Plant fatty acid synthases and use in improved methods for production of medium-chain fatty acids

Abstract

By this invention, compositions and methods of use related to β-ketoacyl-ACP synthases. Of special interest are synthases obtainable from Cuphea species. Amino acid and nucleic acid for synthase protein factors are provided, as well as methods to utilize such sequences in constructs for production of genetically engineered plants having altered fatty acid compositions. Of particular interest is the expression of synthase protein factors in conjunction with expression of plant medium-chain acyl-ACP thioesterases for production of increased levels and/or modified ratios of medium-chain fatty acids in oils of transgenic plant seeds.

Description

INTRODUCTION

1. Field of Invention

The present invention is directed to genes encoding plant fatty acid synthase enzymes relevant to fatty acid synthesis in plants, and to methods of using such genes in combination with genes encoding plant medium-chain preferring thioesterase proteins. Such uses provide a method to increase the levels of medium-chain fatty acids that may be produced in seed oils of transgenic plants.

2. Background

Higher plants synthesize fatty acids via a common metabolic pathway. In developing seeds, where fatty acids attached to triglycerides are stored as a source of energy for further germination, the fatty acid synthesis pathway is located in the plastids. The first step is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by a short chain preferring condensing enzyme, β-ketoacyl-ACP synthase (KAS) III. Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a longer β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (β-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). β-ketoacyl-ACP synthase I (KAS I), is primarily responsible for elongation up to palmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II (KAS II) is predominantly responsible for the final elongation to stearoyl-ACP (C18:0).

Genes encoding peptide components of β-ketoacyl-ACP synthases I and II have been cloned from a number of higher plant species, including castor ( Ricinus communis ) and Brassica species (U.S. Pat. No. 5,510,255). KAS I activity was associated with a single synthase protein factor having an approximate molecular weight of 50 kD (synthase factor B) and KAS II activity was associated with a combination of two synthase protein factors, the 50 kD synthase factor B and a 46 kd protein designated synthase factor A. Cloning and sequence of a plant gene encoding a KAS III protein has been reported by Tai and Jaworski ( Plant Physiol. (1993) 103:1361-1367).

The end products of plant fatty acid synthetase activities are usually 16- and 18-carbon fatty acids. There are, however, several plant families that store large amounts of 8- to 14-carbon (medium-chain) fatty acids in their oilseeds. Recent studies with Umbellularia californica (California bay), a plant that produces seed oil rich in lauric acid (12:0), have demonstrated the existence of a medium-chain-specific isozyme of acyl-ACP thioesterase in the seed plastids. Subsequent purification of the 12:0-ACP thioesterase from Umbellularia californica led to the cloning of a thioesterase cDNA which was expressed in seeds of Arabidopsis and Brassica resulting in a substantial accumulation of lauric acid in the triglyceride pools of these transgenic seeds (U.S. Pat. No. 5,512,482). These results and subsequent studies with medium-chain thioesterases from other plant species have confirmed the chain-length-determining role of acyl-ACP thioesterases during de novo fatty acid biosynthesis (T. Voelker (1996) Genetic Engineering, Ed. J. K. Setlow, Vol. 18, pgs. 111-133).

DESCRIPTION OF THE FIGURES

FIG. 1 . DNA (SEQ ID NO: 1) and translated amino acid sequence (SEQ ID NO:2) of Cuphea hookeriana KAS factor B clone chKAS B-2 are provided.

FIG. 2 . DNA (SEQ ID NO: 3) and translated amino acid sequence (SEQ ID NO:4) of Cuphea hookeriana KAS factor B clone chKAS B-31-7 are provided.

FIG. 3 . DNA (SEQ ID NO:5) and translated amino acid sequence (SEQ ID NO:6) of Cuphea hookeriana KAS factor A clone chKAS A-2-7 are provided.

FIG. 4 . DNA (SEQ ID NO:7) and translated amino acid sequence (SEQ ID NO:8) of Cuphea hookeriana KAS factor A clone chKAS A-1-6 are provided.

FIG. 5 . DNA (SEQ ID NO:9) and translated amino acid sequence (SEQ ID NO: 10) of Cuphea pullcherrima KAS factor B clone cpuKAS B/7-8 are provided.

FIG. 6 . DNA (SEQ ID NO: 11) and translated amino acid sequence (SEQ ID NO:12) of Cuphea pullcherrima KAS factor B clone cpuKAS B/8-7A are provided.

FIG. 7 . DNA (SEQ ID NO: 13) and translated amino acid sequence (SEQ ID NO:14) of Cuphea pullcherrima KAS factor A clone cpuKAS A/p7-6A are provided.

FIG. 8 . Preliminary DNA sequence (SEQ ID NO:15) of Cuphea pullcherrima KAS factor A clone cpuKAS A/p8-9A is provided.

FIG. 9 . DNA (SEQ ID NO:16) and translated amino acid sequence (SEQ ID NO:17) of Cuphea hookeriana KASIII clone chKASIII-27 are provided.

FIG. 10 . The activity profile for purified cpuKAS B/8-7A using various acyl-ACP substrates is provided.

FIG. 11 . The activity profile for purified chKAS A-2-7 and chKAS A-1-6 using various acyl-ACP substrates is provided.

FIG. 12 . The activity profile for purified castor KAS factor A using various acyl-ACP substrates is provided.

FIG. 13 . The activity profile for purified castor KAS factor B using various acyl-ACP substrates is provided.

FIG. 14. A graph showing the number of plants arranged according to C8:0 content for transgenic plants containing CpFatB1 versus transgenic plants containing CpFatB1+chKAS A-2-7 is provided.

FIG. 15 A. Graphs showing the %C10/%C8 ratios in transgenic plants resulting from cross of plants containing ChFatB2 (4804-22-357) and 5401-9 (chKAS A-2-7 plants) is provided.

FIG. 15 B. Graph showing the %C10/%C8 ratio in transgenic plants containing ChFatB2 (4804-22-357) is provided.

FIG. 16 A. Graphs showing the %C10+%C8 content in transgenic plants resulting from cross of plants containing (ChFatB2 (4804-22-357) and 5401-9 (chKAS A-2-7 plants) is provided.

FIG. 16 B. Graph showing the %C10+%C8 content in transgenic plants containing ChFatB2 (4804-22-357) is provided.

FIG. 17 A. Graphs showing the %C10/%C8 ratios in transgenic plants resulting from cross of plants containing ChFatB2 (4804-22-357) and 5413-17 (chKAS A-2-7+CpFatB1 plants) is provided.

FIG. 17 B. Graph showing showing the %C10/%C8 ratio in transgenic plants containing ChFatB2 (4804-22-357) is provided.

FIG. 18 A. Graphs showing the %C10+%C8 content in transgenic plants resulting from cross of plants containing ChFatB2 (4804-22-357) and 5413-17 (chKAS A-2-7+CpFatB1 plants) is provided.

FIG. 18 B. Graph showing the %C10+%C8 content in transgenic plants containing ChFatB2 (4804-22-357) is provided.

FIG. 19 A. Graphs showing the %C12:0 in transgenic plants containing Uc FatB1 (LA86DH186) is provided.

FIG. 19 B. Graph showing the %C12:0 in transgenic plants resulting from cross of plants containing Uc FatB1 (LA86DH186) and wild type (X WT) is provided.

FIG. 19 C. Graph showing the %C12:0 in transgenic plants resulting from cross of plants containing Uc FatB1 (LA86DH186) and lines expressing Ch KAS A-2-7 is provided.

FIG. 20 . Graph showing the relative proportions of C12:0 and C14:0 fatty acids in the seeds of transgenic plants containing Uc FatB1 (LA86DH186) and in plants resulting from crosses with wild type (X WT) and with lines expressing Ch KAS A-2-7.

FIG. 21 A. Graphs showing the %C18:0 in transgenic plants containing Garm FatB1 (5266) is provided.

FIG. 21 B. Graph showing the %C18:0 in transgenic seeds resulting from cross of plants containing Garm FatB1 (5266) and wild type (X WT) is provided.

FIG. 21 C. Graph showing the %C18:0 in transgenic seeds of plants resulting from cross of plants containing Garm FatB1 (5266) and lines expressing Ch KAS A-2-7 is provided.

FIG. 22 . The activity profile of Ch KAS A in protein extracts from transgenic plants containing Ch KAS A-2-7. Extracts were preptreated with the indicated concentrations of cerulenin.

SUMMARY OF THE INVENTION

By this invention, compositions and methods of use related to β-ketoacyl-ACP synthase (KAS) are provided. Also of interest are methods and compositions of amino acid and nucleic acid sequences related to biologically active plant synthase(s).

In particular, genes encoding KAS protein factors A and B from Cuphea species are provided. The KAS genes are of interest for use in a variety of applications, and may be used to provide synthase I and/or synthase II activities in transformed host cells, including bacterial cells, such as E. coli, and plant cells. Synthase activities are distinguished by the preferential activity towards longer and shorter acyl-ACPs as well as by the sensitivity towards the KAS specific inhibitor, cerulenin. Synthase protein preparations having preferential activity towards medium chain length acyl-ACPs are synthase I-type or KAS I. The KAS I class is sensitive to inhibition by cerulenin at concentrations as low as 1 μM. Synthases having preferential activity towards longer chain length acyl-ACPs are synthase II-type or KAS II. The KAS enzymes of the II-type are also sensitive to cerulenin, but at higher concentrations (50 μM). Synthase III-type enzymes have preferential activity towards short chain length acyl-ACPs and are insensitive to cerulenin inhibition.

Nucleic acid sequences encoding a synthase protein may be employed in nucleic acid constructs to modulate the amount of synthase activity present in the host cell, especially the relative amounts of synthase I-type, synthase II-type and synthase III-type activity when the host cell is a plant host cell. In addition, nucleic acid constructs may be designed to decrease expression of endogenous synthase in a plant cell as well. One example is the use of an anti-sense synthase sequence under the control of a promoter capable of expression in at least those plant cells which normally produce the enzyme.

Of particular interest in the present invention is the coordinate expression of a synthase protein with the expression of thioesterase proteins. For example, coordinated expression of synthase factor A and a medium-chain thioesterase provides a method for increasing the level of medium-chain fatty acids which may be harvested from transgenic plant seeds. Furthermore, coordinated expression of a synthase factor A gene with plant medium-chain thioesterase proteins also provides a method by which the ratios of various medium-chain fatty acids produced in a transgenic plant may be modified. For example, by expression of a synthase factor A, it is possible to increase the ratio of C10/C8 fatty acids which are produced in plant seed oils as the result of expression of a thioesterase having activity on C8 and C10 fatty acids.

DETAILED DESCRIPTION OF THE INVENTION

A plant synthase factor protein of this invention includes a sequence of amino acids or polypeptide which is required for catalyzation of a condensation reaction between an acyl-ACP having a chain length of C 2 to C 16 and malonyl-ACP in a plant host cell. A particular plant synthase factor protein may be capable of catalyzing a synthase reaction in a plant host cell (for example as a monomer or homodimer) or may be one component of a multiple peptide enzyme which is capable of catalyzing a synthase reaction in a plant host cell, i.e. one peptide of a heterodimer.

Synthase I (KAS I) demonstrates preferential activity towards acyl-ACPs having shorter carbon chains, C 2 -C 14 and is sensitive to inhibition by cerulenin at concentrations of 1 μM. Synthase II (KAS II) demonstrates preferential activity towards acyl-ACPs having longer carbon chains, C 14 -C 16 , and is inhibited by concentrations of cerulenin (50 μM). Synthase III demonstrates preferential activity towards acyl-CoAs having very short carbon chains, C 2 to C 6 , and is insensitive to inhibition by cerulenin.

Synthase factors A and B, and synthase III proteins obtained from medium-chain fatty acid producing plant species of the genus Cuphea are described herein. As described in the following Examples, synthase A from C. hookeriana is naturally expressed at a high level and only in the seeds. C. hookeriana synthase B is expressed at low levels in all tissues examined. Expression of synthase A and synthase B factors in E. coli and purification of the resulting proteins is employed to determine activity of the various synthase factors. Results of these analyses indicate that synthase factor A from Cuphea hookeriana has the greatest activity on 6:0-ACP substrates, whereas synthase factor B from Cuphea pullcherrima has greatest activity on 14:0-ACP. Similar studies with synthase factors A and B from castor demonstrate similar activity profiles between the factor B synthase proteins from Cuphea and castor. The synthase A clone from castor, however, demonstrates a preference for 14:0-ACP substrate.

Expression of a Cuphea hookeriana KAS A protein in transgenic plant seeds which normally do not produce medium-chain fatty acids does not result in any detectable modification of the fatty acid types and contents produced in such seeds. However, when Cuphea hookeriana KAS A protein is expressed in conjunction with expression of a medium-chain acyl-ACP thioesterase capable of providing for production of C8 and C10 fatty acids in plant seed oils, increases in the levels of medium-chain fatty acids over the levels obtainable by expression of the medium-chain thioesterase alone are observed. In addition, where significant amounts of C8 and C10 fatty acids are produced as the result of medium-chain thioesterase expression, co-expression of a Cuphea KAS A protein also results in an alteration of the proportion of the C8 and C10 fatty acids that are obtained. For example, an increased proportion of C10 fatty acids may be obtained by co-expression of Cuphea hookeriana ChFatB2 thioesterase and a chKAS A synthase factor proteins.

Furthermore, when Cuphea hookeriana KAS A protein is expressed in conjunction with expression of a medium-chain acyl-ACP thioesterase capable of providing for production of C12 fatty acids in plant seed oils, increases in the levels of medium-chain fatty acids over the levels obtainable by expression of the medium-chain thioesterase alone are also observed. In addition, where significant amounts of C12 and C14 fatty acids are produced as the result of medium-chain thioesterase expression, co-expression of a Cuphea KAS A protein also results in an alteration of the proportion of the C12 and C14 fatty acids that are obtained. For example, an increased proportion of C12 fatty acids may be obtained by co-expression of Uc FatB1 thioesterase and a chKAS A synthase factor proteins.

However, when Cuphea hookeriana KAS A protein is expressed in conjunction with the expression of a long-chain acyl-ACP thioesterase capable of providing for production of C18 and C18:1 fatty acids in plant seed oils, no effect on the production of long chain fatty acids was observed. Furthermore, when plants transformed to express a long chain acyl-ACP thioesterase from mangosteen (GarmFatA1, U.S. patent application Ser. No. 08/440,845), which preferentially hydrolyzes C18:0 and C18:1 fatty acyl-ACPs, are crossed with nontransformed control plants, a significant reduction in the levels of C18:0 is obtained. Similar reductions are also observed in the levels of C18:0 in the seeds of plants resulting from crosses between plants transformed to express the GarmFatA1 and plants expressing the Cuphea hookeriana KAS A protein.

Thus, the instant invention provides methods of increasing and/or altering the medium-chain fatty acid compositions in transgenic plant seed oils by co-expression of medium-chain acyl-ACP thioesterases with synthase factor proteins. Furthermore, various combinations of synthase factors and medium-chain thioesterases may be achieved depending upon the particular fatty acids desired. For example, for increased production of C14 fatty acids, synthase protein factors may be expressed in combination with a C14 thioesterase, for example from Cuphea palustris or nutmeg may be employed (WO 96/23892). In addition, thioesterase expression may be combined with a number of different synthase factor proteins for additional effects on medium-chain fatty acid composition.

Synthases of use in the present invention include modified amino acid sequences, such as sequences which have been mutated, truncated, increased and the like, as well as such sequences which are partially or wholly artificially synthesized. The synthase protein encoding sequences provided herein may be employed in probes for further screening or used in genetic engineering constructs for transcription or transcription and translation in host cells, especially plant host cells. One skilled in the art will readily recognize that antibody preparations, nucleic acid probes (DNA and RNA) and the like may be prepared and used to screen and recover synthases and/or synthase nucleic acid sequences from other sources. Typically, a homologously related nucleic acid sequence will show at least about 60% homology, and more preferably at least about 70% homology, between the R. communis synthase and the given plant synthase of interest, excluding any deletions which may be present. Homology is determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions.

Recombinant constructs containing a nucleic acid sequence encoding a synthase protein factor or nucleic acid sequences encoding a synthase protein factor and a medium-chain acyl-ACP thioesterase may be prepared by methods well known in the art. Constructs may be designed to produce synthase in either prokaryotic or eukaryotic cells. The increased expression of a synthase in a plant cell, particularly in conjunction with expression of medium-chain thioesterases, or decreasing the amount of endogenous synthase observed in plant cells are of special interest.

Synthase protein factors may be used, alone or in combination, to catalyze the elongating condensation reactions of fatty acid synthesis depending upon the desired result. For example, rate influencing synthase activity may reside in synthase I-type, synthase II-type, synthase III-type or in a combination of these enzymes. Furthermore, synthase activities may rely on a combination of the various synthase factors described herein.

Constructs which contain elements to provide the transcription and translation of a nucleic acid sequence of interest in a host cell are “expression cassettes”. Depending upon the host, the regulatory regions will vary, including regions from structural genes from viruses, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters may be employed. Among transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, including genes such as β-galactosidase, T7 polymerase, trp-lac (tac), trp E and the like.

An expression cassette for expression of synthase in a plant cell will include, in the 5′ to 3′ direction of transcription, a transcription and translation initiation control regulatory region (also known as a “promoter”) functional in a plant cell, a nucleic acid sequence encoding a synthase, and a transcription termination region. Numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the desaturase structural gene. Among transcriptional initiation regions used for plants are such regions associated with cauliflower mosaic viruses (35S, 19S), and structural genes such as for nopaline synthase or mannopine synthase or napin and ACP promoters, etc. The transcription/translation initiation regions corresponding to such structural genes are found immediately 5′ upstream to the respective start codons. Thus, depending upon the intended use, different promoters may be desired.

Of special interest in this invention are the use of promoters which are capable of preferentially expressing the synthase in seed tissue, in particular, at early stages of seed oil formation. Examples of such seed-specific promoters include the region immediately 5′ upstream of a napin or seed ACP genes such as described in U.S. Pat. No. 5,420,034, desaturase genes such as described in Thompson et al ( Proc. Nat. Acad. Sci. (1991) 88:2578-2582), or a Bce-4 gene such as described in U.S. Pat. No. 5,530,194. Alternatively, the use of the 5′ regulatory region associated with the plant synthase structural gene, i.e., the region immediately 5′ upstream to a plant synthase structural gene and/or the transcription termination regions found immediately 3′ downstream to the plant synthase structural gene, may often be desired. In general, promoters will be selected based upon their expression profile which may change given the particular application.

In addition, one may choose to provide for the transcription or transcription and translation of one or more other sequences of interest in concert with the expression or anti-sense of the synthase sequence, particularly medium-chain plant thioesterases such as described in U.S. Pat. No. 5,512,482, to affect alterations in the amounts and/or composition of plant oils.

When one wishes to provide a plant transformed for the combined effect of more than one nucleic acid sequence of interest, a separate nucleic acid construct may be provided for each or the constructs may both be present on the same plant transformation construct. The constructs may be introduced into the host cells by the same or different methods, including the introduction of such a trait by crossing transgenic plants via traditional plant breeding methods, so long as the resulting product is a plant having both characteristics integrated into its genome.

Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformed cells. The gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species into which the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts.

The manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. Various methods for plant cell transformation include the use of Ti- or Ri-plasmids, microinjection, electroporation, liposome fusion, DNA bombardment or the like. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation.

The expression constructs may be employed with a wide variety of plant life, particularly plant life involved in the production of vegetable oils. These plants include, but are not limited to rapeseed, peanut, sunflower, safflower, cotton, soybean, corn and oilseed palm.

For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.

The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.

EXAMPLES

Example 1

Cuphea KAS Factor A and B Gene Cloning

Total RNA isolated from developing seeds of Cuphea hookeriana and Cuphea pullcherrima was used for cDNA synthesis in commercial l-based cloning vectors. For cloning each type of KAS gene, approximately 400,000-500,000 unamplified recombinant phage were plated and the plaques transferred to nitrocellulose. For KAS factor B cloning from C. hookeriana, a mixed probe containing Brassica napus KAS factor B and Ricinus communis (Castor) KAS factor B radiolabeled cDNA's was used. Similarly, a mixed probe containing Brassica napus KAS factor A and Ricinus communis KAS factor A cDNA clones was used to obtain C. hookeriana KAS factor A genes. For KASIII, a spinach KASIII cDNA clone obtained from Dr. Jan Jaworski was radiolabeled and used as a probe to isolate a KASIII clone from C. hookeriana. For KAS B and KAS A cloning from C. pullcherrima, C. hookeriana KAS B and KAS A genes chKAS B-2 and chKAS A-2-7 (see below) were radiolabeled and used as probes.

DNA sequence and translated amino acid sequence for Cuphea KAS clones are provided in FIGS. 1-9 . Cuphea hookeriana KAS factor B clones chKAS B-2 and chKAS B-31-7 are provided in FIGS. 1 and 2 . Neither of the clones is full length. Cuphea hookeriana KAS Factor A clones chKAS A-2-7 and chKAS A-1-6 are provided in FIGS. 3 and 4 . chKAS A-2-7 contains the entire encoding sequence for the KAS factor protein. Based on comparison with other plant synthase proteins, the transit peptide is believed to be represented in the amino acids encoded by nucleotides 125-466. chKAS A-1-6 is not a full length clone although some transit peptide encoding sequence is present. Nucleotides 1-180 represent transit peptide encoding sequence, and the mature protein encoding sequence is believed to begin at nucleotide 181.

Cuphea pullcherrima KAS factor B clones cpuKAS B/7-8 and cpuKAS B/8-7A are provided in FIGS. 5 and 6 . Both of the clones contain the entire encoding sequences for the KAS factor B proteins. The first 35 amino acids of cpuKAS B/7-8 are believed to represent the transit peptide, with the mature protein encoding sequence beginning at nucleotide 233. The first 39 amino acids of cpuKAS B/8-7A are believed to represent the transit peptide, with the mature protein encoding sequence beginning at nucleotide 209. Cuphea pullcherrima KAS factor A clones cpuKAS A/p7-6A and cpuKAS A-p8-9A are provided in FIGS. 7 and 8 . Both of the clones contain the entire encoding sequences for the KAS factor A proteins. Translated amino acid sequence of cpuKAS A/p7-6A is provided. The mature protein is believed to begin at the lysine residue encoded 595-597, and the first 126 amino acids are believed to represent the transit peptide. The DNA sequence of KAS A clone cpuKAS Ap-8-9A is preliminary. Further analysis will be conducted to determine final DNA sequence and reveal the amino acid sequence encoded by this gene.

DNA and translated amino acid sequence of Cuphea hookeriana KASIII clone chKASIII-27 is provided in FIG. 9 . The encoding sequence from nucleotides 37-144 of chKASIII-27 are believed to encode a transit peptide, and the presumed mature protein encoding sequence is from nucleotides 145-1233.

Deduced amino acid sequence of the C. hookeriana KAS factor B and KAS factor A cDNA's reveals strong homology to the Brassica napus and Ricinus communis clones previously reported. The C. hookeriana KAS factor B clone is more homologous to the Ricinus and Brassica KAS factor B clones (94% and 91% respectively) than it is to the Ricinus and Brassica KAS factor A clones (60% for both). Furthermore, the C. hookeriana KAS factor A clone is more homologous to the Ricinus and Brassica KAS factor A clones (85% and 82% respectively) than it is the Ricinus and Brassica KAS factor B clone (60% for both). The C. hookeriana KAS factor B cDNAs designated as chKAS B-2 and chKAS B-31-7 are 96% identical within the mature portion of the polypeptide. Similarly, the deduced amino acid sequence of the mature protein regions of the C. hookeriana KAS factor A clones chKAS A-2-7 and chKAS A-1-6 are 96% identical. The C. pullcherrima KAS clones also demonstrate homology to the R. communis and Brassica napus KAS clones. The mature protein portion of all of the KAS factor A family members in the different Cuphea species are 95% identical. Similarly the mature protein portion of the KAS factor B genes in Cuphea are also 95-97% identical with each other. However there is only approximately 60% sequence identity between KAS factor B and KAS factor A clones either within the same or different species of Cuphea.

Example 2

Levels and Patterns of Expression

To examine tissue specificity of KAS expression in Cuphea hookeriana, Northern blot analysis was conducted using total RNA isolated from seed, root, leaf and flower tissue. Two separate but identical blots were hybridized with either chKAS B-31-7 or chKAS A-2-7 coding region probes. The data from this RNA blot analysis indicate that KAS B is expressed at a similar level in all tissues examined, whereas KAS A expression is detected only in the seed. These results also demonstrate a different level of expression for each of the syntheses. KAS A is an abundant message, whereas KAS B is expressed at low levels. Furthermore, even under highly stringent hybridization conditions (65_C, 0.1×SSC, 0.5% SDS), the KAS A probe hybridizes equally well with two seed transcripts of 2.3 and 1.9 kb. The larger hybridizing band is likely the transcript of the KAS A-2-7 gene since the size of its cDNA is 2046 bp, and the number of clones obtained from cDNA screening corresponds well with the apparent mobility of the mRNA and its abundance on the blot.

Example 3

Expression of Plant KAS Genes in E. coli

DNA fragments encoding the mature polypeptide of the Cuphea hookeriana KAS A cDNAs and the Cuphea pullcherrima KAS B cDNAs were obtained by PCR and cloned into a QIAexpress expression vector (Qiagene). Experimental conditions for maximum level of expression were determined for all of these clones and the parameters for highest level of soluble fraction were identified. Cells are grown in ECLB media containing 1M sorbitol and 2.5 mM betaine overnight and subcultured as a 1:4 dilution in the same medium. Cells are then grown for 2 hours (to approximately 0.6-0.8 O.D.) and induced with 0.4 mM IPTG and allowed to grow for 5 more hours.

Enzyme activity of the affinity purified recombinant enzymes obtained from over-expression of the chKAS A-2-7 and cpuKAS B/8-7A clones was measured using a wide range of acyl-ACP substrates (6:0- to 16:1-ACP). The activity profile for cpuKAS B/8-7A is provided in FIG. 10 . The data demonstrate that the enzyme is active with all acyl-ACP substrates examined, although activity on 6:0 to 14:0-ACP substrates is substantially greater than the activity on 16:0 and 16:1 substrates.

The activity profile of the C. hookeriana KAS A clones chKAS A-2-7 and chKAS A-1-6 is provided in FIG. 11 . The C. hookeriana KAS A clones are most active with C:6, and have the least activity with C:16:0 substrates. However, the activity of this clone on even the preferred C6:0 substrate is 50 fold lower than the activity of the C. pullcherrima KAS B clones.

A fragment containing the mature protein encoding portion of a R. communis KAS factor A clone was also cloned into a QIAexpress expression vector, expressed in E. coli and the enzyme affinity purified as described above. The activity profile for castor KAS A is provided in FIG. 12 . Highest activity is observed with C14:0 substrates, although some activity is also seen with C6:0 and C16:1. In comparison, the activity profile obtained from purified R. communis KAS factor B also using the QIAexpress expression system is provided in FIG. 13 . The KAS B clone demonstrates substantially higher levels of activity (10 fold and higher) than the R. communis KAS A clone. The preference of the KAS factor B for 6:0- to 14:0-ACP substrates is consistent with the previous observations that this protein provides KAS I activity.

Example 4

KAS and TE Expression in Transgenic Seed

Both the CpFatB1 ( C. hookeriana thioesterase cDNA; Dehesh et al. (1996) Plant Physiol. 110:203-210) and the chKAS A-2-7 were PCR amplified, sequenced, and cloned into a napin expression cassette. The napin/cp FatB1 and the napin/KAS A-2-7 fusions were ligated separately into the binary vector pCGN1558 (McBride and Summerfelt ( Pl.Mol.Biol. (1990) 14:269-276) and transformed into A. tumefaciens, EHA101. The resulting cpFatB1 binary construct is pCGN5400 and the chKAS A-2-7 construct is pCGN5401. Agrobacterium mediated transformation of a Brassica napus canola variety was carried out as described by Radke et al. ( Theor. Appl. Genet. (1988) 75:685-694; Plant Cell Reports (1992) 11:499-505). Several transgenic events were produced for each of the pCGN5400 and pCGN5401 constructs.

A double gene construct containing a napin/cpFatB1 expression construct in combination with a napin/chKAS A-2-7 expression construct was also assembled, ligated into a binary vector and used for co-cultivation of a canola Brassica variety. The binary construct containing the chFatB1 and chKAS A-2-7 expression constructs is pCGN5413.

Fatty acid analysis of 26 transgenic lines containing chKAS A-2-7 (5401 lines) showed no significant changes in the oil content or profile as compared to similar analyses of wild type canola seeds of the transformed variety.

Fatty acid analysis of 36 transgenic lines containing cpFatB1 (5400 lines) showed increased levels of C:8 and C:10 in transgenic seeds. The highest level of C:8 observed in a pool seed sample was 4.2 mol %. The C:10 levels were between 30 and 35% of the C:8 content. Fatty acid analysis of 25 transgenic lines containing the TE/KAS A tandem (5413 lines) demonstrated an overall increase in both C:8 and C:10 levels relative to those observed with TE containing lines (5400) alone. In lines containing the cpFatB1 construct alone, the average level of C:8 average were 1.5 mol %, whereas the C:8 average levels in TE/KAS A tandem containing lines was 2.37 mol %. The ratio of C:8 to C:10 remained constant in both populations. The number of transgenic events relative to the C:8 content are presented in FIG. 14 . These data show that the transgenic events with tandem TE/KAS A construct yield more lines with higher levels of C:8 than those events with single TE construct. For example, several lines containing nearly 7 mole % C8 were obtained with the TE/KAS A pCGN5413 construct, whereas the highest C8 containing line from the pCGN5400 TE alone transformation contained 4.2 mole % C8.

Half seed analysis of the T3 generation of transgenic canola plants expressing a ChFatB2 ( C. hookeriana thioesterase; Dehesh et al. (1996) The Plant Journal 9:167-172) indicate that these plant can accumulate up to 22 weight % (33 mol %) of 8:0 and 10:0 fatty acids (48044-22-357). Segregation analysis shows that these transformants contain two loci and that they are now homozygous. Selected plants grown from these half seeds were transferred into the greenhouse and later crossed with T1 transformants that had been transformed with either Cuphea hookeriana KAS A (5401) alone or KAS A/CpFatB1 double constructs (5413).

Fatty acid analysis of several events resulting from the crosses between transgenic lines containing ChFatB2 (4804-22-357) and chKAS A-2-7 (5401-9), reveal an increase in the ratio of C:10/C:8 levels (FIGS. 15 A and 15 B). This C:10/C:8 ratio in nearly all of the transgenic events containing ChFatB2 TE alone fluctuates between 3 and 6, whereas in the F1 generation of transgenic containing both the TE and the KAS A-2-7, the ratio can be as high as 22. This increase in C:10 levels is accompanied by an increase in the total C:8 and C:10 content (FIGS. 16 A and 16 B). The sum of the C:8 and C:10 fatty acids in the heterozygous F1 lines is as high as those in the homozygous parent line (4804-22-357), whereas the heterozygous lines usually contain substantially less C:8 and C:10 than the homozygous lines.

Similar results were observed in F1 generation seeds resulting from crosses performed between 4804-22-357 (ChFatB2 ) and the 5413-17 event (CpFatB1 and chKAS A-2-7 tandem). Levels of C:8 and C:10 in the 5413-17 line were 6.3 and 2.8 mol % respectively. Data presented in FIGS. 17A and 17B show that there is shift towards C:10 fatty acids as was observed with the 4804-22-357 (ChFatB2)×5401-9 (chKAS A-2-7) crosses. Furthermore, FIGS. 18A and 18B indicates the presence of two separate populations of heterozygotes. Those containing approximately 9-11 weight percent C:10+C:8 are believed to represent offspring containing a single copy of the ChFatB1 TE gene and no copies of the CpFatB1 and chKAS A genes from 5413. Those plants containing approximately 15-20 weight percent C:10+C:8 are believed to represent the heterozygotes containing a single ChFatB1 TE gene as well as the CpFatB1 and chKAS A genes from 5413. Thus, the level of the C:10+C:8 fatty acids does not decrease to 50% of that detected in parent lines when a copy of the ChKAS A gene is present.

To further characterize the chain length specificity of the Cuphea hookeriana KAS A enzyme, crosses between transgenic Brassica napus lines containing a California Bay ( Umbellularia californica ) 12:0 specific thioesterase, Uc FatB1 (U.S. Pat. No. 5,344,771) and chKAS A-2-7 (5401-9) were made. Half seed analysis of transgenic plants containing Uc fatB1 have previuosly indicated that these plants can accumulate up to 52 mol % C12:0 in the seed oil of homozygous dihaploid lines (LA86DH186). Crosses between the line LA86DH186 and untransformed control Brassica demonstrated a decrease in the C12:0 levels.

However, crosses between LA86DH186 and the 5401-9 hemizygous line led to an accumulation of up to 57 mol % C12:0 in the seed oil of F1 progeny (FIGS. 19 A and 19 B). Interestingly, in crosses with LA86DH186×untransformed control line and LA86DH186×5401-9, levels of C14:0 in the seeds of the F1 progeny decreased to 50% of the levels obtained in homozygous LA86DH186 lines (FIG. 20 ). Furthermore, increases in the proportion of C12:0 fatty acid resulted in a substantial decline in the proportions of all the long-chain fatty acyl groups (C16:0, C18:0, C18:2, and C18:3). These results indicate that the ChKAS A-2-7 is an enzyme with substrate specificity ranging from C6:0 to C10:0-ACP, and that its over-expression ultimately reduces the longer chain acyl-ACP pools.

Further evidence is obtained in support of the chain length specificity of the ChKAS A-2-7 in crosses of the 5401-9 line with a transgenic line (5266) expressing an 18:1/18:0 TE from Garcinia mangostana (GarmFatA1 , U.S. patent application Ser. No. 08/440,845). Transgenic Brassica line 5266 has been shown to accumulate up to 24 mol % C18:0 in the seed oil of homozygous lines ( FIGS. 21A , 21 B and 21 C). However, in the seed oil of F1 progeny of crosses between 5266 and 5401-9 levels of C18:0 were reduced to approximately 12 mol %. Furthermore, levels of C16:0 generated from these crosses was similar to the levels obtained from the seed oil of nontransgenic control plants.

Example 5

In vitro Analysis of Plant KAS Enzymes

Seed extracts were prepared from developing seeds of nontransgenic controls or transgenic Brassica expressing chKAS A-2-7 as described in Slabaugh et al. ( Plant Journal, 1998 in press) and Leonard et al. ( Plant Journal, 1998, in press). In vitro fatty acid synthesis assays were performed as described by Post-Beittenmiller ( J. Biol. Chem. (1991), 266:1858-1865). Extracts were concentrated by ammonium sulfate precipitation and desalting using P-6 columns (Bio-Rad, Hercules, Calif.). Reactions (65 μl) contained 0.1M Tris/HCl (pH 8.0), 1 mM dithiothreitol, 25 mM recombinant spinach ACP1, 1 mM NADH, 2 mM NADPH, 50 μM malonyl-CoA, 10 μM [ 1- 14 C]acetyl-CoA (50 mCi/mmol), 1 mg/ml BSA, and 0.25 mg/ml seed protein. Selected seed extracts were preincubated with cerulenin at 23° C. for 10 min. Reaction products were separated on an 18% acrlamide gel containing 2.25M urea, electroblotted onto to nitrocellulose and quntitated by phosporimaging using Image QuaNT software (Molecular Dynamics, Sunnyvale, Calif.). Authentic acyl-ACPs were run in parallel, immunoblotted and finally detected by anti-ACP serum to confirm fatty acid chain lengths.

The results ( FIG. 22 ) indicate that the fatty acid synthesis capabilities of transgenic Brasica (5401-9) seed extracts was greater than that obtained from in the nontransgenic controls as measured by the relative abundance of C8:0- and C10:0-ACP at all time points tested. In addition, pretreatment of the extracts with cerulenin, markedly reduced the synthesis of longer chain fatty acids in both the transgenic and nontransgenic control seed extracts. However, the extension of the spinach-ACP was much less inhibited in the seed extracts from the transgenic lines than in the seed extracts of nontransgenic control Brassica.

These data further support that Ch KAS A-2-7 is a condensing enzyme active on medium chain acyl-ACPs, and that expression of this enzyme in plants results in enlarged substrate pools to be hydrolyzed by medium-chain specific thioesterases. Furthermore, these data suggest that chKAS A-2-7 also is a cerulenin-resistant condensing enzyme.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim.

17 1 1348 DNA Cuphea Hookeriana 1agctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg caggaattcg 60gcacgagccg atctcggtgc cgaccgcctc tccaagatcg acaaggagag agccggagtg 120ctggtcggaa caggaatggg tggtctgact gtcttctctg acggggttca gtctcttatc 180gagaagggtc accggaaaat cacccctttc ttcatcccct atgccattac aaacatgggg 240tctgccctgc tcgctatcga atttggtctc atgggcccaa actattcaat ttccactgca 300tgtgccactt ccaactactg cttccatgct gccgctaatc atatccgccg tggtgaggct 360gatcttatga ttgctggagg cactgaggcc gcaatcattc caattgggtt gggaggcttt 420gtggcttgca gggctttgtc tcaaaggaac gatgacccgc agactgcctc taggccctgg 480gataaagacc gtgatggttt tgtgatgggt gaaggtgctg gagtgttggt gatggagagc 540ttggaacatg caatgagacg aggagcaccg attattgcag agtatttggg aggtgcaatc 600aactgtgatg cttatcacat gactgatcca agggctgatg gtcttggtgt ctcttcttgc 660attgagagta gccttgaaga tgctggcgtc tcacctgaag aggtcaatta cataaatgct 720catgcgactt ctactctagc tggggatctc gccgagataa atgccatcaa gaaggttttc 780aagaacacaa aggatatcaa aattaatgca actaagtcaa tgatcggaca ctgtcttgga 840gcatctggag gtcttgaagc tatagcgact attaagggaa taaacaccgg ctggcttcat 900cccagcatta atcaattcaa tcctgagcca tcggtggagt tcgacactgt tgccaacaag 960aagcagcaac acgaagttaa cgttgcgatc tcgaattcat tcggatttgg aggccacaac 1020tcagtcgtgg ctttctcggc tttcaagcca tgattaccca tttcacaagg tacttgtcat 1080tgagaatacg gattatggac ttgcagagta atttccccat gtttgtcgga agagcatatt 1140accacggttg tccgtcaaac ccatttagga tactgttcta tgtaataaaa ctaaggatta 1200ttaatttccc ttttaatcct gtctccagtt tgagcatgaa attatattta ttttatctta 1260gaaaggtcaa ataagatttt gttttacctc tgtaaaactt ttgtttgtat tggaaaggaa 1320gtgccgtctc aaaaaaaaaa aaaaaaaa 1348 2 350 PRT Cuphea Hookeriana 2Ser Ser Thr Ala Val Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly 1 5 10 15Cys Arg Asn Ser Ala Arg Ala Asp Leu Gly Ala Asp Arg Leu Ser Lys 20 25 30Ile Asp Lys Glu Arg Ala Gly Val Leu Val Gly Thr Gly Met Gly Gly 35 40 45Leu Thr Val Phe Ser Asp Gly Val Gln Ser Leu Ile Glu Lys Gly His 50 55 60Arg Lys Ile Thr Pro Phe Phe Ile Pro Tyr Ala Ile Thr Asn Met Gly65 70 75 80Ser Ala Leu Leu Ala Ile Glu Phe Gly Leu Met Gly Pro Asn Tyr Ser 85 90 95Ile Ser Thr Ala Cys Ala Thr Ser Asn Tyr Cys Phe His Ala Ala Ala 100 105 110Asn His Ile Arg Arg Gly Glu Ala Asp Leu Met Ile Ala Gly Gly Thr 115 120 125Glu Ala Ala Ile Ile Pro Ile Gly Leu Gly Gly Phe Val Ala Cys Arg 130 135 140Ala Leu Ser Gln Arg Asn Asp Asp Pro Gln Thr Ala Ser Arg Pro Trp145 150 155 160Asp Lys Asp Arg Asp Gly Phe Val Met Gly Glu Gly Ala Gly Val Leu 165 170 175Val Met Glu Ser Leu Glu His Ala Met Arg Arg Gly Ala Pro Ile Ile 180 185 190Ala Glu Tyr Leu Gly Gly Ala Ile Asn Cys Asp Ala Tyr His Met Thr 195 200 205Asp Pro Arg Ala Asp Gly Leu Gly Val Ser Ser Cys Ile Glu Ser Ser 210 215 220Leu Glu Asp Ala Gly Val Ser Pro Glu Glu Val Asn Tyr Ile Asn Ala225 230 235 240His Ala Thr Ser Thr Leu Ala Gly Asp Leu Ala Glu Ile Asn Ala Ile 245 250 255Lys Lys Val Phe Lys Asn Thr Lys Asp Ile Lys Ile Asn Ala Thr Lys 260 265 270Ser Met Ile Gly His Cys Leu Gly Ala Ser Gly Gly Leu Glu Ala Ile 275 280 285Ala Thr Ile Lys Gly Ile Asn Thr Gly Trp Leu His Pro Ser Ile Asn 290 295 300Gln Phe Asn Pro Glu Pro Ser Val Glu Phe Asp Thr Val Ala Asn Lys305 310 315 320Lys Gln Gln His Glu Val Asn Val Ala Ile Ser Asn Ser Phe Gly Phe 325 330 335Gly Gly His Asn Ser Val Val Ala Phe Ser Ala Phe Lys Pro 340 345 350 3 1703 DNA Cuphea Hookeriana misc_feature (1)...(1703) n = A,T,C or G 3aaattaaccc tcactaaagg gaacaaaagc tggagctcca ccgnggtggc ggccgctcta 60gaactagtgg atcccccggg ctgcaggaat tcggcacgag ccggcatggg cctcgtctcc 120gtattcggct ccgacgtcga ctcttattac gaaaagctcc tctccggcga gagcgggatc 180agcttaatcg accgcttcga cgcttccaag ttccccacca ggttcggcgg ccagatccgg 240ggattcaacg cgacgggata catcgacggg aagaacgaca ggaggctcga cgattgcctc 300gctactgcat tgtcgccggg aagaaggctc tcgaaaattc cgatctcggc ggtgaaagcc 360tctccaagat tgataaggag agagctggag tgctagttgg aactggtatg ggtggcctaa 420ccgtcttctc tgacggggtt cagaatctca tcgagaaagg tcaccggaag atctccccgt 480ttttcattcc ctatgccatt acaaacatgg ggtctgctct gcttgccatc gatttgggtc 540tgatgggccc aaactattcg atttcaactg catgtgctac ttccaactac tgcttttatg 600ccgctgccaa tcatatccgc cgaggcgagg ctgacctcat gattgctgga ggaactgagg 660ctgcaatcat tccaattggg ttaggaggat tcgttgcctg cagggcttta tctcaaagga 720atgatgaccc tcagactgcc tcaaggccgt gggataagga ccgtgatggt tttgtgatgg 780gcgaaggggc tggagtattg gttatggaga gcttggaaca tgcaatgaaa cgaggagcgc 840cgattattgc agaatatttg ggaggtgcag tcaattgtga tgcttatcat atgactgatc 900caagggctga tgggcttggt gtctcctctt gcattgagag cagtctggaa gatgctgggg 960tctcacctga agaggtcaat tacataaatg ctcatgcgac ttccactctt gctggggatc 1020ttgccgagat aaatgccatc aagaaggttt tcaagaacac caaggaaatc acaatcaatg 1080caactaagtc gatgatcgga cactgtcttg gagcatcagg gggtcttgaa gccattgcga 1140caattaaggg aataaccacc ggctggcttc atcccagcat aaaccaattc aatcccgagc 1200catcagtgga attcgacaca gttgccaaca agaagcagca acatgaagtg aatgttgcta 1260tctcaaattc attcggattc ggaggccaca actcagttgt agctttctca gccttcaagc 1320catgattact cggttcaaat gcaaatttgt tgctgagaca gtgagcttca acttgcagag 1380caatttttta catgccttgt cgtcggaaga gcgtaatacc gggatagttc cttgatagtt 1440catttaggat gttttactgc aataatcgaa gattatttcc attctaatcc agtctccgnc 1500gagtttgaga atctatctgt ttgtattaga aagaacgagg caagattttg tttcatgttt 1560gtgtttgtat tactttcttt ttgcccttgt caatggcatt taagataagc ttataaaaaa 1620aaaaaaaaaa aaaaaaactc gagggggggc ccggtaccca attcgcccta tagtgagtcg 1680tatgacaatt cactgtccgt cgg 1703 4 441 PRT Cuphea Hookeriana VARIANT (1)...(441) Xaa = Any Amino Acid 4Lys Leu Thr Leu Thr Lys Gly Asn Lys Ser Trp Ser Ser Thr Xaa Val 1 5 10 15Ala Ala Ala Leu Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser Ala 20 25 30Arg Ala Gly Met Gly Leu Val Ser Val Phe Gly Ser Asp Val Asp Ser 35 40 45Tyr Tyr Glu Lys Leu Leu Ser Gly Glu Ser Gly Ile Ser Leu Ile Asp 50 55 60Arg Phe Asp Ala Ser Lys Phe Pro Thr Arg Phe Gly Gly Gln Ile Arg65 70 75 80Gly Phe Asn Ala Thr Gly Tyr Ile Asp Gly Lys Asn Asp Arg Arg Leu 85 90 95Asp Asp Cys Leu Arg Tyr Cys Ile Val Ala Gly Lys Lys Ala Leu Glu 100 105 110Asn Ser Asp Leu Gly Gly Glu Ser Leu Ser Lys Ile Asp Lys Glu Arg 115 120 125Ala Gly Val Leu Val Gly Thr Gly Met Gly Gly Leu Thr Val Phe Ser 130 135 140Asp Gly Val Gln Asn Leu Ile Glu Lys Gly His Arg Lys Ile Ser Pro145 150 155 160Phe Phe Ile Pro Tyr Ala Ile Thr Asn Met Gly Ser Ala Leu Leu Ala 165 170 175Ile Asp Leu Gly Leu Met Gly Pro Asn Tyr Ser Ile Ser Thr Ala Cys 180 185 190Ala Thr Ser Asn Tyr Cys Phe Tyr Ala Ala Ala Asn His Ile Arg Arg 195 200 205Gly Glu Ala Asp Leu Met Ile Ala Gly Gly Thr Glu Ala Ala Ile Ile 210 215 220Pro Ile Gly Leu Gly Gly Phe Val Ala Cys Arg Ala Leu Ser Gln Arg225 230 235 240Asn Asp Asp Pro Gln Thr Ala Ser Arg Pro Trp Asp Lys Asp Arg Asp 245 250 255Gly Phe Val Met Gly Glu Gly Ala Gly Val Leu Val Met Glu Ser Leu 260 265 270Glu His Ala Met Lys Arg Gly Ala Pro Ile Ile Ala Glu Tyr Leu Gly 275 280 285Gly Ala Val Asn Cys Asp Ala Tyr His Met Thr Asp Pro Arg Ala Asp 290 295 300Gly Leu Gly Val Ser Ser Cys Ile Glu Ser Ser Leu Glu Asp Ala Gly305 310 315 320Val Ser Pro Glu Glu Val Asn Tyr Ile Asn Ala His Ala Thr Ser Thr 325 330 335Leu Ala Gly Asp Leu Ala Glu Ile Asn Ala Ile Lys Lys Val Phe Lys 340 345 350Asn Thr Lys Glu Ile Thr Ile Asn Ala Thr Lys Ser Met Ile Gly His 355 360 365Cys Leu Gly Ala Ser Gly Gly Leu Glu Ala Ile Ala Thr Ile Lys Gly 370 375 380Ile Thr Thr Gly Trp Leu His Pro Ser Ile Asn Gln Phe Asn Pro Glu385 390 395 400Pro Ser Val Glu Phe Asp Thr Val Ala Asn Lys Lys Gln Gln His Glu 405 410 415Val Asn Val Ala Ile Ser Asn Ser Phe Gly Phe Gly Gly His Asn Ser 420 425 430Val Val Ala Phe Ser Ala Phe Lys Pro 435 440 5 2046 DNA Cuphea Hookeriana 5actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgctctaga actagtggat 60cccccgggct gcaggaattc ggcacgagtt ttcttacttg ggtcggctca gctcaggtgt 120tccaatggcg accgcttctt gcatggttgc gtcccctttc tgtacgtggc tcgtagctgc 180atgcatgccc acttcatccg acaacgaccc acgttccctt tcccacaagc ggctccgcct 240ctcccgtcgc cggaggactc tctcctccca ttgctccctc cgcggatcca ccttccaatg 300cctcgatcct tgcaaccagc aacgcttcct cggggataac ggattcgctt ccctcttcgg 360atccaagcct cttcgttcaa atcgcggcca cctgaggctc ggccgcactt cccattccgg 420ggaggtcatg gctgtggcta tgcaacctgc acaggaagtc tccacaaata agaaacctgc 480taccaagcaa aggcgagtag ttgtgacagg tatgggcgtg gtgactcctc taggccatga 540ccccgatgtt tactacaaca atctcctaga cggaataagt ggcataagtg agatagagaa 600cttcgactgc tctcagtttc ccacgagaat tgccggagag atcaagtctt tttccacaga 660tggctgggtg gccccaaagt tctccgagag gatggacaag ttcatgcttt acatgctgac 720tgcaggcaag aaagcattag cagatggtgg aatcactgaa gatgcgatga aagagctcaa 780taaaagaaag tgtggagttc tcattggctc cggattgggc ggtatgaagg tattcagcga 840ttccattgaa gctctgagga cttcatataa gaagatcagt cccttttgtg tacctttttc 900taccacaaat atgggatccg ctattcttgc aatggacttg ggatggatgg gccctaacta 960ttcgatatca actgcctgtg caacaagtaa cttctgtata ctgaatgctg cgaaccacat 1020aatcaaaggc gaagcagaca tgatgctttg tggtggctcg gatgcggccg ttttacctgt 1080tggtttggga ggtttcgtag catgccgagc tttgtcacag aggaataatg accctaccaa 1140agcttcgaga ccatgggaca gtaatcgtga tggatttgtg atgggagaag gagctggagt 1200tttacttctt gaggagttag agcatgcaaa gaaaagaggt gcaaccattt atgcggaatt 1260tctaggtggg agtttcactt gcgacgccta ccacatgacc gagcctcacc ctgaaggagc 1320tggtgtgatc ctctgcatag agaaggcctt ggctcagtcc ggagtctcga gggaagacgt 1380aaattacata aatgcgcatg caacttccac tcctgctgga gatatcaagg aataccaagc 1440tctcgcccac tgtttcggcc aaaacagtga gctgagagtg aattccacca aatcgatgat 1500cggtcacctt cttggaggag ctggtggcgt agaagcagtt gcagtagttc aggcaataag 1560gacaggatgg atccatccaa atattaattt ggaagacccg gacgaaggcg tggatgcaaa 1620actgctcgtc ggccctaaga aggagaaact gaaggtcaag gtcggtttgt ccaattcatt 1680tgggttcggc ggccataact catccatact atttgccccc tgcaactaga aaagagtctg 1740tggaagccga gagtctttga gaactcatgc acgttagtag cttcttatgc ctctgaaacc 1800gagatagacc ggctactcga ggggatgcca aagatactcc ttgccggtat tggtgttaag 1860agatcactgc ttgtcccttt tattttcttc ttcttttgag agctttaacc gaggtagtcg 1920tattttcgag cttttcgaat acatgttcgt tatcggatca atgtgtttct tctaagatca 1980tttgtaatgc atattttgaa aaaccacatc tcagtatgca aaataaaaaa aaaaaaaaaa 2040aaaaaa 2046 6 534 PRT Cuphea Hookeriana 6Met Ala Thr Ala Ser Cys Met Val Ala Ser Pro Phe Cys Thr Trp Leu 1 5 10 15Val Ala Ala Cys Met Pro Thr Ser Ser Asp Asn Asp Pro Arg Ser Leu 20 25 30Ser His Lys Arg Leu Arg Leu Ser Arg Arg Arg Arg Thr Leu Ser Ser 35 40 45His Cys Ser Leu Arg Gly Ser Thr Phe Gln Cys Leu Asp Pro Cys Asn 50 55 60Gln Gln Arg Phe Leu Gly Asp Asn Gly Phe Ala Ser Leu Phe Gly Ser65 70 75 80Lys Pro Leu Arg Ser Asn Arg Gly His Leu Arg Leu Gly Arg Thr Ser 85 90 95His Ser Gly Glu Val Met Ala Val Ala Met Gln Pro Ala Gln Glu Val 100 105 110Ser Thr Asn Lys Lys Pro Ala Thr Lys Gln Arg Arg Val Val Val Thr 115 120 125Gly Met Gly Val Val Thr Pro Leu Gly His Asp Pro Asp Val Tyr Tyr 130 135 140Asn Asn Leu Leu Asp Gly Ile Ser Gly Ile Ser Glu Ile Glu Asn Phe145 150 155 160Asp Cys Ser Gln Phe Pro Thr Arg Ile Ala Gly Glu Ile Lys Ser Phe 165 170 175Ser Thr Asp Gly Trp Val Ala Pro Lys Phe Ser Glu Arg Met Asp Lys 180 185 190Phe Met Leu Tyr Met Leu Thr Ala Gly Lys Lys Ala Leu Ala Asp Gly 195 200 205Gly Ile Thr Glu Asp Ala Met Lys Glu Leu Asn Lys Arg Lys Cys Gly 210 215 220Val Leu Ile Gly Ser Gly Leu Gly Gly Met Lys Val Phe Ser Asp Ser225 230 235 240Ile Glu Ala Leu Arg Thr Ser Tyr Lys Lys Ile Ser Pro Phe Cys Val 245 250 255Pro Phe Ser Thr Thr Asn Met Gly Ser Ala Ile Leu Ala Met Asp Leu 260 265 270Gly Trp Met Gly Pro Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Ser 275 280 285Asn Phe Cys Ile Leu Asn Ala Ala Asn His Ile Ile Lys Gly Glu Ala 290 295 300Asp Met Met Leu Cys Gly Gly Ser Asp Ala Ala Val Leu Pro Val Gly305 310 315 320Leu Gly Gly Phe Val Ala Cys Arg Ala Leu Ser Gln Arg Asn Asn Asp 325 330 335Pro Thr Lys Ala Ser Arg Pro Trp Asp Ser Asn Arg Asp Gly Phe Val 340 345 350Met Gly Glu Gly Ala Gly Val Leu Leu Leu Glu Glu Leu Glu His Ala 355 360 365Lys Lys Arg Gly Ala Thr Ile Tyr Ala Glu Phe Leu Gly Gly Ser Phe 370 375 380Thr Cys Asp Ala Tyr His Met Thr Glu Pro His Pro Glu Gly Ala Gly385 390 395 400Val Ile Leu Cys Ile Glu Lys Ala Leu Ala Gln Ser Gly Val Ser Arg 405 410 415Glu Asp Val Asn Tyr Ile Asn Ala His Ala Thr Ser Thr Pro Ala Gly 420 425 430Asp Ile Lys Glu Tyr Gln Ala Leu Ala His Cys Phe Gly Gln Asn Ser 435 440 445Glu Leu Arg Val Asn Ser Thr Lys Ser Met Ile Gly His Leu Leu Gly 450 455 460Gly Ala Gly Gly Val Glu Ala Val Ala Val Val Gln Ala Ile Arg Thr465 470 475 480Gly Trp Ile His Pro Asn Ile Asn Leu Glu Asp Pro Asp Glu Gly Val 485 490 495Asp Ala Lys Leu Leu Val Gly Pro Lys Lys Glu Lys Leu Lys Val Lys 500 505 510Val Gly Leu Ser Asn Ser Phe Gly Phe Gly Gly His Asn Ser Ser Ile 515 520 525Leu Phe Ala Pro Cys Asn 530 7 1921 DNA Cuphea Hookeriana 7cggcacgagg tcacctctta cctcgcctgc ttcgagccct gccatgacta ctacacctcc 60gcatccttgt tcggatccag gcccatccgc accacccgca ggcaccggag gctcaatcga 120gcttcccctt ccggggaggc aatggctgtg gctctgcaac ctgcacagga agttaccaca 180aagaagaagc caagtatcaa acagcggcga gtagttgtga ctggaatggg tgtggtgact 240cctctaggcc atgaccctga tgttttctac aataatctgc ttgatggaac gagtggcata 300agtgagatag agacctttga ttgtgctcaa tttcctacga gaattgctgg agagatcaag 360tctttctcca cagatggttg ggtggccccg aagctctcca agaggatgga caagttcatg 420ctttacatgc tgactgccgg caagaaagca ttaacaaatg gtggaatcac cgaagatgtg 480atgaaagagc tagataaaag aaaatgcgga gttctcattg gctcagcaat gggtggaatg 540aaggtattca atgatgccat tgaagcccta aggatttcat ataagaagat gaatcccttt 600tgtgtacctt tcgctaccac aaatatggga tcagctatgc ttgcaatgga cttgggatgg 660atgggcccca actactcgat atctactgct tgtgcaacga gtaacttttg tatcctgaat 720gctgcgaacc acataatcag aggcgaagca gatgtgatgc tttgcggggg ctcagatgcg 780gtaatcatac ctattggtat gggaggtttt gttgcatgcc gagctttgtc acagagaaat 840gccgacccta ctaaagcttc aagaccatgg gacagtaatc gtgatggatt tgttatgggg 900gaaggagctg gagtgctact actagaggag ttagagcatg caaagaaaag aggtgcgact 960atttacgcag aatttctagg tggaagtttc acttgcgatg cctaccacat gaccgagcct 1020caccctgatg gagctggagt gattctctgc atagagaagg ctttggctca gtcaggagtc 1080tctagggaag acgtaaatta cataaatgca catgccacat ccactccagc tggagatatc 1140aaagagtacc aagctcttat ccactgtttc ggccaaaaca acgagttaaa agtgaattct 1200accaaatcaa tgattggtca ccttctcgga gcagccggtg gtgtggaagc agtttcagta 1260gttcaggcaa taaggactgg gtggatccat ccgaatatta atttggaaaa cccagatgaa 1320ggcgtggata ccaaattgct cgtgggccct aagaaggaga gactgaacat taaggtcggt 1380ttgtctaatt cattcgggtt tggtgggcac aactcgtcca tactcttcgc cccttacaac 1440tagggcgttt catgtgtgga attctactca atctatcaaa gctgaagttt tgaggactcc 1500agcatgttgg tagctcctta cgtctctaga catgcccatg agttttgtgt cgggagctgt 1560agtcggaacc atgacggatt gagtactcat ggcgacacag gatatactcc ttgctagaat 1620tgttagagca ctattcatta tcccattttt tttctgaaat ctccctcctt acggtagttg 1680tactttcgag cgtttcatcg agtcagtgaa gaagagaaca aagctaactc gggcacgtag 1740taaccatttg ccctttgttt tgctctctat tttatcgccg ttttgtgggt taaaatttgt 1800aaaactagac gactggtttg ttttctcttg atcattggag atgtatggcc atatttgcct 1860ttcattgatg ataaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920a 1921 8 420 PRT Cuphea Hookeriana 8Lys Lys Lys Pro Ser Ile Lys Gln Arg Arg Val Val Val Thr Gly Met 1 5 10 15Gly Val Val Thr Pro Leu Gly His Asp Pro Asp Val Phe Tyr Asn Asn 20 25 30Leu Leu Asp Gly Thr Ser Gly Ile Ser Glu Ile Glu Thr Phe Asp Cys 35 40 45Ala Gln Phe Pro Thr Arg Ile Ala Gly Glu Ile Lys Ser Phe Ser Thr 50 55 60Asp Gly Trp Val Ala Pro Lys Leu Ser Lys Arg Met Asp Lys Phe Met65 70 75 80Leu Tyr Met Leu Thr Ala Gly Lys Lys Ala Leu Thr Asn Gly Gly Ile 85 90 95Thr Glu Asp Val Met Lys Glu Leu Asp Lys Arg Lys Cys Gly Val Leu 100 105 110Ile Gly Ser Ala Met Gly Gly Met Lys Val Phe Asn Asp Ala Ile Glu 115 120 125Ala Leu Arg Ile Ser Tyr Lys Lys Met Asn Pro Phe Cys Val Pro Phe 130 135 140Ala Thr Thr Asn Met Gly Ser Ala Met Leu Ala Met Asp Leu Gly Trp145 150 155 160Met Gly Pro Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Ser Asn Phe 165 170 175Cys Ile Leu Asn Ala Ala Asn His Ile Ile Arg Gly Glu Ala Asp Val 180 185 190Met Leu Cys Gly Gly Ser Asp Ala Val Ile Ile Pro Ile Gly Met Gly 195 200 205Gly Phe Val Ala Cys Arg Ala Leu Ser Gln Arg Asn Ala Asp Pro Thr 210 215 220Lys Ala Ser Arg Pro Trp Asp Ser Asn Arg Asp Gly Phe Val Met Gly225 230 235 240Glu Gly Ala Gly Val Leu Leu Leu Glu Glu Leu Glu His Ala Lys Lys 245 250 255Arg Gly Ala Thr Ile Tyr Ala Glu Phe Leu Gly Gly Ser Phe Thr Cys 260 265 270Asp Ala Tyr His Met Thr Glu Pro His Pro Asp Gly Ala Gly Val Ile 275 280 285Leu Cys Ile Glu Lys Ala Leu Ala Gln Ser Gly Val Ser Arg Glu Asp 290 295 300Val Asn Tyr Ile Asn Ala His Ala Thr Ser Thr Pro Ala Gly Asp Ile305 310 315 320Lys Glu Tyr Gln Ala Leu Ile His Cys Phe Gly Gln Asn Asn Glu Leu 325 330 335Lys Val Asn Ser Thr Lys Ser Met Ile Gly His Leu Leu Gly Ala Ala 340 345 350Gly Gly Val Glu Ala Val Ser Val Val Gln Ala Ile Arg Thr Gly Trp 355 360 365Ile His Pro Asn Ile Asn Leu Glu Asn Pro Asp Glu Gly Val Asp Thr 370 375 380Lys Leu Leu Val Gly Pro Lys Lys Glu Arg Leu Asn Ile Lys Val Gly385 390 395 400Leu Ser Asn Ser Phe Gly Phe Gly Gly His Asn Ser Ser Ile Leu Phe 405 410 415Ala Pro Tyr Asn 420 9 1713 DNA Cuphea pullcherrima 9ctggtacgcc tgcaggtacc ggtccggaat tcccgggtcg acccacgcgt ccgtcttccc 60actccgatcg ttcttcttcc accgcatctc ttctcttctc ttggcttctc cgccatcctc 120cgccgccatg cattccctcc agtcaccctc ccttcgggcc tccccgctcg accccttccg 180ccccaaatca tccaccgtcc gccccctcca ccgagcatca attcccaacg tccgggccgc 240ttcccccacc gtctccgctc ccaagcgcga gaccgacccc aagaagcgcg tcgtgatcac 300cggaatgggc cttgtctccg ttttcggctc cgacgtcgat gcgtactacg acaagctcct 360gtcaggcgag agcgggatcg gcccaatcga ccgcttcgac gcctccaagt tccccaccag 420gttcggcggc cagattcgtg gcttcaactc catgggatac attgacggca aaaacgacag 480gcggcttgat gattgccttc gctactgcat tgtcgccggg aagaagtctc ttgaggacgc 540cgatctcggt gccgaccgcc tctccaagat cgacaaggag agagccggag tgctggttgg 600gacaggaatg ggtggtctga ctgtcttctc tgacggggtt caatctctta tcgagaaggg 660tcaccggaaa atcacccctt tcttcatccc ctatgccatt acaaacatgg ggtctgccct 720gctcgctatt gaactcggtc tgatgggccc aaactattca atttccactg catgtgccac 780ttccaactac tgcttccatg ctgctgctaa tcatatccgc cgtggtgagg ctgatcttat 840gattgctgga ggcactgagg ccgcaatcat tccaattggg ttgggaggct ttgtggcttg 900cagggctctg tctcaaagga acgatgaccc tcagactgcc tctaggccct gggataaaga 960ccgtgatggt tttgtgatgg gtgaaggtgc tggagtgttg gtgctggaga gcttggaaca 1020tgcaatgaaa cgaggagcac ctattattgc agagtatttg ggaggtgcaa tcaactgtga 1080tgcttatcac atgactgacc caagggctga tggtctcggt gtctcctctt gcattgagag 1140tagccttgaa gatgctggcg tctcacctga agaggtcaat tacataaatg ctcatgcgac 1200ttctactcta gctggggatc tcgccgagat aaatgccatc aagaaggttt tcaagaacac 1260aaaggatatc aaaattaatg caactaagtc aatgatcgga cactgtcttg gagcctctgg 1320aggtcttgaa gctatagcga ctattaaggg aataaacacc ggctggcttc atcccagcat 1380taatcaattc aatcctgagc catccgtgga gttcgacact gttgccaaca agaagcagca 1440acacgaagtt aatgttgcga tctcgaattc atttggattc ggaggccaca actcagtcgt 1500ggctttctcg gctttcaagc catgattacc catttcacaa ggcacttgtc attgagagta 1560cggttgttcg tcaaacccat ttaggatact gttctatgta aaaaaaagta aggattatca 1620ctttcccttc taatcctgtc tccagtttga gaatgaaatt atatttattt taaaaaaaaa 1680aaaaaagggc ggccgctcta gaggatccaa gct 1713 10 465 PRT Cuphea pullcherrima 10Met His Ser Leu Gln Ser Pro Ser Leu Arg Ala Ser Pro Leu Asp Pro 1 5 10 15Phe Arg Pro Lys Ser Ser Thr Val Arg Pro Leu His Arg Ala Ser Ile 20 25 30Pro Asn Val Arg Ala Ala Ser Pro Thr Val Ser Ala Pro Lys Arg Glu 35 40 45Thr Asp Pro Lys Lys Arg Val Val Ile Thr Gly Met Gly Leu Val Ser 50 55 60Val Phe Gly Ser Asp Val Asp Ala Tyr Tyr Asp Lys Leu Leu Ser Gly65 70 75 80Glu Ser Gly Ile Gly Pro Ile Asp Arg Phe Asp Ala Ser Lys Phe Pro 85 90 95Thr Arg Phe Gly Gly Gln Ile Arg Gly Phe Asn Ser Met Gly Tyr Ile 100 105 110Asp Gly Lys Asn Asp Arg Arg Leu Asp Asp Cys Leu Arg Tyr Cys Ile 115 120 125Val Ala Gly Lys Lys Ser Leu Glu Asp Ala Asp Leu Gly Ala Asp Arg 130 135 140Leu Ser Lys Ile Asp Lys Glu Arg Ala Gly Val Leu Val Gly Thr Gly145 150 155 160Met Gly Gly Leu Thr Val Phe Ser Asp Gly Val Gln Ser Leu Ile Glu 165 170 175Lys Gly His Arg Lys Ile Thr Pro Phe Phe Ile Pro Tyr Ala Ile Thr 180 185 190Asn Met Gly Ser Ala Leu Leu Ala Ile Glu Leu Gly Leu Met Gly Pro 195 200 205Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Ser Asn Tyr Cys Phe His 210 215 220Ala Ala Ala Asn His Ile Arg Arg Gly Glu Ala Asp Leu Met Ile Ala225 230 235 240Gly Gly Thr Glu Ala Ala Ile Ile Pro Ile Gly Leu Gly Gly Phe Val 245 250 255Ala Cys Arg Ala Leu Ser Gln Arg Asn Asp Asp Pro Gln Thr Ala Ser 260 265 270Arg Pro Trp Asp Lys Asp Arg Asp Gly Phe Val Met Gly Glu Gly Ala 275 280 285Gly Val Leu Val Leu Glu Ser Leu Glu His Ala Met Lys Arg Gly Ala 290 295 300Pro Ile Ile Ala Glu Tyr Leu Gly Gly Ala Ile Asn Cys Asp Ala Tyr305 310 315 320His Met Thr Asp Pro Arg Ala Asp Gly Leu Gly Val Ser Ser Cys Ile 325 330 335Glu Ser Ser Leu Glu Asp Ala Gly Val Ser Pro Glu Glu Val Asn Tyr 340 345 350Ile Asn Ala His Ala Thr Ser Thr Leu Ala Gly Asp Leu Ala Glu Ile 355 360 365Asn Ala Ile Lys Lys Val Phe Lys Asn Thr Lys Asp Ile Lys Ile Asn 370 375 380Ala Thr Lys Ser Met Ile Gly His Cys Leu Gly Ala Ser Gly Gly Leu385 390 395 400Glu Ala Ile Ala Thr Ile Lys Gly Ile Asn Thr Gly Trp Leu His Pro 405 410 415Ser Ile Asn Gln Phe Asn Pro Glu Pro Ser Val Glu Phe Asp Thr Val 420 425 430Ala Asn Lys Lys Gln Gln His Glu Val Asn Val Ala Ile Ser Asn Ser 435 440 445Phe Gly Phe Gly Gly His Asn Ser Val Val Ala Phe Ser Ala Phe Lys 450 455 460Pro465 11 1802 DNA Cuphea pullcherrima 11ggtcgaccca cgcgtccggg ctttccgacc acatttcatt tcttgcctcg ttatctccgc 60cgctcctccg ccgtcgttcg ccgccgccgc catgcaatcc ctccactccc cttccctccg 120cccctcccct ctcgagccct tccgcctcaa ttccccctcc tccgccgccg ctctccgccc 180cctccgtcgc gccagcctcc ccgtcatccg tgctgccacc gcctccgccc ccaagcgcga 240gtccgacccc aagaagcggg tcgtcatcac cggcatgggc ctcgtctccg tcttcggctc 300cgacgtcgac gcctactacg acaagctgct ctccggcgag agcggcatca gcctaatcga 360ccgcttcgac gcttccaaat tccccaccag gttcgccggc cagatccgtg gcttcaacgc 420gacgggctac atcgacggca agaacgaccg gcggctcgac gattgcctcc gctactgcat 480tgtcgccggc aagaaggctc tcgaagacgc cgatctcgcc ggccaatccc tctccaagat 540tgataaggag agggccggag tgctagttgg aaccggtatg ggtggcctaa ctgtcttctc 600tgacggggtt cagaatctca tcgagaaagg tcaccggaag atctccccgt ttttcattcc 660atatgccatt acaaacatgg ggtctgcgct gcttgccatc gatttgggtc tgatgggccc 720aaactattcg atttcaactg catgtgctac ttccaactac tgcttttatg ctgccgccaa 780tcatatccgc cgaggtgagg ctgacctgat gattgctgga ggaactgagg ctgcggtcat 840tccaattggt ttaggaggat tcgttgcctg cagggcttta tctcaaagga atgatgatcc 900tcagactgcc tcaaggccgt gggataagga ccgtgatggc tttgtgatgg gtgaaggggc 960tggagtattg gttatggaga gcttggagca tgcaatgaaa cggggagcgc cgattattgc 1020agaatatttg ggaggtgcag tcaactgtga tgcttatcat atgactgatc caagggctga 1080tgggcttggt gtctcctcgt gcattgagag cagtctcgaa gatgccgggg tctcacctga 1140agaggtcaat tacataaatg ctcatgcgac ttctactctt gctggggatc ttgccgagat 1200aaatgccatt aagaaagttt tcaagaacac caaggaaatc aaaatcaatg caactaagtc 1260aatgatcgga cactgtcttg gagcatcagg aggtcttgaa gccatcgcaa ccattaaggg 1320aataaccacc ggctggcttc atcccagcat taatcaattt aatcccgagc catcggtgga 1380cttcaacact gttgccaaca aaaagcagca acatgaagtg aacgtcgcta tctcgaattc 1440ttttggattt ggagggcaca actcggttgt ggcattctca gctttcaagc catgaattct 1500acttggttca aaatgcacac cagttgctga gatagggctt caacttgcag agcaattttt 1560taaatgcctt gtcggaagag cgtaataccg gaataggtcg gtcctttgat agttcctcga 1620agccatttag gatgatgttt tactgtaata atcgaagatg attcccattt taaatctagt 1680ctctgattta tgtattagaa agaccaatga aagattttgt gtcatgtttg tgttgtcaat 1740gttatttaag ataaagcaaa aaaaaaaaaa aagggcggcc gctctagagg atccagctta 1800ct 1802 12 467 PRT Cuphea pullcherrima 12Met Gln Ser Leu His Ser Pro Ser Leu Arg Pro Ser Pro Leu Glu Pro 1 5 10 15Phe Arg Leu Asn Ser Pro Ser Ser Ala Ala Ala Leu Arg Pro Leu Arg 20 25 30Arg Ala Ser Leu Pro Val Ile Arg Ala Ala Thr Ala Ser Ala Pro Lys 35 40 45Arg Glu Ser Asp Pro Lys Lys Arg Val Val Ile Thr Gly Met Gly Leu 50 55 60Val Ser Val Phe Gly Ser Asp Val Asp Ala Tyr Tyr Asp Lys Leu Leu65 70 75 80Ser Gly Glu Ser Gly Ile Ser Leu Ile Asp Arg Phe Asp Ala Ser Lys 85 90 95Phe Pro Thr Arg Phe Ala Gly Gln Ile Arg Gly Phe Asn Ala Thr Gly 100 105 110Tyr Ile Asp Gly Lys Asn Asp Arg Arg Leu Asp Asp Cys Leu Arg Tyr 115 120 125Cys Ile Val Ala Gly Lys Lys Ala Leu Glu Asp Ala Asp Leu Ala Gly 130 135 140Gln Ser Leu Ser Lys Ile Asp Lys Glu Arg Ala Gly Val Leu Val Gly145 150 155 160Thr Gly Met Gly Gly Leu Thr Val Phe Ser Asp Gly Val Gln Asn Leu 165 170 175Ile Glu Lys Gly His Arg Lys Ile Ser Pro Phe Phe Ile Pro Tyr Ala 180 185 190Ile Thr Asn Met Gly Ser Ala Leu Leu Ala Ile Asp Leu Gly Leu Met 195 200 205Gly Pro Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Ser Asn Tyr Cys 210 215 220Phe Tyr Ala Ala Ala Asn His Ile Arg Arg Gly Glu Ala Asp Leu Met225 230 235 240Ile Ala Gly Gly Thr Glu Ala Ala Val Ile Pro Ile Gly Leu Gly Gly 245 250 255Phe Val Ala Cys Arg Ala Leu Ser Gln Arg Asn Asp Asp Pro Gln Thr 260 265 270Ala Ser Arg Pro Trp Asp Lys Asp Arg Asp Gly Phe Val Met Gly Glu 275 280 285Gly Ala Gly Val Leu Val Met Glu Ser Leu Glu His Ala Met Lys Arg 290 295 300Gly Ala Pro Ile Ile Ala Glu Tyr Leu Gly Gly Ala Val Asn Cys Asp305 310 315 320Ala Tyr His Met Thr Asp Pro Arg Ala Asp Gly Leu Gly Val Ser Ser 325 330 335Cys Ile Glu Ser Ser Leu Glu Asp Ala Gly Val Ser Pro Glu Glu Val 340 345 350Asn Tyr Ile Asn Ala His Ala Thr Ser Thr Leu Ala Gly Asp Leu Ala 355 360 365Glu Ile Asn Ala Ile Lys Lys Val Phe Lys Asn Thr Lys Glu Ile Lys 370 375 380Ile Asn Ala Thr Lys Ser Met Ile Gly His Cys Leu Gly Ala Ser Gly385 390 395 400Gly Leu Glu Ala Ile Ala Thr Ile Lys Gly Ile Thr Thr Gly Trp Leu 405 410 415His Pro Ser Ile Asn Gln Phe Asn Pro Glu Pro Ser Val Asp Phe Asn 420 425 430Thr Val Ala Asn Lys Lys Gln Gln His Glu Val Asn Val Ala Ile Ser 435 440 445Asn Ser Phe Gly Phe Gly Gly His Asn Ser Val Val Ala Phe Ser Ala 450 455 460Phe Lys Pro465 13 2369 DNA Cuphea pullcherrima 13gtacgcctgc aggtaccggt ccggaattcc cgggtcgacc cacgcgtccg cataaaagag 60agagagaggg atccatcgaa tgcggccacc ctcctttcat cttcgattca ttaccatacc 120attccgctga tccattttcc gccttttccg ggtctttcat cccaaagggt atccttttct 180atcctatctt ctcaaagggt cagtcagttc cctccaatgc ctgccgcctc ttccctgctc 240gcttcccctc tctgtacgtg gctccttgcc gcctgcatgt ctacctcctt ccacccctcc 300gaccctcttc cgccttccat ctcctctcct cgccgacgcc tctcccgccg ccggattctc 360tcccaatgcg ccccactacc ttctgcttcc tccgccctcc gcggatccag tttccatacc 420ctcgtcacct cttacctcgc ctgcttcgag ccctgccatg actactatac atccgcatcc 480ttgttcggat ccagacccat tcgcaccacc cgcaggcacc ggaggctcaa tcgagcttcc 540ccttccaggg aggcaatggc cgtggctctg caacctgaac aggaagttac cacaaagaag 600aagccaagta tcaaacagcg gcgagtagtt gtgactggaa tgggtgtggt gactcctcta 660ggccatgacc ctgatgtttt ctacaataat ctgcttgatg gaacgagtgg cataagcgag 720atagagacct ttgattgtgc tcaatttcct acgagaattg ctggagagat caagtctttc 780tccacagatg gttgggtggc cccgaagctc tctaagagga tggacaagtt catgctatac 840atgctgaccg ctggcaagaa agcattaaca gatggtggaa tcaccgaaga tgtgatgaaa 900gagctagata aaagaaaatg cggagttctc attggctcag caatgggtgg aatgaaggta 960ttcaatgatg ccattgaagc cctaaggatt tcatataaga agatgaatcc cttttgtgta 1020cctttcgcta ccacaaatat gggatcagct atgcttgcaa tggacttggg atggatgggg 1080cccaactact cgatatctac tgcttgtgca acgagtaact tttgtataat gaatgctgcg 1140aaccatataa tcagaggcga agcagatgtg atgctttgcg ggggctcaga tgcggtaatc 1200atacctattg gtatgggagg ttttgttgca tgccgagctt tgtcccagag aaattccgac 1260cctactaaag cttcaagacc atgggacagt aatcgtgatg gatttgttat gggggaagga 1320gctggagtgc tactactaga ggagttggag catgcaaaga aaagaggtgc gactatttac 1380gcagaatttc taggtgggag tttcacttgc gatgcctacc acatgaccga gcctcaccct 1440gatggagctg gagtgattct ctgcatagag aaggctttgg ctcagtcagg agtctctagg 1500gaagacgtaa attacataaa tgcccatgcc acatccactc cggctggaga tatcaaagag 1560taccaagctc ttatccactg tttcggccaa aacagagagt taaaagttaa ttcaaccaaa 1620tcaatgattg gtcaccttct cggagcagcc ggtggtgtgg aagcagtttc agtagttcag 1680gcaataagga ctgggtggat ccatccgaat attaatttgg aaaacccaga tgaaggcgtg 1740gatacaaaat tgctcgtggg tcctaagaag gagagactga acgttaaggt cggtttgtct 1800aattcatttg ggtttggtgg gcacaactcg tccatactct tcgcccctta catctaggac 1860gtttccgtgt gtggaattct actcaacata tcaaagctga agttttgagg actccagcat 1920gttggtagct ccttacgtct ctagacatgc ccatgagttt tgtgtccgga gctttagtcg 1980gaaccatgac ggattgagta ctcatggcga cacttgatat actccttgct agaattgttg 2040gtagagcaat attcattatc tcatattttt tttttctctg aaatctccct ccttgcaata 2100gttgtacttt cgagcttttc atcgagtcag tgaagaagag aacaaagctg ttaactcggg 2160cacgtagtaa ccatttgccc tttgttttgc tctctatttc atcaccgttt tgtggtttta 2220aaatttgtaa aactagaaga ctggtttaga ttggtttgtt ttctcattga taattggggr 2280atgtatgttt tggaaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340aaaaaaaaaa agggcggccg ctctagagg 2369 14 546 PRT Cuphea pullcherrima 14Met Pro Ala Ala Ser Ser Leu Leu Ala Ser Pro Leu Cys Thr Trp Leu 1 5 10 15Leu Ala Ala Cys Met Ser Thr Ser Phe His Pro Ser Asp Pro Leu Pro 20 25 30Pro Ser Ile Ser Ser Pro Arg Arg Arg Leu Ser Arg Arg Arg Ile Leu 35 40 45Ser Gln Cys Ala Pro Leu Pro Ser Ala Ser Ser Ala Leu Arg Gly Ser 50 55 60Ser Phe His Thr Leu Val Thr Ser Tyr Leu Ala Cys Phe Glu Pro Cys65 70 75 80His Asp Tyr Tyr Thr Ser Ala Ser Leu Phe Gly Ser Arg Pro Ile Arg 85 90 95Thr Thr Arg Arg His Arg Arg Leu Asn Arg Ala Ser Pro Ser Arg Glu 100 105 110Ala Met Ala Val Ala Leu Gln Pro Glu Gln Glu Val Thr Thr Lys Lys 115 120 125Lys Pro Ser Ile Lys Gln Arg Arg Val Val Val Thr Gly Met Gly Val 130 135 140Val Thr Pro Leu Gly His Asp Pro Asp Val Phe Tyr Asn Asn Leu Leu145 150 155 160Asp Gly Thr Ser Gly Ile Ser Glu Ile Glu Thr Phe Asp Cys Ala Gln 165 170 175Phe Pro Thr Arg Ile Ala Gly Glu Ile Lys Ser Phe Ser Thr Asp Gly 180 185 190Trp Val Ala Pro Lys Leu Ser Lys Arg Met Asp Lys Phe Met Leu Tyr 195 200 205Met Leu Thr Ala Gly Lys Lys Ala Leu Thr Asp Gly Gly Ile Thr Glu 210 215 220Asp Val Met Lys Glu Leu Asp Lys Arg Lys Cys Gly Val Leu Ile Gly225 230 235 240Ser Ala Met Gly Gly Met Lys Val Phe Asn Asp Ala Ile Glu Ala Leu 245 250 255Arg Ile Ser Tyr Lys Lys Met Asn Pro Phe Cys Val Pro Phe Ala Thr 260 265 270Thr Asn Met Gly Ser Ala Met Leu Ala Met Asp Leu Gly Trp Met Gly 275 280 285Pro Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Ser Asn Phe Cys Ile 290 295 300Met Asn Ala Ala Asn His Ile Ile Arg Gly Glu Ala Asp Val Met Leu305 310 315 320Cys Gly Gly Ser Asp Ala Val Ile Ile Pro Ile Gly Met Gly Gly Phe 325 330 335Val Ala Cys Arg Ala Leu Ser Gln Arg Asn Ser Asp Pro Thr Lys Ala 340 345 350Ser Arg Pro Trp Asp Ser Asn Arg Asp Gly Phe Val Met Gly Glu Gly 355 360 365Ala Gly Val Leu Leu Leu Glu Glu Leu Glu His Ala Lys Lys Arg Gly 370 375 380Ala Thr Ile Tyr Ala Glu Phe Leu Gly Gly Ser Phe Thr Cys Asp Ala385 390 395 400Tyr His Met Thr Glu Pro His Pro Asp Gly Ala Gly Val Ile Leu Cys 405 410 415Ile Glu Lys Ala Leu Ala Gln Ser Gly Val Ser Arg Glu Asp Val Asn 420 425 430Tyr Ile Asn Ala His Ala Thr Ser Thr Pro Ala Gly Asp Ile Lys Glu 435 440 445Tyr Gln Ala Leu Ile His Cys Phe Gly Gln Asn Arg Glu Leu Lys Val 450 455 460Asn Ser Thr Lys Ser Met Ile Gly His Leu Leu Gly Ala Ala Gly Gly465 470 475 480Val Glu Ala Val Ser Val Val Gln Ala Ile Arg Thr Gly Trp Ile His 485 490 495Pro Asn Ile Asn Leu Glu Asn Pro Asp Glu Gly Val Asp Thr Lys Leu 500 505 510Leu Val Gly Pro Lys Lys Glu Arg Leu Asn Val Lys Val Gly Leu Ser 515 520 525Asn Ser Phe Gly Phe Gly Gly His Asn Ser Ser Ile Leu Phe Ala Pro 530 535 540Tyr Ile545 15 2372 DNA Cuphea pullcherrima misc_feature (1)...(2372) n = A,T,C or G 15acntggtccg gaattcccgg gtcgacccac gcgtccgcga cgccaaccca caccaaactt 60cctcagcttc tcttctcaag acggacgcca ttggcagcag acagacagac agacagaccc 120ataaaagaga gagagaggga tccatcgaat gcggccaccc tcctttcatc ttcgattcat 180taccatacca ttccgctgat ccattttccg ccttttccgg gtctttcatc ccaaagggta 240tccttttcta tcctatcttc tcaaagggtc agtcagttcc ctccaatgcc tgccgcctct 300tccctgctcg cttcccctct ctgtacgtgg ctccttgccg cctgcatgtc tacctccttc 360cacccctccg accctcttcc gccttccatc tcctctcctc gccgacgcct ctcccgccgc 420cggattctct cccaatgcgc cccactacct tctgcttcct ccgccctccg cggatccagt 480ttccataccc tcgtcacctc ttacctcgcc tgcttcgagc cctgccatga ctactataca 540tccgcatcct tgttcggatc cagacccatt cgcaccaccc gcaggcaccg gaggctcaat 600cgagcttccc cttccagggg aggcaatggc cgtggctctg caacctgaac aggaagttac 660cacaaagaag aagccaagta tcaaacagcg gcgagtagtt gtgactggaa tgggtgtggt 720gactcctcta ggccatgaac ctgatgtttt tctacaataa tctgcttgat ggaacgagtg 780gcataagcga gatagagacc tttgattgtg ctcaatttcc tacgagaatt gctggagaga 840tcaagtcttt ctccacagat ggttgggtgg ccccgaagct ctctaagagg atggacaagt 900tcatgctata catgctgact gctggcaaga aagcattaac agatggtgga atcaccgaag 960atgtgatgaa agagctagat aaaagaaaat gcggagttct cattggctca gcaatgggtg 1020gaatgaaggt attcaatgat gccattgaag ccctaaggat ttcatataag aagatgaatc 1080ccttttgtgt acctttcgct accacaaata tgggatcagc tatgcttgca atggacttgg 1140gatggatggg gcccaactac tcgatatcta ctgcttgtgc aacgagtaac ttttgtataa 1200tgaatgctgc gaaccatata atcagaggcg aagcagatgt gatgctttgc gggggctcag 1260atgcggtaat catacctatt ggtatgggag gttttgttgc atgccgagct ttgtcccaga 1320gaaattccga ccctactaaa gcttcaagac catgggacag taatcgtgat ggatttgtta 1380tgggggaagg agctggagtg ctactactag aggagttgga gcatgcaaag aaaagaggtg 1440cgactattta cgcagaattt ctaggtggga gtttcacttg cgatgcctac cacatgaccg 1500agcctcaccc tgatggagct ggagtgattc tctgcataga gaaggctttg gctcagtcag 1560gagtctctag ggaagacgta aattacataa atgcccatgc cacatccact ccggctggag 1620atatcaaaga gtaccaagct cttatccact gtttcggcca aaacagagag ttaaaagtta 1680attcaaccaa atcaatgatt ggtcaccttc tcggagcagc cggtggtgtg gaagcagttt 1740cagtagttca ggcaataagg actgggtgga tccatccgaa tattaatttg gaaaacccag 1800atgaaggcgt ggatacaaaa ttgctcgtgg gtcctaagaa ggagagactg aacgttaagg 1860tcggtttgtc taattcattt gggtttggtg ggcacaactc gtccatactc ttcgcccctt 1920acatctagga cgtttcgtgt gtggaattct actcaacata tcaaagctga agttttgagg 1980actccagcat gttggtagct ccttacgtct ctagacatgc ccatgagttt tgtgtccgga 2040gctttagtcg gaaccatgac ggattgagta ctcatggcga cacttgatat actccttgct 2100agaattgttg gtagagcaat attcattatc tcatattttt tttttctctg aaatctccct 2160ccttgcaata gttgtacttt cgagcttttc atcgagtcag tgaagaagag aacaaagctg 2220ttaactcggg cacgtagtaa ccatttgccc tttgttttgc tctctatttc atcaccgttt 2280tgtggtttta aaatttgtaa aactagaaga ctggtttaga ttggtttgtt ttctcaaaaa 2340aaaaaaaaaa gggcggccgc tctagaggat cc 2372 16 1580 DNA Cuphea Hookeriana misc_feature (1)...(1580) n = A,T,C or G 16cctgaatcgg attcaagaga gagtttcgtt gctgggatgg cgaatgcatc tgggtttctg 60ggttcttcag ttcctgccct gagaagggca actcagcatt cgatttcatc gtctcgtgga 120tcttcctcgg agtttgtctc caaaagggtg ttttgctgta gtgccgttca ggattctgac 180aggcagtctt tgggtgattc tcgctcgccg aggcttgtga gtagaggatg caaattaatt 240ggatctggtt ctgctatacc agctcttcaa gtctcaaatg atgatcttgc taaaattgtc 300gacaccaatg atgaatggat tactgtccga acggggatcc gcaaccgaag ggttctctca 360ggtaaagata gtcttacaaa tttagcatca gaggcagcaa ggaaagctct agagatggca 420caggtagacg caaatgatgt ggatatggtt ttgatgtgta cttctacccc tgaggacctt 480ttcggcagtg ctcctcagat atcgaaagca cttggctgca aaaagaatcc tttgtcttac 540gacattaccg ctgcatgcag tggatttgtg ttgggtttag tctcagctgc ttgccacatt 600agaggtgggg gttttaacaa tattctagtg attggtgctg attctctttc tcggtatgtt 660gactggaccg atcggggaac atgtattctc tttggagatg ctgctggagc tgtagtggtg 720cagtcatgtg atgctgagga agatgggctc tttgcttttg atttgcatag cgatggagat 780gggcaaaggc atctaaaagc tgcaatcaaa gaagatgaag ttgataaagc cctgggacat 840aatgggtcca tcagagattt tccaccaagg cgttcttcat actcttgcat ccaaatgaac 900ggtaaagagg tattccgctt tgcttgccgc tctgtgcctc agtcaatcga atcagcactt 960ggaaaggccg gtcttaatgg atccaacatc gactggttgc tgcttcatca ggcaaatcag 1020aggatcattg atgcagtagc aacacgtcta gaggttcctc aagaacgaat tatctcaaac 1080ttggcaaatt acgggaacac tagtgcggca tccattccct tggcactaga cgaagctgtg 1140aggagtggaa atgtgaagcc gggtcacgtg attgcaaccg caggatttgg cgccggactc 1200acatggggtt ctgctattat caggtgggga taagactgaa gccgagccag cactgcagct 1260tcctctcaaa ccgatgtttc acgaaatttt gcttccatga ccanaaaaag aagaagtcag 1320tcttttatgg agcaagcaac acgacacgat cttcatcaca ttgccctttt tcgttcccct 1380tttccattag tttgatgatt ttgctgacaa tacaataccc atagtttctt ttgtccccaa 1440taagttattt gtttcttgtt taattgttca gcttttactt cattttgtct cgggacattg 1500gagatgacag cataaacatc atgtttatat tttgctaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa 1580 17 382 PRT Cuphea Hookeriana 17Met Ala Asn Ala Ser Gly Phe Leu Gly Ser Ser Val Pro Ala Leu Arg 1 5 10 15Arg Ala Thr Gln His Ser Ile Ser Ser Ser Arg Gly Ser Ser Ser Glu 20 25 30Phe Val Ser Lys Arg Val Phe Cys Cys Ser Ala Val Gln Asp Ser Asp 35 40 45Arg Gln Ser Leu Gly Asp Ser Arg Ser Pro Arg Leu Val Ser Arg Gly 50 55 60Cys Lys Leu Ile Gly Ser Gly Ser Ala Ile Pro Ala Leu Gln Val Ser65 70 75 80Asn Asp Asp Leu Ala Lys Ile Val Asp Thr Asn Asp Glu Trp Ile Thr 85 90 95Val Arg Thr Gly Ile Arg Asn Arg Arg Val Leu Ser Gly Lys Asp Ser 100 105 110Leu Thr Asn Leu Ala Ser Glu Ala Ala Arg Lys Ala Leu Glu Met Ala 115 120 125Gln Val Asp Ala Asn Asp Val Asp Met Val Leu Met Cys Thr Ser Thr 130 135 140Pro Glu Asp Leu Phe Gly Ser Ala Pro Gln Ile Ser Lys Ala Leu Gly145 150 155 160Cys Lys Lys Asn Pro Leu Ser Tyr Asp Ile Thr Ala Ala Cys Ser Gly 165 170 175Phe Val Leu Gly Leu Val Ser Ala Ala Cys His Ile Arg Gly Gly Gly 180 185 190Phe Asn Asn Ile Leu Val Ile Gly Ala Asp Ser Leu Ser Arg Tyr Val 195 200 205Asp Trp Thr Asp Arg Gly Thr Cys Ile Leu Phe Gly Asp Ala Ala Gly 210 215 220Ala Val Val Val Gln Ser Cys Asp Ala Glu Glu Asp Gly Leu Phe Ala225 230 235 240Phe Asp Leu His Ser Asp Gly Asp Gly Gln Arg His Leu Lys Ala Ala 245 250 255Ile Lys Glu Asp Glu Val Asp Lys Ala Leu Gly His Asn Gly Ser Ile 260 265 270Arg Asp Phe Pro Pro Arg Arg Ser Ser Tyr Ser Cys Ile Gln Met Asn 275 280 285Gly Lys Glu Val Phe Arg Phe Ala Cys Arg Ser Val Pro Gln Ser Ile 290 295 300Glu Ser Ala Leu Gly Lys Ala Gly Leu Asn Gly Ser Asn Ile Asp Trp305 310 315 320Leu Leu Leu His Gln Ala Asn Gln Arg Ile Ile Asp Ala Val Ala Thr 325 330 335Arg Leu Glu Val Pro Gln Glu Arg Ile Ile Ser Asn Leu Ala Asn Tyr 340 345 350Gly Asn Thr Ser Ala Ala Pro Gly His Val Ile Ala Thr Ala Gly Phe 355 360 365Gly Ala Gly Leu Thr Trp Gly Ser Ala Ile Ile Arg Trp Gly 370 375 380

Claims

1. A DNA construct comprising a coding sequence as set forth in SEQ ID NO:5.

2. A DNA construct comprising a coding sequence for a β-keto acyl-ACP synthase factor A protein encoding an amino acid sequence as set forth in SEQ ID NO:6.