488Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys 2485 2490 2495caa gcc act atg tta aaa acc ctg aac gat atc att ttg cat att cgc 7536Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg 2500 2505 2510tac acc att aaa taa 7551Tyr Thr Ile Lys 2515 2 7515 DNA Photorhabdus luminescens CDS (1)..(7512) 2atg caa aac tca tta tca agc act atc gat act att tgt cag aaa ctg 48Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu 1 5 10 15caa tta act tgt ccg gcg gaa att gct ttg tat ccc ttt gat act ttc 96Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe 20 25 30cgg gaa aaa act cgg gga atg gtt aat tgg ggg gaa gca aaa cgg att 144Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile 35 40 45tat gaa att gca caa gcg gaa cag gat aga aac cta ctt cat gaa aaa 192Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys 50 55 60cgt att ttt gcc tat gct aat ccg ctg ctg aaa aac gct gtt cgg ttg 240Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu 65 70 75 80ggt acc cgg caa atg ttg ggt ttt ata caa ggt tat agt gat ctg ttt 288Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe 85 90 95ggt aat cgt gct gat aac tat gcc gcg ccg ggc tcg gtt gca tcg atg 336Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met 100 105 110ttc tca ccg gcg gct tat ttg acg gaa ttg tac cgt gaa gcc aaa aac 384Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn 115 120 125ttg cat gac agc agc tca att tat tac cta gat aaa cgt cgc ccg gat 432Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp 130 135 140tta gca agc tta atg ctc agc cag aaa aat atg gat gag gaa att tca 480Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser145 150 155 160acg ctg gct ctc tct aat gaa ttg tgc ctt gcc ggg atc gaa aca aaa 528Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys 165 170 175aca gga aaa tca caa gat gaa gtg atg gat atg ttg tca act tat cgt 576Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg 180 185 190tta agt gga gag aca cct tat cat cac gct tat gaa act gtt cgt gaa 624Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu 195 200 205atc gtt cat gaa cgt gat cca gga ttt cgt cat ttg tca cag gca ccc 672Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro 210 215 220att gtt gct gct aag ctc gat cct gtg act ttg ttg ggt att agc tcc 720Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser225 230 235 240cat att tcg cca gaa ctg tat aac ttg ctg att gag gag atc ccg gaa 768His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu 245 250 255aaa gat gaa gcc gcg ctt gat acg ctt tat aaa aca aac ttt ggc gat 816Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp 260 265 270att act act gct cag tta atg tcc cca agt tat ctg gcc cgg tat tat 864Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr 275 280 285ggc gtc tca ccg gaa gat att gcc tac gtg acg act tca tta tca cat 912Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His 290 295 300gtt gga tat agc agt gat att ctg gtt att ccg ttg gtc gat ggt gtg 960Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val305 310 315 320ggt aag atg gaa gta gtt cgt gtt acc cga aca cca tcg gat aat tat 1008Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr 325 330 335acc agt cag acg aat tat att gag ctg tat cca cag ggt ggc gac aat 1056Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn 340 345 350tat ttg atc aaa tac aat cta agc aat agt ttt ggt ttg gat gat ttt 1104Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe 355 360 365tat ctg caa tat aaa gat ggt tcc gct gat tgg act gag att gcc cat 1152Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His 370 375 380aat ccc tat cct gat atg gtc ata aat caa aag tat gaa tca cag gcg 1200Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala385 390 395 400aca atc aaa cgt agt gac tct gac aat ata ctc agt ata ggg tta caa 1248Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln 405 410 415aga tgg cat agc ggt agt tat aat ttt gcc gcc gcc aat ttt aaa att 1296Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile 420 425 430gac caa tac tcc ccg aaa gct ttc ctg ctt aaa atg aat aag gct att 1344Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile 435 440 445cgg ttg ctc aaa gct acc ggc ctc tct ttt gct acg ttg gag cgt att 1392Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile 450 455 460gtt gat agt gtt aat agc acc aaa tcc atc acg gtt gag gta tta aac 1440Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn465 470 475 480aag gtt tat cgg gta aaa ttc tat att gat cgt tat ggc atc agt gaa 1488Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu 485 490 495gag aca gcc gct att ttg gct aat att aat atc tct cag caa gct gtt 1536Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val 500 505 510ggc aat cag ctt agc cag ttt gag caa cta ttt aat cac ccg ccg ctc 1584Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu 515 520 525aat ggt att cgc tat gaa atc agt gag gac aac tcc aaa cat ctt cct 1632Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro 530 535 540aat cct gat ctg aac ctt aaa cca gac agt acc ggt gat gat caa cgc 1680Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg545 550 555 560aag gcg gtt tta aaa cgc gcg ttt cag gtt aac gcc agt gag ttg tat 1728Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr 565 570 575cag atg tta ttg atc act gat cgt aaa gaa gac ggt gtt atc aaa aat 1776Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn 580 585 590aac tta gag aat ttg tct gat ctg tat ttg gtt agt ttg ctg gcc cag 1824Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln 595 600 605att cat aac ctg act att gct gaa ttg aac att ttg ttg gtg att tgt 1872Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys 610 615 620ggc tat ggc gac acc aac att tat cag att acc gac gat aat tta gcc 1920Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala625 630 635 640aaa ata gtg gaa aca ttg ttg tgg atc act caa tgg ttg aag acc caa 1968Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln 645 650 655aaa tgg aca gtt acc gac ctg ttt ctg atg acc acg gcc act tac agc 2016Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser 660 665 670acc act tta acg cca gaa att agc aat ctg acg gct acg ttg tct tca 2064Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser 675 680 685act ttg cat ggc aaa gag agt ctg att ggg gaa gat ctg aaa aga gca 2112Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala 690 695 700atg gcg cct tgc ttc act tcg gct ttg cat ttg act tct caa gaa gtt 2160Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val705 710 715 720gcg tat gac ctg ctg ttg tgg ata gac cag att caa ccg gca caa ata 2208Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile 725 730 735act gtt gat ggg ttt tgg gaa gaa gtg caa aca aca cca acc agc ttg 2256Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu 740 745 750aag gtg att acc ttt gct cag gtg ctg gca caa ttg agc ctg atc tat 2304Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr 755 760 765cgt cgt att ggg tta agt gaa acg gaa ctg tca ctg atc gtg act caa 2352Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln 770 775 780tct tct ctg cta gtg gca ggc aaa agc ata ctg gat cac ggt ctg tta 2400Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu785 790 795 800acc ctg atg gcc ttg gaa ggt ttt cat acc tgg gtt aat ggc ttg ggg 2448Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly 805 810 815caa cat gcc tcc ttg ata ttg gcg gcg ttg aaa gac gga gcc ttg aca 2496Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr 820 825 830gtt acc gat gta gca caa gct atg aat aag gag gaa tct ctc cta caa 2544Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln 835 840 845atg gca gct aat cag gtg gag aag gat cta aca aaa ctg acc agt tgg 2592Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp 850 855 860aca cag att gac gct att ctg caa tgg tta cag atg tct tcg gcc ttg 2640Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu865 870 875 880gcg gtt tct cca ctg gat ctg gca ggg atg atg gcc ctg aaa tat ggg 2688Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly 885 890 895ata gat cat aac tat gct gcc tgg caa gct gcg gcg gct gcg ctg atg 2736Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met 900 905 910gct gat cat gct aat cag gca cag aaa aaa ctg gat gag acg ttc agt 2784Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser 915 920 925aag gca tta tgt aac tat tat att aat gct gtt gtc gat agt gct gct 2832Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala 930 935 940gga gta cgt gat cgt aac ggt tta tat acc tat ttg ctg att gat aat 2880Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn945 950 955 960cag gtt tct gcc gat gtg atc act tca cgt att gca gaa gct atc gcc 2928Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala 965 970 975ggt att caa ctg tac gtt aac cgg gct tta aac cga gat gaa ggt cag 2976Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln 980 985 990ctt gca tcg gac gtt agt acc cgt cag ttc ttc act gac tgg gaa cgt 3024Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg 995 1000 1005tac aat aaa cgt tac agt act tgg gct ggt gtc tct gaa ctg gtc tat 3072Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr 1010 1015 1020tat cca gaa aac tat gtt gat ccc act cag cgc att ggg caa acc aaa 3120Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys1025 1030 1035 1040atg atg gat gcg ctg ttg caa tcc atc aac cag agc cag cta aat gcg 3168Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala 1045 1050 1055gat acg gtg gaa gat gct ttc aaa act tat ttg acc agc ttt gag cag 3216Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln 1060 1065 1070gta gca aat ctg aaa gta att agt gct tac cac gat aat gtg aat gtg 3264Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val 1075 1080 1085gat caa gga tta act tat ttt atc ggt atc gac caa gca gct ccg ggt 3312Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly 1090 1095 1100acg tat tac tgg cgt agt gtt gat cac agc aaa tgt gaa aat ggc aag 3360Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys1105 1110 1115 1120ttt gcc gct aat gct tgg ggt gag tgg aat aaa att acc tgt gct gtc 3408Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val 1125 1130 1135aat cct tgg aaa aat atc atc cgt ccg gtt gtt tat atg tcc cgc tta 3456Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu 1140 1145 1150tat ctg cta tgg ctg gag cag caa tca aag aaa agt gat gat ggt aaa 3504Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys 1155 1160 1165acc acg att tat caa tat aac tta aaa ctg gct cat att cgt tac gac 3552Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp 1170 1175 1180ggt agt tgg aat aca cca ttt act ttt gat gtg aca gaa aag gta aaa 3600Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys1185 1190 1195 1200aat tac acg tcg agt act gat gct gct gaa tct tta ggg ttg tat tgt 3648Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys 1205 1210 1215act ggt tat caa ggg gaa gac act cta tta gtt atg ttc tat tcg atg 3696Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met 1220 1225 1230cag agt agt tat agc tcc tat acc gat aat aat gcg ccg gtc act ggg 3744Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly 1235 1240 1245cta tat att ttc gct gat atg tca tca gac aat atg acg aat gca caa 3792Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln 1250 1255 1260gca act aac tat tgg aat aac agt tat ccg caa ttt gat act gtg atg 3840Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met1265 1270 1275 1280gca gat ccg gat agc gac aat aaa aaa gtc ata acc aga aga gtt aat 3888Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn 1285 1290 1295aac cgt tat gcg gag gat tat gaa att cct tcc tct gtg aca agt aac 3936Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn 1300 1305 1310agt aat tat tct tgg ggt gat cac agt tta acc atg ctt tat ggt ggt 3984Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly 1315 1320 1325agt gtt cct aat att act ttt gaa tcg gcg gca gaa gat tta agg cta 4032Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu 1330 1335 1340tct acc aat atg gca ttg agt att att cat aat gga tat gcg gga acc 4080Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr1345 1350 1355 1360cgc cgt ata caa tgt aat ctt atg aaa caa tac gct tca tta ggt gat 4128Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp 1365 1370 1375aaa ttt ata att tat gat tca tca ttt gat gat gca aac cgt ttt aat 4176Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn 1380 1385 1390ctg gtg cca ttg ttt aaa ttc gga aaa gac gag aac tca gat gat agt 4224Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser 1395 1400 1405att tgt ata tat aat gaa aac cct tcc tct gaa gat aag aag tgg tat 4272Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr 1410 1415 1420ttt tct tcg aaa gat gac aat aaa aca gcg gat tat aat ggt gga act 4320Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr1425 1430 1435 1440caa tgt ata gat gct gga acc agt aac aaa gat ttt tat tat aat ctc 4368Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu 1445 1450 1455cag gag att gaa gta att agt gtt act ggt ggg tat tgg tcg agt tat 4416Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr 1460 1465 1470aaa ata tcc aac ccg att aat atc aat acg ggc att gat agt gct aaa 4464Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys 1475 1480 1485gta aaa gtc acc gta aaa gcg ggt ggt gac gat caa atc ttt act gct 4512Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala 1490 1495 1500gat aat agt acc tat gtt cct cag caa ccg gca ccc agt ttt gag gag 4560Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu1505 1510 1515 1520atg att tat cag ttc aat aac ctg aca ata gat tgt aag aat tta aat 4608Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn 1525 1530 1535ttc atc gac aat cag gca cat att gag att gat ttc acc gct acg gca 4656Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala 1540 1545 1550caa gat ggc cga ttc ttg ggt gca gaa act ttt att atc ccg gta act 4704Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr 1555 1560 1565aaa aaa gtt ctc ggt act gag aac gtg att gcg tta tat agc gaa aat 4752Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn 1570 1575 1580aac ggt gtt caa tat atg caa att ggc gca tat cgt acc cgt ttg aat 4800Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn1585 1590 1595 1600acg tta ttc gct caa cag ttg gtt agc cgt gct aat cgt ggc att gat 4848Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp 1605 1610 1615gca gtg ctc agt atg gaa act cag aat att cag gaa ccg caa tta gga 4896Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly 1620 1625 1630gcg ggc aca tat gtg cag ctt gtg ttg gat aaa tat gat gag tct att 4944Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile 1635 1640 1645cat ggc act aat aaa agc ttt gct att gaa tat gtt gat ata ttt aaa 4992His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys 1650 1655 1660gag aac gat agt ttt gtg att tat caa gga gaa ctt agc gaa aca agt 5040Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser1665 1670 1675 1680caa act gtt gtg aaa gtt ttc tta tcc tat ttt ata gag gcg act gga 5088Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly 1685 1690 1695aat aag aac cac tta tgg gta cgt gct aaa tac caa aag gaa acg act 5136Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr 1700 1705 1710gat aag atc ttg ttc gac cgt act gat gag aaa gat ccg cac ggt tgg 5184Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp 1715 1720 1725ttt ctc agc gac gat cac aag acc ttt agt ggt ctc tct tcc gca cag 5232Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln 1730 1735 1740gca tta aag aac gac agt gaa ccg atg gat ttc tct ggc gcc aat gct 5280Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala1745 1750 1755 1760ctc tat ttc tgg gaa ctg ttc tat tac acg ccg atg atg atg gct cat 5328Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His 1765 1770 1775cgt ttg ttg cag gaa cag aat ttt gat gcg gcg aac cat tgg ttc cgt 5376Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg 1780 1785 1790tat gtc tgg agt cca tcc ggt tat atc gtt gat ggt aaa att gct atc 5424Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile 1795 1800 1805tac cac tgg aac gtg cga ccg ctg gaa gaa gac acc agt tgg aat gca 5472Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala 1810 1815 1820caa caa ctg gac tcc acc gat cca gat gct gta gcc caa gat gat ccg 5520Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro1825 1830 1835 1840atg cac tac aag gtg gct acc ttt atg gcg acg ttg gat ctg cta atg 5568Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met 1845 1850 1855gcc cgt ggt gat gct gct tac cgc cag tta gag cgt gat acg ttg gct 5616Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala 1860 1865 1870gaa gct aaa atg tgg tat aca cag gcg ctt aat ctg ttg ggt gat gag 5664Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu 1875 1880 1885cca caa gtg atg ctg agt acg act tgg gct aat cca aca ttg ggt aat 5712Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn 1890 1895 1900gct gct tca aaa acc aca cag cag gtt cgt cag caa gtg ctt acc cag 5760Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln1905 1910 1915 1920ttg cgt ctc aat agc agg gta aaa acc ccg ttg cta gga aca gcc aat 5808Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn 1925 1930 1935tcc ctg acc gct tta ttc ctg ccg cag gaa aat agc aag ctc aaa ggc 5856Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly 1940 1945 1950tac tgg cgg aca ctg gcg cag cgt atg ttt aat tta cgt cat aat ctg 5904Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu 1955 1960 1965tcg att gac ggc cag ccg ctc tcc ttg ccg ctg tat gct aaa ccg gct 5952Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala 1970 1975 1980gat cca aaa gct tta ctg agt gcg gcg gtt tca gct tct caa ggg gga 6000Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly1985 1990 1995 2000gcc gac ttg ccg aag gcg ccg ctg act att cac cgc ttc cct caa atg 6048Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met 2005 2010 2015cta gaa ggg gca cgg ggc ttg gtt aac cag ctt ata cag ttc ggt agt 6096Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser 2020 2025 2030tca cta ttg ggg tac agt gag cgt cag gat gcg gaa gct atg agt caa 6144Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln 2035 2040 2045cta ctg caa acc caa gcc agc gag tta ata ctg acc agt att cgt atg 6192Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met 2050 2055 2060cag gat aac caa ttg gca gag ctg gat tcg gaa aaa acc gcc ttg caa 6240Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln2065 2070 2075 2080gtc tct tta gct gga gtg caa caa cgg ttt gac agc tat agc caa ctg 6288Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu 2085 2090 2095tat gag gag aac atc aac gca ggt gag cag cga gcg ctg gcg tta cgc 6336Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg 2100 2105 2110tca gaa tct gct att gag tct cag gga gcg cag att tcc cgt atg gca 6384Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala 2115 2120 2125ggc gcg ggt gtt gat atg gca cca aat atc ttc ggc ctg gct gat ggc 6432Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly 2130 2135 2140ggc atg cat tat ggt gct att gcc tat gcc atc gct gac ggt att gag 6480Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu2145 2150 2155 2160ttg agt gct tct gcc aag atg gtt gat gcg gag aaa gtt gct cag tcg 6528Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser 2165 2170 2175gaa ata tat cgc cgt cgc cgt caa gaa tgg aaa att cag cgt gac aac 6576Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn 2180 2185 2190gca caa gcg gag att aac cag tta aac gcg caa ctg gaa tca ctg tct 6624Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser 2195 2200 2205att cgc cgt gaa gcc gct gaa atg caa aaa gag tac ctg aaa acc cag 6672Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln 2210 2215 2220caa gct cag gcg cag gca caa ctt act ttc tta aga agc aaa ttc agt 6720Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser2225 2230 2235 2240aat caa gcg tta tat agt tgg tta cga ggg cgt ttg tca ggt att tat 6768Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr 2245 2250 2255ttc cag ttc tat gac ttg gcc gta tca cgt tgc ctg atg gca gag caa 6816Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln 2260 2265 2270tcc tat caa tgg gaa gct aat gat aat tcc att agc ttt gtc aaa ccg 6864Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro 2275 2280 2285ggt gca tgg caa gga act tac gcc ggc tta ttg tgt gga gaa gct ttg 6912Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu 2290 2295 2300ata caa aat ctg gca caa atg gaa gag gca tat ctg aaa tgg gaa tct 6960Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser2305 2310 2315 2320cgc gct ttg gaa gta gaa cgc acg gtt tca ttg gca gtg gtt tat gat 7008Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp 2325 2330 2335tca ctg gaa ggt aat gat cgt ttt aat tta gcg gaa caa ata cct gca 7056Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala 2340 2345 2350tta ttg gat aag ggg gag gga aca gca gga act aaa gaa aat ggg tta 7104Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu 2355 2360 2365tca ttg gct aat gct atc ctg tca gct tcg gtc aaa ttg tcc gac ttg 7152Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu 2370 2375 2380aaa ctg gga acg gat tat cca gac agt atc gtt ggt agc aac aag gtt 7200Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val2385 2390 2395 2400cgt cgt att aag caa atc agt gtt tcg cta cct gca ttg gtt ggg cct 7248Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro 2405 2410 2415tat cag gat gtt cag gct atg ctc agc tat ggt ggc agt act caa ttg 7296Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu 2420 2425 2430ccg aaa ggt tgt tca gcg ttg gct gtg tct cat ggt acc aat gat agt 7344Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser 2435 2440 2445ggt cag ttc cag ttg gat ttc aat gac ggc aaa tac ctg cca ttt gaa 7392Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu 2450 2455 2460ggt att gct ctt gat gat cag ggt aca ctg aat ctt caa ttt ccg aat 7440Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn2465 2470 2475 2480gct acc gac aag cag aaa gca ata ttg caa act atg agc gat att att 7488Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile 2485 2490 2495ttg cat att cgt tat acc atc cgt taa 7515Leu His Ile Arg Tyr Thr Ile Arg 2500 3 7577 DNA Artificial Sequence CDS (3)..(7553) Description of Artificial Sequencehemicot tcdA 3cc atg gct aac gag tcc gtc aag gag atc cca gac gtc ctc aag tcc 47 Met Ala Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser 1 5 10 15caa tgc ggt ttc aac tgc ctc act gac atc tcc cac agc tcc ttc aac 95Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn 20 25 30gag ttc aga caa caa gtc tct gag cac ctc tcc tgg tcc gag acc cat 143Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His 35 40 45gac ctc tac cat gac gct cag caa gct cag aag gac aac agg ctc tac 191Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr 50 55 60gag gct agg atc ctc aag agg gct aac cca caa ctc cag aac gct gtc 239Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val 65 70 75cac ctc gcc atc ttg gct cca aac gct gag ttg att ggt tac aac aac 287His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn 80 85 90 95cag ttc tct ggc aga gct agc cag tac gtg gct cct ggt aca gtc tcc 335Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser 100 105 110tcc atg ttc agc cca gcc gct tac ctc act gag ttg tac cgc gag gct 383Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala 115 120 125agg aac ctt cat gct tct gac tcc gtc tac tac ttg gac aca cgc aga 431Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg 130 135 140cca gac ctc aag agc atg gcc ctc agc caa cag aac atg gac att gag 479Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu 145 150 155ttg tcc acc ctc tcc ttg agc aac gag ctt ctc ttg gag tcc atc aag 527Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys160 165 170 175act gag agc aag ttg gag aac tac acc aag gtc atg gag atg ctc tcc 575Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser 180 185 190acc ttc aga cca agc ggt gca act cca tac cat gat gcc tac gag aac 623Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn 195 200 205gtc agg gag gtc atc caa ctt caa gac cct ggt ctt gag caa ctc aac 671Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn 210 215 220gct tct cca gcc att gct ggt ttg atg cac cag gca tcc ttg ctc ggt 719Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly 225 230 235atc aac gcc tcc atc tct cct gag ttg ttc aac atc ttg act gag gag 767Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu240 245 250 255atc act gag ggc aac gct gag gag ttg tac aag aag aac ttc ggc aac 815Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn 260 265 270att gag cca gcc tct ctt gca atg cct gag tac ctc aag agg tac tac 863Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr 275 280 285aac ttg tct gat gag gag ctt tct caa ttc att ggc aag gct tcc aac 911Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn 290 295 300ttc ggt caa cag gag tac agc aac aac cag ctc atc act cca gtt gtg 959Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val 305 310 315aac tcc tct gat ggc act gtg aag gtc tac cgc atc aca cgt gag tac 1007Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr320 325 330 335acc aca aac gcc tac caa atg gat gtt gag ttg ttc cca ttc ggt ggt 1055Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly 340 345 350gag aac tac aga ctt gac tac aag ttc aag aac ttc tac aac gcc tcc 1103Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser 355 360 365tac ctc tcc atc aag ttg aac gac aag agg gag ctt gtc agg act gag 1151Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu 370 375 380ggt gct cct caa gtg aac att gag tac tct gcc aac atc acc ctc aac 1199Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn 385 390 395aca gct gac atc tct caa cca ttc gag att ggt ttg acc aga gtc ctt 1247Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu400 405 410 415ccc tct ggc tcc tgg gcc tac gct gca gcc aag ttc act gtt gag gag 1295Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu 420 425 430tac aac cag tac tct ttc ctc ttg aag ctc aac aag gca att cgt ctc 1343Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu 435 440 445agc aga gcc act gag ttg tct ccc acc atc ttg gag ggc att gtg agg 1391Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg 450 455 460tct gtc aac ctt caa ctt gac atc aac act gat gtg ctt ggc aag gtc 1439Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val 465 470 475ttc ctc acc aag tac tac atg caa cgc tac gcc atc cat gct gag act 1487Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr480 485 490 495gca ctc atc ctc tgc aac gca ccc atc tct caa cgc tcc tac gac aac 1535Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn 500 505 510cag cct tcc cag ttc gac agg ctc ttc aac act cct ctc ttg aac ggc 1583Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly 515 520 525cag tac ttc tcc act ggt gat gag gag att gac ctc aac tct ggc tcc 1631Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser 530 535 540aca ggt gac tgg aga aag acc atc ttg aag agg gcc ttc aac att gat 1679Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp 545 550 555gat gtc tct ctc ttc cgt ctc ttg aag atc aca gat cac gac aac aag 1727Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys560 565 570 575gat ggc aag atc aag aac aac ttg aag aac ctt tcc aac ctc tac att 1775Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile 580 585 590ggc aag ttg ctt gca gac atc cac caa ctc acc att gat gag ttg gac 1823Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp 595 600 605ctc ttg ctc att gca gtc ggt gag ggc aag acc aac ctc tct gca atc 1871Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile 610 615 620tct gac aag cag ttg gca acc ctc atc agg aag ttg aac acc atc acc 1919Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr 625 630 635tcc tgg ctt cac acc cag aag tgg tct gtc ttc caa ctc ttc atc atg 1967Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met640 645 650 655acc agc acc tcc tac aac aag acc ctc act cct gag atc aag aac ctc 2015Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu 660 665 670ttg gac aca gtc tac cac ggt ctc caa ggc ttc gac aag gac aag gct 2063Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala 675 680 685gac ttg ctt cat gtc atg gct ccc tac att gca gcc acc ctc caa ctc 2111Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu 690 695 700tcc tct gag aac gtg gct cac tct gtc ttg ctc tgg gct gac aag ctc 2159Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu 705 710 715caa cct ggt gat ggt gcc atg act gct gag aag ttc tgg gac tgg ctc 2207Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu720 725 730 735aac acc aag tac aca cca ggc tcc tct gag gct gtt gag act caa gag 2255Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu 740 745 750cac att gtg caa tac tgc cag gct ctt gca cag ttg gag atg gtc tac 2303His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr 755 760 765cac tcc act ggc atc aac gag aac gct ttc aga ctc ttc gtc acc aag 2351His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys 770 775 780cct gag atg ttc ggt gct gcc aca ggt gct gca cct gct cat gat gct 2399Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala 785 790 795ctc tcc ctc atc atg ttg acc agg ttc gct gac tgg gtc aac gct ctt 2447Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu800 805 810 815ggt gag aag gct tcc tct gtc ttg gct gcc ttc gag gcc aac tcc ctc 2495Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu 820 825 830act gct gag caa ctt gct gat gcc atg aac ctt gat gcc aac ctc ttg 2543Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu 835 840 845ctc caa gct tcc att caa gct cag aac cac caa cac ctc cca cct gtc 2591Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val 850 855 860act cca gag aac gct ttc tcc tgc tgg acc tcc atc aac acc atc ctc 2639Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu 865 870 875caa tgg gtc aac gtg gct cag caa ctc aac gtg gct cca caa ggt gtc 2687Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val880 885 890 895tct gct ttg gtc ggt ctt gac tac atc cag tcc atg aag gag aca cca 2735Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro 900 905 910acc tac gct caa tgg gag aac gca gct ggt gtc ttg act gct ggt ctc 2783Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu 915 920 925aac tcc caa cag gcc aac acc ctc cat gct ttc ttg gat gag tct cgc 2831Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg 930 935 940tct gct gcc ctc tcc acc tac tac atc agg caa gtc gcc aag gca gct 2879Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala 945 950 955gct gcc atc aag tct cgc gat gac ctc tac caa tac ctc ctc att gac 2927Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp960 965 970 975aac cag gtc tct gct gcc atc aag acc acc agg atc gct gag gcc atc 2975Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile 980 985 990gct tcc atc caa ctc tac gtc aac cgc gct ctt gag aac gtt gag gag 3023Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu 995 1000 1005aac gcc aac tct ggt gtc atc tct cgc caa ttc ttc atc gac tgg gac 3071Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp 1010 1015 1020aag tac aac aag agg tac tcc acc tgg gct ggt gtc tct caa ctt gtc 3119Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val 1025 1030 1035tac tac cca gag aac tac att gac cca acc atg agg att ggt cag acc 3167Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr1040 1045 1050 1055aag atg atg gat gct ctc ttg caa tct gtc tcc caa agc caa ctc aac 3215Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn 1060 1065 1070gct gac act gtg gag gat gcc ttc atg agc tac ctc acc tcc ttc gag 3263Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu 1075 1080 1085caa gtt gcc aac ctc aag gtc atc tct gct tac cat gac aac atc aac 3311Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn 1090 1095 1100aac gac caa ggt ctc acc tac ttc att ggt ctc tct gag act gat gct 3359Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala 1105 1110 1115ggt gag tac tac tgg aga tcc gtg gac cac agc aag ttc aac gat ggc 3407Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly1120 1125 1130 1135aag ttc gct gca aac gct tgg tct gag tgg cac aag att gac tgc cct 3455Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro 1140 1145 1150atc aac cca tac aag tcc acc atc aga cct gtc atc tac aag agc cgc 3503Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg 1155 1160 1165ctc tac ttg ctc tgg ctt gag cag aag gag atc acc aag caa act ggc 3551Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly 1170 1175 1180aac tcc aag gat ggt tac caa act gag act gac tac cgc tac gag ttg 3599Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu 1185 1190 1195aag ttg gct cac atc cgc tac gat ggt acc tgg aac act cca atc acc 3647Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr1200 1205 1210 1215ttc gat gtc aac aag aag atc agc gag ttg aag ttg gag aag aac cgt 3695Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg 1220 1225 1230gct cct ggt ctc tac tgc gct ggt tac caa ggt gag gac acc ctc ttg 3743Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu 1235 1240 1245gtc atg ttc tac aac cag caa gac acc ctt gac tcc tac aag aac gct 3791Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala 1250 1255 1260tcc atg caa ggt ctc tac atc ttc gct gac atg gct tcc aag gac atg 3839Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met 1265 1270 1275act cca gag caa agc aac gtc tac cgt gac aac tcc tac caa cag ttc 3887Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe1280 1285 1290 1295gac acc aac aac gtc agg cgt gtc aac aac aga tac gct gag gac tac 3935Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr 1300 1305 1310gag atc cca agc tct gtc agc tct cgc aag gac tac ggc tgg ggt gac 3983Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp 1315 1320 1325tac tac ctc agc atg gtg tac aac ggt gac atc cca acc atc aac tac 4031Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr 1330 1335 1340aag gct gcc tct tcc gac ctc aaa atc tac atc agc cca aag ctc agg 4079Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg 1345 1350 1355atc atc cac aac ggc tac gag ggt cag aag agg aac cag tgc aac ttg 4127Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu1360 1365 1370 1375atg aac aag tac ggc aag ttg ggt gac aag ttc att gtc tac acc tct 4175Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser 1380 1385 1390ctt ggt gtc aac cca aac aac agc tcc aac aag ctc atg ttc tac cca 4223Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro 1395 1400 1405gtc tac caa tac tct ggc aac acc tct ggt ctc aac cag ggt aga ctc 4271Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu 1410 1415 1420ttg ttc cac agg gac acc acc tac cca agc aag gtg gag gct tgg att 4319Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile 1425 1430 1435cct ggt gcc aag agg tcc ctc acc aac cag aac gct gcc att ggt gat 4367Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp1440 1445 1450 1455gac tac gcc aca gac tcc ctc aac aag cct gat gac ctc aag cag tac 4415Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr 1460 1465 1470atc ttc atg act gac tcc aag ggc aca gcc act gat gtc tct ggt cca 4463Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro 1475 1480 1485gtg gag atc aac act gca atc agc cca gcc aag gtc caa atc att gtc 4511Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val 1490 1495 1500aag gct ggt ggc aag gag caa acc ttc aca gct gac aag gat gtc tcc 4559Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser 1505 1510 1515atc cag cca agc cca tcc ttc gat gag atg aac tac caa ttc aac gct 4607Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala1520 1525 1530 1535ctt gag att gat ggt tct ggc ctc aac ttc atc aac aac tct gct tcc 4655Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser 1540 1545 1550att gat gtc acc ttc act gcc ttc gct gag gat ggc cgc aag ttg ggt 4703Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly 1555 1560 1565tac gag agc ttc tcc atc cca gtc acc ctt aag gtt tcc act gac aac 4751Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn 1570 1575 1580gca ctc acc ctt cat cac aac gag aac ggt gct cag tac atg caa tgg 4799Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp 1585 1590 1595caa agc tac cgc acc agg ttg aac acc ctc ttc gca agg caa ctt gtg 4847Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val1600 1605 1610 1615gcc cgt gcc acc aca ggc att gac acc atc ctc agc atg gag acc cag 4895Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln 1620 1625 1630aac atc caa gag cca cag ttg ggc aag ggt ttc tac gcc acc ttc gtc 4943Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val 1635 1640 1645atc cca cct tac aac ctc agc act cat ggt gat gag agg tgg ttc aag 4991Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys 1650 1655 1660ctc tac atc aag cac gtg gtt gac aac aac tcc cac atc atc tac tct 5039Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser 1665 1670 1675ggt caa ctc act gac acc aac atc aac atc acc ctc ttc atc cca ctt 5087Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu1680 1685 1690 1695gac gat gtc cca ctc aac cag gac tac cat gcc aag gtc tac atg acc 5135Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr 1700 1705 1710ttc aag aag tct cca tct gat ggc acc tgg tgg ggt cca cac ttc gtc 5183Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val 1715 1720 1725cgt gat gac aag ggc atc gtc acc atc aac cca aag tcc atc ctc acc 5231Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr 1730 1735 1740cac ttc gag tct gtc aac gtt ctc aac aac atc tcc tct gag cca atg 5279His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met 1745 1750 1755gac ttc tct ggt gcc aac tcc ctc tac ttc tgg gag ttg ttc tac tac 5327Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr1760 1765 1770 1775aca cca atg ctt gtg gct caa agg ttg ctc cat gag cag aac ttc gat 5375Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp 1780 1785 1790gag gcc aac agg tgg ctc aag tac gtc tgg agc cca tct ggt tac att 5423Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile 1795 1800 1805gtg cat ggt caa atc cag aac tac caa tgg aac gtc agg cca ttg ctt 5471Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu 1810 1815 1820gag gac acc tcc tgg aac tct gac cca ctt gac tct gtg gac cct gat 5519Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp 1825 1830 1835gct gtg gct caa cat gac cca atg cac tac aag gtc tcc acc ttc atg 5567Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met1840 1845 1850 1855agg acc ttg gac ctc ttg att gcc aga ggt gac cat gct tac cgc caa 5615Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln 1860 1865 1870ttg gag agg gac acc ctc aac gag gca aag atg tgg tac atg caa gct 5663Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala 1875 1880 1885ctc cac ctc ttg ggt gac aag cca tac ctc cca ctc agc acc act tgg 5711Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp 1890 1895 1900tcc gac cca agg ttg gac cgt gct gct gac atc acc act cag aac gct 5759Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala 1905 1910 1915cat gac tct gcc att gtt gct ctc agg cag aac atc cca act cct gct 5807His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala1920 1925 1930 1935cca ctc tcc ctc aga tct gct aac acc ctc act gac ttg ttc ctc cca 5855Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro 1940 1945 1950cag atc aac gag gtc atg atg aac tac tgg caa acc ttg gct caa agg 5903Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg 1955 1960 1965gtc tac aac ctc aga cac aac ctc tcc att gat ggt caa cca ctc tac 5951Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr 1970 1975 1980ctc cca atc tac gcc aca cca gct gac cca aag gct ctt ctc tct gct 5999Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala 1985 1990 1995gct gtg gct acc agc caa ggt ggt ggc aag ctc cca gag tcc ttc atg 6047Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met2000 2005 2010 2015tcc ctc tgg agg ttc cca cac atg ttg gag aac gcc cgt ggc atg gtc 6095Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val 2020 2025 2030tcc caa ctc acc cag ttc ggt tcc acc ctc cag aac atc att gag agg 6143Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg 2035 2040 2045caa gat gct gag gct ctc aac gct ttg ctc cag aac cag gca gct gag 6191Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu 2050 2055 2060ttg atc ctc acc aac ttg tcc atc caa gac aag acc att gag gag ctt 6239Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu 2065 2070 2075gat gct gag aag aca gtc ctt gag aag agc aag gct ggt gcc caa tct 6287Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser2080 2085 2090 2095cgc ttc gac tcc tac ggc aag ctc tac gat gag aac atc aac gct ggt 6335Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly 2100 2105 2110gag aac cag gcc atg acc ctc agg gct tcc gca gct ggt ctc acc act 6383Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr 2115 2120 2125gct gtc caa gcc tct cgc ttg gct ggt gca gct gct gac ctc gtt cca 6431Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro 2130 2135 2140aac atc ttc ggt ttc gct ggt ggt ggc tcc aga tgg ggt gcc att gct 6479Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala 2145 2150 2155gag gct acc ggt tac gtc atg gag ttc tct gcc aac gtc atg aac act 6527Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr2160 2165 2170 2175gag gct gac aag atc agc caa tct gag acc tac aga agg cgc cgt caa 6575Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln 2180 2185 2190gag tgg gag atc caa agg aac aac gct gag gca gag ttg aag caa atc 6623Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile 2195 2200 2205gat gct caa ctc aag tcc ttg gct gtc aga agg gag gct gct gtc ctc 6671Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu 2210 2215 2220cag aag acc tcc ctc aag acc caa cag gag caa acc cag tcc cag ttg 6719Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu 2225 2230 2235gct ttc ctc caa agg aag ttc tcc aac cag gct ctc tac aac tgg ctc 6767Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu2240 2245 2250 2255aga ggc cgc ttg gct gcc atc tac ttc caa ttc tac gac ctt gct gtg 6815Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val 2260 2265 2270gcc agg tgc ctc atg gct gag caa gcc tac cgc tgg gag ttg aac gat 6863Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp 2275 2280 2285gac tcc gcc agg ttc atc aag cca ggt gct tgg caa ggc acc tac gct 6911Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala 2290 2295 2300ggt ctc ctt gct ggt gag acc ctc atg ctc tcc ttg gct caa atg gag 6959Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu 2305 2310 2315gat gct cac ctc aag agg gac aag agg gct ttg gag gtg gag agg aca 7007Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr2320 2325 2330 2335gtc tcc ctt gct gag gtc tac gct ggt ctc cca aag gac aac ggt cca 7055Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro 2340 2345 2350ttc tcc ctt gct caa gag att gac aag ttg gtc agc caa ggt tct ggt 7103Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly 2355 2360 2365tct gct ggt tct ggt aac aac aac ttg gct ttc ggc gct ggt act gac 7151Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp 2370 2375 2380acc aag acc tcc ctc caa gcc tct gtc tcc ttc gct gac ctc aag atc 7199Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile 2385 2390 2395agg gag gac tac cca gct tcc ctt ggc aag atc agg cgc atc aag caa 7247Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln2400 2405 2410 2415atc tct gtc acc ctc cca gct ctc ttg ggt cca tac caa gat gtc caa 7295Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln 2420 2425 2430gca atc ctc tcc tac ggt gac aag gct ggt ttg gcg aac ggt tgc gag 7343Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu 2435 2440 2445gct ctt gct gtc tct cat ggc atg aac gac tct ggt caa ttc caa ctt 7391Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu 2450 2455 2460gac ttc aac gat ggc aag ttc ctc cca ttc gag ggc att gcc att gac 7439Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp 2465 2470 2475caa ggc acc ctc acc ctc tcc ttc cca aac gct tcc atg cca gag aag 7487Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys2480 2485 2490 2495gga aag caa gcc acc atg ctc aag acc ctc aac gat atc atc ctc cac 7535Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His 2500 2505 2510atc agg tac acc atc aag tgagctcgag aggcctgcgg ccgc 7577Ile Arg Tyr Thr Ile Lys 2515 4 7541 DNA Artificial Sequence CDS (3)..(7517) Description of Artificial Sequencehemicot tcbA 4cc atg gct cag aac tcc ctc agc tcc acc att gac acc atc tgc cag 47 Met Ala Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln 1 5 10 15aag ctt caa ctc acc tgc cca gct gag atc gcc ctc tac cca ttc gac 95Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp 20 25 30acc ttc cgt gag aag acc aga ggc atg gtc aac tgg ggt gag gcc aag 143Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys 35 40 45agg atc tac gag att gct caa gct gag caa gac agg aac ctc ctt cat 191Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His 50 55 60gag aag agg atc ttc gcc tac gct aac cca ttg ctc aag aac gct gtc 239Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val 65 70 75agg ctt ggt acc agg caa atg ttg ggt ttc atc caa ggt tac tct gac 287Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp 80 85 90 95ttg ttc ggc aac agg gct gac aac tac gca gct cct ggt tct gtt gct 335Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala 100 105 110agc atg ttc agc cca gct gcc tac ctc act gag ttg tac cgt gag gcc 383Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala 115 120 125aag aac ctc cat gac agc tcc agc atc tac tac ctt gac aag agg cgc 431Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg 130 135 140cca gac ctt gct tcc ttg atg ctc tcc cag aag aac atg gat gag gag 479Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu 145 150 155atc agc acc ttg gct ctc tcc aac gag ctt tgc ttg gct ggc att gag 527Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu160 165 170 175acc aag act ggc aag tcc caa gat gag gtc atg gac atg ctc tcc acc 575Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr 180 185 190tac cgc ctc tct ggt gag act cca tac cac cat gct tac gag act gtc 623Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val 195 200 205agg gag att gtc cat gag agg gac cca ggt ttc cgc cac ctc tcc caa 671Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln 210 215 220gct ccc att gtg gct gcc aag ttg gac cca gtc acc ctc ttg ggc atc 719Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile 225 230 235tcc agc cac atc agc cca gag ttg tac aac ctt ctc att gag gag atc 767Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile240 245 250 255cca gag aag gat gag gca gct ttg gac acc ctc tac aag acc aac ttc 815Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe 260 265 270ggt gac atc acc act gct caa ctc atg agc cca tcc tac ttg gcc agg 863Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg 275 280 285tac tac ggt gtc tct cca gag gac att gct tac gtc acc aca agc ctc 911Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu 290 295 300tcc cat gtg ggt tac tcc tct gac atc ctt gtc atc cca ctc gtg gat 959Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp 305 310 315ggt gtg ggc aag atg gag gtt gtc agg gtc acc agg act cca tct gac 1007Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp320 325 330 335aac tac acc tcc cag acc aac tac att gag ttg tac cca caa ggt ggt 1055Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly 340 345 350gac aac tac ctc atc aag tac aac ctc tcc aac tct ttc ggt ttg gat 1103Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp 355 360 365gac ttc tac ctc cag tac aag gat ggt tct gct gac tgg act gag att 1151Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile 370 375 380gct cac aac cca tac cca gac atg gtc atc aac cag aag tac gag tcc 1199Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser 385 390 395caa gcc acc atc aag aga tct gac tct gac aac atc ctc tcc att ggt 1247Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly400 405 410 415ctc caa agg tgg cac tct ggt tcc tac aac ttc gct gct gcc aac ttc 1295Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe 420 425 430aag att gac caa tac tct cca aag gct ttc ctc ttg aag atg aac aag 1343Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys 435 440 445gcc atc agg ctc ttg aag gcc act ggt ctc tcc ttc gcc acc ctt gag 1391Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu 450 455 460agg att gtg gac tct gtc aac tcc acc aag tcc atc act gtg gag gtc 1439Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val 465 470 475ctc aac aag gtc tac aga gtc aag ttc tac att gac cgc tac ggc atc 1487Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile480 485 490 495tct gag gag act gct gcc atc ctt gcc aac atc aac atc tcc cag caa 1535Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln 500 505 510gct gtc ggc aac cag ctc tcc caa ttc gag caa ctc ttc aac cac cct 1583Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro 515 520 525cca ctc aac ggc atc cgc tac gag atc agc gag gac aac tcc aag cac 1631Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His 530 535 540ctc cca aac cca gac ctc aac ctc aag cca gac tcc act ggt gat gac 1679Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp 545 550 555caa agg aag gct gtc ctc aag agg gct ttc caa gtc aac gct tct gag 1727Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu560 565 570 575ctt tac caa atg ctc ttg atc act gac agg aag gag gat ggt gtc atc 1775Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile 580 585 590aag aac aac ttg gag aac ctc tct gac ctc tac ctt gtc tcc ctc ttg 1823Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu 595 600 605gcc caa atc cac aac ttg acc att gct gag ttg aac atc ctc ttg gtc 1871Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val 610 615 620atc tgc ggt tac ggt gac acc aac atc tac caa atc act gac gac aac 1919Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn 625 630 635ctt gcc aag att gtg gag acc ctc ttg tgg atc acc caa tgg ctc aag 1967Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys640 645 650 655acc cag aag tgg act gtc aca gac ctc ttc ctc atg acc act gcc acc 2015Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr 660 665 670tac tcc acc act ctc act cca gag att tcc aac ctc act gcc acc ctc 2063Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu 675 680 685agc tcc acc ctc cac ggc aag gag tcc ctc att ggt gag gac ctc aag 2111Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys 690 695 700agg gca atg gct cca tgc ttc acc tct gct ctc cac ctc acc tcc caa 2159Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln 705 710 715gag gtg gct tac gac ctc ctt ctc tgg att gac caa atc caa cca gct 2207Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala720 725 730 735caa atc act gtg gat ggt ttc tgg gag gag gtc caa acc act cca acc 2255Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr 740 745 750tcc ctc aag gtc atc acc ttc gct caa gtc ttg gct caa ctc tcc ctc 2303Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu 755 760 765atc tac aga agg att ggt ctc tct gag act gag ttg tcc ctc att gtc 2351Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val 770 775 780acc caa tcc agc ctc ttg gtc gct ggc aag tcc atc ctt gat cat ggt 2399Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly 785 790 795ctc ttg acc ctc atg gct ctt gag ggt ttc cac acc tgg gtc aac ggt 2447Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly800 805 810 815ttg ggt caa cat gct tcc ctc atc ttg gct gca ctc aag gat ggt gct 2495Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala 820 825 830ctc acc gtc acc gat gtg gct caa gcc atg aac aag gag gag tcc ctc 2543Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu 835 840 845ttg caa atg gct gcc aac cag gtg gag aag gac ctc acc aag ctc acc 2591Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr 850 855 860tcc tgg acc caa atc gat gcc atc ctc caa tgg ctc caa atg tcc tct 2639Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser 865 870 875gct ctt gct gtc agc cca ttg gac ctt gct ggc atg atg gct ctc aag 2687Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys880 885 890 895tac ggc att gat cac aac tac gct gcc tgg caa gca gct gcc gct gcc 2735Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala 900 905 910ctc atg gct gac cat gcc aac cag gct cag aag aag ttg gat gag acc 2783Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr 915 920 925ttc tcc aag gct ctc tgc aac tac tac atc aac gcc gtg gtt gac tct 2831Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser 930 935 940gct gcc ggt gtc agg gac agg aac ggt ctc tac acc tac ctc ttg att 2879Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile 945 950 955gac aac cag gtc tct gct gat gtc atc acc tcc aga att gct gag gcc 2927Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala960 965 970 975att gct ggc atc caa ctc tac gtc aac agg gct ctc aac agg gat gag 2975Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu 980 985 990ggt cag ttg gct tct gat gtc tcc acc agg caa ttc ttc acc gac tgg 3023Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp 995 1000 1005gag agg tac aac aag agg tac tcc acc tgg gct ggt gtc tct gag ttg 3071Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu 1010 1015 1020gtc tac tac cca gag aac tac gtg gac cca acc caa agg att ggt cag 3119Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln 1025 1030 1035acc aag atg atg gat gct ttg ctc caa tcc atc aac cag tcc caa ctc 3167Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu1040 1045 1050 1055aac gct gac act gtg gag gat gct ttc aag acc tac ctc acc tcc ttc 3215Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe 1060 1065 1070gag caa gtg gcc aac ctc aag gtc atc tct gct tac cat gac aac gtc 3263Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val 1075 1080 1085aac gtg gac caa ggt ctc acc tac ttc att ggc att gac caa gcc gct 3311Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala 1090 1095 1100cct ggc acc tac tac tgg agg tct gtg gac cac tcc aag tgc gag aac 3359Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn 1105 1110 1115ggc aag ttc gct gcc aac gct tgg ggt gag tgg aac aag atc acc tgc 3407Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys1120 1125 1130 1135gct gtc aac cct tgg aag aac atc atc agg cca gtg gtc tac atg tcc 3455Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser 1140 1145 1150aga ctc tac ttg ctc tgg ctt gag caa cag tcc aag aag tct gat gac 3503Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp 1155 1160 1165ggc aag aca act atc tac cag tac aac ctc aag ttg gct cac atc cgc 3551Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg 1170 1175 1180tac gat ggt tcc tgg aac act cca ttc acc ttc gat gtc act gag aag 3599Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys 1185 1190 1195gtc aag aac tac acc tcc agc act gat gca gct gag tcc ctt ggt ctc 3647Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu1200 1205 1210 1215tac tgc act ggt tac caa ggt gag gac acc ctc ttg gtc atg ttc tac 3695Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr 1220 1225 1230tcc atg caa tcc agc tac tcc agc tac act gac aac aac gct cca gtc 3743Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val 1235 1240 1245act ggt ctc tac atc ttc gct gac atg tcc tct gac aac atg acc aac 3791Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn 1250 1255 1260gct caa gcc acc aac tac tgg aac aac tcc tac cca caa ttc gac act 3839Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr 1265 1270 1275gtc atg gct gac cca gac tct gac aac aag aag gtc atc acc agg cgt 3887Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg1280 1285 1290 1295gtc aac aac cgc tac gct gag gac tac gag atc cca agc tct gtc acc 3935Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr 1300 1305 1310tcc aac agc aac tac tcc tgg ggt gac cac tcc ctc acc atg ctc tac 3983Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr 1315 1320 1325ggt ggc tct gtc cca aac atc acc ttc gag tct gca gct gag gac ctc 4031Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu 1330 1335 1340agg ctc tcc acc aac atg gct ctc tcc atc att cac aac ggt tac gct 4079Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala 1345 1350 1355ggc acc agg cgc atc caa tgc aac ctc atg aag caa tac gct tcc ctt 4127Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu1360 1365 1370 1375ggt gac aag ttc att atc tac gac tcc agc ttc gat gac gcc aac agg 4175Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg 1380 1385 1390ttc aac ttg gtc cca ctc ttc aag ttc ggc aag gat gag aac tct gat 4223Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp 1395 1400 1405gac tcc atc tgc atc tac aac gag aac cca agc tct gag gac aag aag 4271Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys 1410 1415 1420tgg tac ttc agc tcc aag gac gac aac aag act gct gac tac aac ggt 4319Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly 1425 1430 1435ggc acc caa tgc att gat gct ggc acc tcc aac aag gac ttc tac tac 4367Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr1440 1445 1450 1455aac ctc caa gag att gag gtc atc tct gtc act ggt ggc tac tgg tcc 4415Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser 1460 1465 1470agc tac aag atc agc aac ccc atc aac atc aac act ggc att gac tct 4463Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser 1475 1480 1485gcc aag gtc aag gtc act gtc aag gct ggt ggc gat gac caa atc ttc 4511Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe 1490 1495 1500act gct gac aac tcc acc tac gtc cca cag caa cct gct cca tcc ttc 4559Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe 1505 1510 1515gag gag atg atc tac caa ttc aac aac ctc acc att gac tgc aag aac 4607Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn1520 1525 1530 1535ctc aac ttc att gac aac cag gct cac att gag att gac ttc act gcc 4655Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala 1540 1545 1550aca gct caa gat ggc cgc ttc ttg ggt gct gag acc ttc atc att cca 4703Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro 1555 1560 1565gtc acc aag aag gtc ctt ggc act gag aac gtc att gct ctc tac tct 4751Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser 1570 1575 1580gag aac aac ggt gtc cag tac atg caa att ggt gct tac aga acc agg 4799Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg 1585 1590 1595ctc aac acc ctc ttc gct caa cag ttg gtc tcc cgt gcc aac aga ggc 4847Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly1600 1605 1610 1615att gat gct gtc ctc agc atg gag act cag aac atc caa gag cca caa 4895Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln 1620 1625 1630ctt ggt gct ggc acc tac gtc caa ctt gtc ttg gac aag tac gat gag 4943Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu 1635 1640 1645tcc att cat ggc acc aac aag tcc ttc gcc att gag tac gtg gac atc 4991Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile 1650 1655 1660ttc aag gag aac gac tcc ttc gtc atc tac caa ggt gag ttg tct gag 5039Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu 1665 1670 1675acc tcc caa act gtg gtc aag gtc ttc ctc tcc tac ttc att gag gcc 5087Thr Ser Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala1680 1685 1690 1695acc ggt aac aag aac cac ctc tgg gtc agg gcc aag tac cag aag gag 5135Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu 1700 1705 1710acc act gac aag atc ctc ttc gac agg act gat gag aag gac cca cat 5183Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His 1715 1720 1725ggt tgg ttc ctc tct gat gac cac aag acc ttc tct ggt ctc agc tct 5231Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser 1730 1735 1740gct caa gct ctc aag aac gac tct gag cca atg gac ttc tct ggt gcc 5279Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala 1745 1750 1755aac gct ctc tac ttc tgg gag ttg ttc tac tac act cca atg atg atg 5327Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met1760 1765 1770 1775gct cac agg ctc ctt caa gag cag aac ttc gat gct gcc aac cac tgg 5375Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp 1780 1785 1790ttc cgc tac gtc tgg agc cca tct ggt tac att gtg gat ggc aag att 5423Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile 1795 1800 1805gcc atc tac cac tgg aac gtc agg cca ttg gag gag gac acc tcc tgg 5471Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp 1810 1815 1820aac gct cag caa ctt gac tcc act gac cca gat gct gtg gct caa gat 5519Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp 1825 1830 1835gac cca atg cac tac aag gtg gcc acc ttc atg gcc acc ttg gac ctt 5567Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu1840 1845 1850 1855ctc atg gcc aga ggt gat gct gcc tac cgc caa ttg gag agg gac acc 5615Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr 1860 1865 1870ttg gct gag gcc aag atg tgg tac acc caa gct ctc aac ttg ctg ggt 5663Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly 1875 1880 1885gat gag cca caa gtc atg ctc tcc aca acc tgg gcc aac cca acc ttg 5711Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu 1890 1895 1900ggc aac gct gcc tcc aag acc aca caa cag gtc agg caa cag gtc ctc 5759Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu 1905 1910 1915acc caa ctc agg ctc aac tct aga gtc aag act cca ctc ttg ggc act 5807Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr1920 1925 1930 1935gcc aac tcc ctc act gct ctc ttc ctc cca caa gag aac tcc aaa ctt 5855Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu 1940 1945 1950aag ggt tac tgg agg acc ctt gct caa cgc atg ttc aac ctc agg cac 5903Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His 1955 1960 1965aac ctc tcc att gat ggt caa cca ctc tcc ttg cca ctc tac gct aag 5951Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys 1970 1975 1980cca gct gac cca aag gct ctc ctt tcc gct gct gtc tcc gca tcc caa 5999Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln 1985 1990 1995ggt ggt gct gac ctc cca aag gct cca ctc acc atc cac agg ttc cca 6047Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro2000 2005 2010 2015caa atg ttg gag ggt gcc cgt ggt ctt gtc aac cag ctc atc caa ttc 6095Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe 2020 2025 2030ggt tcc tct ctc ctt ggt tac tct gag agg caa gat gct gag gcc atg 6143Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met 2035 2040 2045tcc caa ctc ttg caa acc cag gct tct gag ttg atc ctc acc tcc atc 6191Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile 2050 2055 2060agg atg caa gac aac cag ctt gct gag ttg gac tct gag aag act gct 6239Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala 2065 2070 2075ctc caa gtc tcc ctt gct ggt gtc caa cag agg ttc gac agc tac tcc 6287Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser2080 2085 2090 2095caa ctc tac gag gag aac atc aac gct ggt gag caa agg gct ttg gct 6335Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala 2100 2105 2110ctc agg tct gag tct gcc att gag tcc caa ggt gct caa atc tcc cgc 6383Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg 2115 2120 2125atg gct ggt gct ggc gtg gac atg gct cca aac atc ttc ggt ctt gct 6431Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala 2130 2135 2140gat ggt ggc atg cac tac ggt gcc att gct tac gcc att gct gat ggc 6479Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly 2145 2150 2155att gag ctt tct gct tct gcc aag atg gtt gat gct gag aag gtg gct 6527Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala2160 2165 2170 2175caa tct gaa atc tac cgt cgc aga cgc caa gaa tgg aag atc caa agg 6575Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg 2180 2185 2190gac aac gct caa gct gag atc aac cag ctc aac gct caa ctt gag tcc 6623Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser 2195 2200 2205ctc agc atc agg cgt gag gct gct gag atg cag aag gag tac ctc aag 6671Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys 2210 2215 2220acc caa cag gct caa gct cag gct caa ctc acc ttc ctc agg tcc aag 6719Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys 2225 2230 2235ttc tcc aac cag gct ctc tac tcc tgg ctc aga ggc cgc ctc tct ggc 6767Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly2240 2245 2250 2255atc tac ttc caa ttc tac gac ttg gct gtc tcc cgc tgc ctc atg gct 6815Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala 2260 2265 2270gag caa tcc tac caa tgg gag gcc aac gac aac agc atc tcc ttc gtc 6863Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val 2275 2280 2285aag cca ggt gct tgg caa ggc acc tac gct ggt ctc ctt tgc ggt gag 6911Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu 2290 2295 2300gct ctc atc cag aac ttg gct caa atg gag gag gct tac ctc aag tgg 6959Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp 2305 2310 2315gag tcc aga gct ttg gag gta gag agg act gtc tcc ctt gct gta gtc 7007Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val2320 2325 2330 2335tac gac tcc ttg gag ggc aac gac agg ttc aac ctt gct gag caa atc 7055Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile 2340 2345 2350cca gct ctc ttg gac aag ggt gag ggc act gct ggc acc aag gag aac 7103Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn 2355 2360 2365ggt ctc tcc ttg gcc aac gcc atc ctc tct gct tct gtc aag ctc tct 7151Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser 2370 2375 2380gac ctc aag ttg ggt act gac tac cca gac tcc att gtg ggt tcc aac 7199Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn 2385 2390 2395aag gtc aga agg atc aag caa atc tct gtc tcc ctc cca gct ttg gtg 7247Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val2400 2405 2410 2415ggt cca tac caa gat gtc caa gcc atg ctc tcc tac ggt ggc tcc acc 7295Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr 2420 2425 2430caa ctc cca aag ggt tgc tct gct ttg gct gtc tcc cac ggc acc aac 7343Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn 2435 2440 2445gac tct ggt caa ttc caa ctt gac ttc aac gat ggc aag tac ctc cca 7391Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro 2450 2455 2460ttc gaa ggc att gct ttg gat gac caa ggc acc ctc aac ctc caa ttc 7439Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe 2465 2470 2475cca aac gcc act gac aag cag aag gcc atc ctc caa acc atg tct gac 7487Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp2480 2485 2490 2495atc atc ctc cac atc agg tac acc atc agg tgagctcgag aggcctgcgg 7537Ile Ile Leu His Ile Arg Tyr Thr Ile Arg 2500 2505ccgc 7541 5 63 DNA Artificial Sequence Description of Artificial Sequencehemicot sequence encoding ER signal from 15 kDa zein from Black Mexican Sweet maize 5atg gct aag atg gtc att gtg ctt gtg gtc tgc ttg gct ctc tct gct 48Met Ala Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser Ala 1 5 10 15gcc tgt gct tca gcc 63Ala Cys Ala Ser Ala 20 6 7621 DNA Artificial Sequence Description of Artificial Sequencehemicot tcdA fused to the modified 15 kDa zein endoplasmic reticulum signal peptide 6ncc atg gct aag atg gtc att gtg ctt gtg gtc tgc ttg gct ctc tct 48 Met Ala Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser 1 5 10 15gct gcc tgt gct tca gcc atg aac gag tcc gtc aag gag atc cca gac 96Ala Ala Cys Ala Ser Ala Met Asn Glu Ser Val Lys Glu Ile Pro Asp 20 25 30gtc ctc aag tcc caa tgc ggt ttc aac tgc ctc act gac atc tcc cac 144Val Leu Lys Ser Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser His 35 40 45agc tcc ttc aac gag ttc aga caa caa gtc tct gag cac ctc tcc tgg 192Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp 50 55 60tcc gag acc cat gac ctc tac cat gac gct cag caa gct cag aag gac 240Ser Glu Thr His Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys Asp 65 70 75aac agg ctc tac gag gct agg atc ctc aag agg gct aac cca caa ctc 288Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu 80 85 90 95cag aac gct gtc cac ctc gcc atc ttg gct cca aac gct gag ttg att 336Gln Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile 100 105 110ggt tac aac aac cag ttc tct ggc aga gct agc cag tac gtg gct cct 384Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro 115 120 125ggt aca gtc tcc tcc atg ttc agc cca gcc gct tac ctc act gag ttg 432Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu 130 135 140tac cgc gag gct agg aac ctt cat gct tct gac tcc gtc tac tac ttg 480Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu 145 150 155gac aca cgc aga cca gac ctc aag agc atg gcc ctc agc caa cag aac 528Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn160 165 170 175atg gac att gag ttg tcc acc ctc tcc ttg agc aac gag ctt ctc ttg 576Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu 180 185 190gag tcc atc aag act gag agc aag ttg gag aac tac acc aag gtc atg 624Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met 195 200 205gag atg ctc tcc acc ttc aga cca agc ggt gca act cca tac cat gat 672Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp 210 215 220gcc tac gag aac gtc agg gag gtc atc caa ctt caa gac cct ggt ctt 720Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu 225 230 235gag caa ctc aac gct tct cca gcc att gct ggt ttg atg cac cag gca 768Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala240 245 250 255tcc ttg ctc ggt atc aac gcc tcc atc tct cct gag ttg ttc aac atc 816Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile 260 265 270ttg act gag gag atc act gag ggc aac gct gag gag ttg tac aag aag 864Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys 275 280 285aac ttc ggc aac att gag cca gcc tct ctt gca atg cct gag tac ctc 912Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu 290 295 300aag agg tac tac aac ttg tct gat gag gag ctt tct caa ttc att ggc 960Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly 305 310 315aag gct tcc aac ttc ggt caa cag gag tac agc aac aac cag ctc atc 1008Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile320 325 330 335act cca gtt gtg aac tcc tct gat ggc act gtg aag gtc tac cgc atc 1056Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile 340 345 350aca cgt gag tac acc aca aac gcc tac caa atg gat gtt gag ttg ttc 1104Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe 355 360 365cca ttc ggt ggt gag aac tac aga ctt gac tac aag ttc aag aac ttc 1152Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe 370 375 380tac aac gcc tcc tac ctc tcc atc aag ttg aac gac aag agg gag ctt 1200Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu 385 390 395gtc agg act gag ggt gct cct caa gtg aac att gag tac tct gcc aac 1248Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn400 405 410 415atc acc ctc aac aca gct gac atc tct caa cca ttc gag att ggt ttg 1296Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu 420 425 430acc aga gtc ctt ccc tct ggc tcc tgg gcc tac gct gca gcc aag ttc 1344Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe 435 440 445act gtt gag gag tac aac cag tac tct ttc ctc ttg aag ctc aac aag 1392Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys 450 455 460gca att cgt ctc agc aga gcc act gag ttg tct ccc acc atc ttg gag 1440Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu 465 470 475ggc att gtg agg tct gtc aac ctt caa ctt gac atc aac act gat gtg 1488Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val480 485 490 495ctt ggc aag gtc ttc ctc acc aag tac tac atg caa cgc tac gcc atc 1536Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile 500 505 510cat gct gag act gca ctc atc ctc tgc aac gca ccc atc tct caa cgc 1584His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg 515 520 525tcc tac gac aac cag cct tcc cag ttc gac agg ctc ttc aac act cct 1632Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro 530 535 540ctc ttg aac ggc cag tac ttc tcc act ggt gat gag gag att gac ctc 1680Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu 545 550 555aac tct ggc tcc aca ggt gac tgg aga aag acc atc ttg aag agg gcc 1728Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala560 565 570 575ttc aac att gat gat gtc tct ctc ttc cgt ctc ttg aag atc aca gat 1776Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp 580 585 590cac gac aac aag gat ggc aag atc aag aac aac ttg aag aac ctt tcc 1824His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser 595 600 605aac ctc tac att ggc aag ttg ctt gca gac atc cac caa ctc acc att 1872Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile 610 615 620gat gag ttg gac ctc ttg ctc att gca gtc ggt gag ggc aag acc aac 1920Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn 625 630 635ctc tct gca atc tct gac aag cag ttg gca acc ctc atc agg aag ttg 1968Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu640 645 650 655aac acc atc acc tcc tgg ctt cac acc cag aag tgg tct gtc ttc caa 2016Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln 660 665 670ctc ttc atc atg acc agc acc tcc tac aac aag acc ctc act cct gag 2064Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu 675 680 685atc aag aac ctc ttg gac aca gtc tac cac ggt ctc caa ggc ttc gac 2112Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp 690 695 700aag gac aag gct gac ttg ctt cat gtc atg gct ccc tac att gca gcc 2160Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala 705 710 715acc ctc caa ctc tcc tct gag aac gtg gct cac tct gtc ttg ctc tgg 2208Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp720 725 730 735gct gac aag ctc caa cct ggt gat ggt gcc atg act gct gag aag ttc 2256Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe 740 745 750tgg gac tgg ctc aac acc aag tac aca cca ggc tcc tct gag gct gtt 2304Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val 755 760 765gag act caa gag cac att gtg caa tac tgc cag gct ctt gca cag ttg 2352Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu 770 775 780gag atg gtc tac cac tcc act ggc atc aac gag aac gct ttc aga ctc 2400Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu 785 790 795ttc gtc acc aag cct gag atg ttc ggt gct gcc aca ggt gct gca cct 2448Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro800 805 810 815gct cat gat gct ctc tcc ctc atc atg ttg acc agg ttc gct gac tgg 2496Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp 820 825 830gtc aac gct ctt ggt gag aag gct tcc tct gtc ttg gct gcc ttc gag 2544Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu 835 840 845gcc aac tcc ctc act gct gag caa ctt gct gat gcc atg aac ctt gat 2592Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp 850 855 860gcc aac ctc ttg ctc caa gct tcc att caa gct cag aac cac caa cac 2640Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His 865 870 875ctc cca cct gtc act cca gag aac gct ttc tcc tgc tgg acc tcc atc 2688Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile880 885 890 895aac acc atc ctc caa tgg gtc aac gtg gct cag caa ctc aac gtg gct 2736Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala 900 905 910cca caa ggt gtc tct gct ttg gtc ggt ctt gac tac atc cag tcc atg 2784Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met 915 920 925aag gag aca cca acc tac gct caa tgg gag aac gca gct ggt gtc ttg 2832Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu 930 935 940act gct ggt ctc aac tcc caa cag gcc aac acc ctc cat gct ttc ttg 2880Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu 945 950 955gat gag tct cgc tct gct gcc ctc tcc acc tac tac atc agg caa gtc 2928Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val960 965 970 975gcc aag gca gct gct gcc atc aag tct cgc gat gac ctc tac caa tac 2976Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr 980 985 990ctc ctc att gac aac cag gtc tct gct gcc atc aag acc acc agg atc 3024Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile 995 1000 1005gct gag gcc atc gct tcc atc caa ctc tac gtc aac cgc gct ctt gag 3072Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu 1010 1015 1020aac gtt gag gag aac gcc aac tct ggt gtc atc tct cgc caa ttc ttc 3120Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe 1025 1030 1035atc gac tgg gac aag tac aac aag agg tac tcc acc tgg gct ggt gtc 3168Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val1040 1045 1050 1055tct caa ctt gtc tac tac cca gag aac tac att gac cca acc atg agg 3216Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg 1060 1065 1070att ggt cag acc aag atg atg gat gct ctc ttg caa tct gtc tcc caa 3264Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln 1075 1080 1085agc caa ctc aac gct gac act gtg gag gat gcc ttc atg agc tac ctc 3312Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu 1090 1095 1100acc tcc ttc gag caa gtt gcc aac ctc aag gtc atc tct gct tac cat 3360Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His 1105 1110 1115gac aac atc aac aac gac caa ggt ctc acc tac ttc att ggt ctc tct 3408Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser1120 1125 1130 1135gag act gat gct ggt gag tac tac tgg aga tcc gtg gac cac agc aag 3456Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys 1140 1145 1150ttc aac gat ggc aag ttc gct gca aac gct tgg tct gag tgg cac aag 3504Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys 1155 1160 1165att gac tgc cct atc aac cca tac aag tcc acc atc aga cct gtc atc 3552Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile 1170 1175 1180tac aag agc cgc ctc tac ttg ctc tgg ctt gag cag aag gag atc acc 3600Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr 1185 1190 1195aag caa act ggc aac tcc aag gat ggt tac caa act gag act gac tac 3648Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr1200 1205 1210 1215cgc tac gag ttg aag ttg gct cac atc cgc tac gat ggt acc tgg aac 3696Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn 1220 1225 1230act cca atc acc ttc gat gtc aac aag aag atc agc gag ttg aag ttg 3744Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu 1235 1240 1245gag aag aac cgt gct cct ggt ctc tac tgc gct ggt tac caa ggt gag 3792Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu 1250 1255 1260gac acc ctc ttg gtc atg ttc tac aac cag caa gac acc ctt gac tcc 3840Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser 1265 1270 1275tac aag aac gct tcc atg caa ggt ctc tac atc ttc gct gac atg gct 3888Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala1280 1285 1290 1295tcc aag gac atg act cca gag caa agc aac gtc tac cgt gac aac tcc 3936Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser 1300 1305 1310tac caa cag ttc gac acc aac aac gtc agg cgt gtc aac aac aga tac 3984Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr 1315 1320 1325gct gag gac tac gag atc cca agc tct gtc agc tct cgc aag gac tac 4032Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr 1330 1335 1340ggc tgg ggt gac tac tac ctc agc atg gtg tac aac ggt gac atc cca 4080Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro 1345 1350 1355acc atc aac tac aag gct gcc tct tcc gac ctc aaa atc tac atc agc 4128Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser1360 1365 1370 1375cca aag ctc agg atc atc cac aac ggc tac gag ggt cag aag agg aac 4176Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn 1380 1385 1390cag tgc aac ttg atg aac aag tac ggc aag ttg ggt gac aag ttc att 4224Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile 1395 1400 1405gtc tac acc tct ctt ggt gtc aac cca aac aac agc tcc aac aag ctc 4272Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu 1410 1415 1420atg ttc tac cca gtc tac caa tac tct ggc aac acc tct ggt ctc aac 4320Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn 1425 1430 1435cag ggt aga ctc ttg ttc cac agg gac acc acc tac cca agc aag gtg 4368Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val1440 1445 1450 1455gag gct tgg att cct ggt gcc aag agg tcc ctc acc aac cag aac gct 4416Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala 1460 1465 1470gcc att ggt gat gac tac gcc aca gac tcc ctc aac aag cct gat gac 4464Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp 1475 1480 1485ctc aag cag tac atc ttc atg act gac tcc aag ggc aca gcc act gat 4512Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp 1490 1495 1500gtc tct ggt cca gtg gag atc aac act gca atc agc cca gcc aag gtc 4560Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val 1505 1510 1515caa atc att gtc aag gct ggt ggc aag gag caa acc ttc aca gct gac 4608Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp1520 1525 1530 1535aag gat gtc tcc atc cag cca agc cca tcc ttc gat gag atg aac tac 4656Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr 1540 1545 1550caa ttc aac gct ctt gag att gat ggt tct ggc ctc aac ttc atc aac 4704Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn 1555 1560 1565aac tct gct tcc att gat gtc acc ttc act gcc ttc gct gag gat ggc 4752Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly 1570 1575 1580cgc aag ttg ggt tac gag agc ttc tcc atc cca gtc acc ctt aag gtt 4800Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val 1585 1590 1595tcc act gac aac gca ctc acc ctt cat cac aac gag aac ggt gct cag 4848Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln1600 1605 1610 1615tac atg caa tgg caa agc tac cgc acc agg ttg aac acc ctc ttc gca 4896Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala 1620 1625 1630agg caa ctt gtg gcc cgt gcc acc aca ggc att gac acc atc ctc agc 4944Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser 1635 1640 1645atg gag acc cag aac atc caa gag cca cag ttg ggc aag ggt ttc tac 4992Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr 1650 1655 1660gcc acc ttc gtc atc cca cct tac aac ctc agc act cat ggt gat gag 5040Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu 1665 1670 1675agg tgg ttc aag ctc tac atc aag cac gtg gtt gac aac aac tcc cac 5088Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His1680 1685 1690 1695atc atc tac tct ggt caa ctc act gac acc aac atc aac atc acc ctc 5136Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu 1700 1705 1710ttc atc cca ctt gac gat gtc cca ctc aac cag gac tac cat gcc aag 5184Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys 1715 1720 1725gtc tac atg acc ttc aag aag tct cca tct gat ggc acc tgg tgg ggt 5232Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly 1730 1735 1740cca cac ttc gtc cgt gat gac aag ggc atc gtc acc atc aac cca aag 5280Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys 1745 1750 1755tcc atc ctc acc cac ttc gag tct gtc aac gtt ctc aac aac atc tcc 5328Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser1760 1765 1770 1775tct gag cca atg gac ttc tct ggt gcc aac tcc ctc tac ttc tgg gag 5376Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu 1780 1785 1790ttg ttc tac tac aca cca atg ctt gtg gct caa agg ttg ctc cat gag 5424Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu 1795 1800 1805cag aac ttc gat gag gcc aac agg tgg ctc aag tac gtc tgg agc cca 5472Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro 1810 1815 1820tct ggt tac att gtg cat ggt caa atc cag aac tac caa tgg aac gtc 5520Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val 1825 1830 1835agg cca ttg ctt gag gac acc tcc tgg aac tct gac cca ctt gac tct 5568Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser1840 1845 1850 1855gtg gac cct gat gct gtg gct caa cat gac cca atg cac tac aag gtc 5616Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val 1860 1865 1870tcc acc ttc atg agg acc ttg gac ctc ttg att gcc aga ggt gac cat 5664Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His 1875 1880 1885gct tac cgc caa ttg gag agg gac acc ctc aac gag gca aag atg tgg 5712Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp 1890 1895 1900tac atg caa gct ctc cac ctc ttg ggt gac aag cca tac ctc cca ctc 5760Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu 1905 1910 1915agc acc act tgg tcc gac cca agg ttg gac cgt gct gct gac atc acc 5808Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr1920 1925 1930 1935act cag aac gct cat gac tct gcc att gtt gct ctc agg cag aac atc 5856Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile 1940 1945 1950cca act cct gct cca ctc tcc ctc aga tct gct aac acc ctc act gac 5904Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp 1955 1960 1965ttg ttc ctc cca cag atc aac gag gtc atg atg aac tac tgg caa acc 5952Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr 1970 1975 1980ttg gct caa agg gtc tac aac ctc aga cac aac ctc tcc att gat ggt 6000Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly 1985 1990 1995caa cca ctc tac ctc cca atc tac gcc aca cca gct gac cca aag gct 6048Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala2000 2005 2010 2015ctt ctc tct gct gct gtg gct acc agc caa ggt ggt ggc aag ctc cca 6096Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro 2020 2025 2030gag tcc ttc atg tcc ctc tgg agg ttc cca cac atg ttg gag aac gcc 6144Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala 2035 2040 2045cgt ggc atg gtc tcc caa ctc acc cag ttc ggt tcc acc ctc cag aac 6192Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn 2050 2055 2060atc att gag agg caa gat gct gag gct ctc aac gct ttg ctc cag aac 6240Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn 2065 2070 2075cag gca gct gag ttg atc ctc acc aac ttg tcc atc caa gac aag acc 6288Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr2080 2085 2090 2095att gag gag ctt gat gct gag aag aca gtc ctt gag aag agc aag gct 6336Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala 2100 2105 2110ggt gcc caa tct cgc ttc gac tcc tac ggc aag ctc tac gat gag aac 6384Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn 2115 2120 2125atc aac gct ggt gag aac cag gcc atg acc ctc agg gct tcc gca gct 6432Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala 2130 2135 2140ggt ctc acc act gct gtc caa gcc tct cgc ttg gct ggt gca gct gct 6480Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala 2145 2150 2155gac ctc gtt cca aac atc ttc ggt ttc gct ggt ggt ggc tcc aga tgg 6528Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp2160 2165 2170 2175ggt gcc att gct gag gct acc ggt tac gtc atg gag ttc tct gcc aac 6576Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn 2180 2185 2190gtc atg aac act gag gct gac aag atc agc caa tct gag acc tac aga 6624Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg 2195 2200 2205agg cgc cgt caa gag tgg gag atc caa agg aac aac gct gag gca gag 6672Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu 2210 2215 2220ttg aag caa atc gat gct caa ctc aag tcc ttg gct gtc aga agg gag 6720Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu 2225 2230 2235gct gct gtc ctc cag aag acc tcc ctc aag acc caa cag gag caa acc 6768Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr2240 2245 2250 2255cag tcc cag ttg gct ttc ctc caa agg aag ttc tcc aac cag gct ctc 6816Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu 2260 2265 2270tac aac tgg ctc aga ggc cgc ttg gct gcc atc tac ttc caa ttc tac 6864Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr 2275 2280 2285gac ctt gct gtg gcc agg tgc ctc atg gct gag caa gcc tac cgc tgg 6912Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp 2290 2295 2300gag ttg aac gat gac tcc gcc agg ttc atc aag cca ggt gct tgg caa 6960Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln 2305 2310 2315ggc acc tac gct ggt ctc ctt gct ggt gag acc ctc atg ctc tcc ttg 7008Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu2320 2325 2330 2335gct caa atg gag gat gct cac ctc aag agg gac aag agg gct ttg gag 7056Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu 2340 2345 2350gtg gag agg aca gtc tcc ctt gct gag gtc tac gct ggt ctc cca aag 7104Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys 2355 2360 2365gac aac ggt cca ttc tcc ctt gct caa gag att gac aag ttg gtc agc 7152Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser 2370 2375 2380caa ggt tct ggt tct gct ggt tct ggt aac aac aac ttg gct ttc ggc 7200Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly 2385 2390 2395gct ggt act gac acc aag acc tcc ctc caa gcc tct gtc tcc ttc gct 7248Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala2400 2405 2410 2415gac ctc aag atc agg gag gac tac cca gct tcc ctt ggc aag atc agg 7296Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg 2420 2425 2430cgc atc aag caa atc tct gtc acc ctc cca gct ctc ttg ggt cca tac 7344Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr 2435 2440 2445caa gat gtc caa gca atc ctc tcc tac ggt gac aag gct ggt ttg gcg 7392Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala 2450 2455 2460aac ggt tgc gag gct ctt gct gtc tct cat ggc atg aac gac tct ggt 7440Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly 2465 2470 2475caa ttc caa ctt gac ttc aac gat ggc aag ttc ctc cca ttc gag ggc 7488Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly2480 2485 2490 2495att gcc att gac caa ggc acc ctc acc ctc tcc ttc cca aac gct tcc 7536Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser 2500 2505 2510atg cca gag aag gga aag caa gcc acc atg ctc aag acc ctc aac gat 7584Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp 2515 2520 2525atc atc ctc cac atc agg tac acc atc aag tgagctc 7621Ile Ile Leu His Ile Arg Tyr Thr Ile Lys 2530 2535

Claims

1. An isolated nucleic acid comprising the sequence of SEQ ID NO: 3.

2. A transgenic monocot slant cell comprising a nucleic acid comprising the sequence of SEQ ID NO: 3.

3. A transgenic dicot plant cell comprising a nucleic acid comprising the sequence of SEQ ID NO: 3.

4. A transgenic plant with a genome comprising a nucleic acid comprising the sequence of SEQ ID NO: 3 that imparts insect resistance.

5. The transgenic plant of claim 4 wherein the plant is rice.

6. The transgenic plant of claim 4 wherein the plant is maize.

7. The transgenic plant of claim 4 wherein the plant is tobacco.