Patent Application
20130014290
The present invention relates to methods and compositions for grain improvement and/or improved digestibility of transgenic plants. In certain embodiments, these plants can contain a UDP-xylose synthase (UXS) nucleic acid that inhibits arabinoxylan and/or hemicellulose production resulting in improved digestibility and/or ease of grain processing. In other embodiments, these plants can contain a UXS nucleic acid over-expressed to increase arabinoxylan and/or hemicellulose production resulting in, for example, harder pericarp when expressed in seed.
Primary plant cell walls contain approximately 15-20% hemicellulose in the vegetative tissue and 40-60% in the grain.
Hemicellulose in monocot walls consists mainly of arabinoxylan (also referred to as glucurono-arabinoxylan or pentosans), a branched polymer consisting of a beta-gylosyl backbone decorated with arabinosyl and glucuronosyl residues. This polymer is often referred to as arabinoxylan because of the relatively low proportion of glucuronosyl residues. Carpita, (1996) Annual Review of Plant Physiol. and Plant Molecular Biology 476:445-476.
Dicot cell walls contain xyloglucan as a major hemicellulosic polymer. Xyloglucan is also a branched polymer consisting of a linear beta-glycosyl backbone decorated with xylosyl residues, some of which are substituted with galactosyl residues.
Cell walls of corn grain constitute about 6-8% of the total dry weight of the seed. Whistler, et al., Hemicelluloses In Industrial Gums: Polysaccharides and Their Derivatives, San Diego Academic Press, pp. 295-308 (1993). The grain cell wall contains 45-65% arabinoxylan, the remainder being mainly cellulose. Arabinoxylans are considered to be anti-nutritional components of animal feed because they can absorb large amounts of water. This leads to increased viscosity and possible sequestering of other digestible feed components, such as starch and polypeptides, away from digestive enzymes.
Arabinoxylans are known to lower the food conversion ratio (FCR) of animal feed. Studies where fungal xylanase were included in cereal-derived feed showed improved FCR and weight gain in broilers and pigs. Veldman and Vahl, (1994) British Poultry Science 35:537-550. The observed improvement in FCR and weight gain was higher than expected from arabinoxylan breakdown and digestion alone. Thus, arabinoxylan appears to reduce the digestion of other feed components. An unresolved need exists for methods and compositions to reduce the concentration of arabinoxylan (and thus hemicellulose) in grain.
Cellulose microfibrils have the highest tensile strength of any of the other cell wall polymers. Increasing the cellulose/arabinoxylan ratio in the cell wall should lead to a harder pericarp and improved ability to handle the grain.
About 12% of the corn processed through wet-milling is fiber (see, Johnson and Can, (2003) Wet milling: the basis of corn biorefineries; in Corn: Chemistry and Technology. P J White and L A Johnson (eds). AACC, St. Paul, Minn. pp. 449-494). Of this 12%, approximately 4% is coarse fiber (mainly pericarp-derived) and the rest is fine fiber. While coarse fiber consists of approximately 9% starch, fine fiber can have up to 30% starch, constituting 2.5% of the total dry mass. The other major component of fine fiber is arabinoxylan, which makes up to 21% of this fraction. Some starch can become intricately associated with arabinoxylan, making it difficult to extract during wet-milling. A need exists for a method to reduce the concentration of arabinoxylan (and thus hemicellulose) in grain to improve starch extractability.
Terms that are not otherwise defined herein are used in accordance with their plain and ordinary meaning.
Units, prefixes and symbols can be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
As used herein, “a” or “an” can mean one or more than one of an item.
“UXS polynucleotide” refers to a nucleic acid construct that comprises at least part or all of the UXS nucleic acid sequence, non-limiting examples of which are provided in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15. A “polynucleotide” can be single, double and/or triple stranded and can comprise part or all of a native or modified UXS nucleic acid sequence. A “polynucleotide” can be designed to produce an inhibitory RNA that decreases expression of the endogenous UXS sequences. A “polynucleotide” can be of any size, from a short oligonucleotide to a full length UXS gene. A UXS “polynucleotide” sequence can be in the sense or anti-sense orientation. A “polynucleotide” can be of genomic or cDNA sequence.
The present invention also provides isolated polynucleotide sequences comprising transcriptional units for gene over-expression and gene-suppression that have been used either as single units or in combination as multiple units to transform plant cells.
A “transgenic plant” is one that contains an introduced segment of nucleic acid, such as a UXS nucleic acid. The introduced nucleic acid can encode a native UXS polynucleotide sequence or a modified polynucleotide sequence of any length, up to a full-length sequence. Any method known in the art can be used to transform one or more plant cells and regenerate a transgenic plant.
As used herein “operably linked” refers to a functional linkage between a promoter and/or other regulatory element and a second nucleic acid sequence, wherein the promoter initiates and mediates transcription of the second sequence.
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. “Plant cell”, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the claimed methods is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the native nucleic acid, i.e., “functional fragments”.
Alternatively, fragments of a nucleotide sequence that can be useful as hybridization probes may not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence are generally greater than 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 or 700 nucleotides and up to and including the entire nucleotide sequence encoding the proteins of the invention. Generally the probes are less than 1000 nucleotides and often less than 500 nucleotides.
Similarly, fragments of a nucleotide sequence that are useful for generating cells, tissues or plants transiently or permanently suppressing a gene or genes may not encode fragment proteins retaining biological activity. Fragments may be in sense or antisense orientation, reverse orientation, complementary or a combination thereof. Thus, for example, fragments of such nucleotide sequence may range from at least about 10 nucleotides, to greater than 25, 50, 100, 200, 300, 400, 500, 600 or 700 nucleotides and up to and including the full-length nucleotide sequence-encoding native UXS proteins.
By “variants” is intended substantially similar sequences. Generally, nucleic acid sequence variants of the invention will have at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native nucleotide sequence, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 analysis using default parameters. Generally, polypeptide sequence variants of the invention will have at least about 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native protein, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 analysis using default parameters. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
As used herein “transformation” can include stable transformation and transient transformation. Unless otherwise stated, “transformation” refers to stable transformation.
As used herein “stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism (this includes both nuclear and organelle genomes) resulting in genetically stable inheritance. In addition to traditional methods, stable transformation includes the alteration of gene expression by any means including chimeraplasty or transposon insertion.
As used herein “transient transformation” refers to the transfer of a nucleic acid fragment or protein into the nucleus (or DNA-containing organelle) of a host organism resulting in gene expression without integration and stable inheritance.
As used herein, the term “co-suppression” is used to collectively designate gene silencing methods based on mechanisms involving the expression of antisense, sense RNA molecules, aberrant RNA molecules, double-stranded RNA molecules, micro RNA molecules and the like.
“Non-ruminant animal” means an animal with a simple stomach divided into the esophageal, cardia, fundus and pylorus regions. A non-ruminant animal additionally implies a species of animal without a functional rumen. A rumen is a section of the digestive system where feedstuff/food is soaked and subjected to digestion by microorganisms before passing on through the digestive tract. This phenomenon does not occur in a non-ruminant animal. The term non-ruminant animal includes, but is not limited to, humans, swine, poultry, cats and dogs.
UDP-xylose (Xyl) is an essential sugar donor for the synthesis of hemicellulose, glycoproteins and oligosaccharides. UDP-Xyl also feedback inhibits upstream enzymes. In plants, the major route by which UDP-Xyl is produced is via UDP-Glucose. The biosynthesis of UDP-Xyl is catalyzed by different UDP-xylose synthase (UXS) isozymes, all of which convert UDP-GlcA to UDP-Xyl by NADH dependent decarboxylation of UDP-glucuronic acid.
A plant UDP-xylose synthase (UXS) was purified from pea by peptide sequencing and enzyme activity was demonstrated with the recombinant polypeptide. The A. thaliana and rice genomes each contain six putative UXS genes that are highly similar to both the pea and fungal UXS genes. The sub-cellular location of UXS activities varies in different species. For example, the only UXS enzyme from rat is localized to the Golgi, while the sole UXS of Cryptococcus is cytosolic. In plants, activities of UXS enzymes are either soluble or membrane associated. Three of the Arabidopsis UXS isoforms (AtUXS 3, 5, 6) appear to be cytosolic and the other three (AtUxs 1, 2, 4) are likely to reside in the endomembrane system. In addition to six UXS isoforms, two A. thaliana genes that are distantly related to the UXS gene family apparently encode UDP-D-apiose/UDP-D-xylose synthase (AXS). Purified recombinant AXS1 generates UDP-D-xylose and UDP-D-apiose using UDP-D-glucuronic acid as its exclusive substrate.
In various embodiments, nucleic acids of the invention can encode a UXS polypeptide sequence, an inhibitory RNA or another type of UXS macromolecule. The nucleic acid can be derived from genomic DNA, complementary DNA (cDNA), RNA or synthetic DNA. Where incorporation into an expression cassette is desired, the nucleic acid can also comprise one or more introns.
A nucleic acid includes single-stranded and double-stranded or triple-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogs. It is contemplated that a nucleic acid can be of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater nucleotide residues in length, up to a full length UXS gene.
UXS polypeptides can be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence. The design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables (see, Table 1 below). The codons selected for encoding each amino acid can be modified to optimize expression of the nucleic acid in the host cell of interest, for example by using codons optimized for expression in maize. Codon preferences for various species of host cell are well known in the art.
Isolated nucleic acids can be made by any method known in the art, for example using standard recombinant methods, synthetic techniques, or combinations thereof. In some embodiments, the nucleic acids can be cloned, amplified, or otherwise constructed.
The nucleic acids can conveniently comprise sequences in addition to UXS nucleic acid sequences. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be added. Regulatory sequences can be added to promote expression of the nucleic acid. Translatable sequences can be inserted to aid in the isolation of expressed polypeptides. For example, a hexa-histidine marker sequence provides a convenient means to purify tagged polypeptides. A nucleic acid can be attached to a cassette, adapter or linker for cloning and/or expression of a nucleic acid. Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the nucleic acid, or to improve the introduction of the nucleic acid into a cell. Use of cloning cassettes, expression cassettes, adapters and linkers is well known in the art.
Isolated nucleic acids, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the nucleic acids are used to identify a sequence in a cDNA or genomic DNA library. Methods for isolation of mRNA and construction of cDNA and genomic libraries are known and any such known methods can be used. (See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Carninci, et al., (1996) Genomics 37:327-336; Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711; Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989); Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego Academic Press, Inc. (1987).]
cDNA or genomic libraries, transgenic cell lines, transgenic plants or tissues or native plants or tissues can be screened for the presence and/or expression levels of UXS nucleic acids using a probe based upon one or more UXS sequences, such as those disclosed in the present invention. Probes also can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Various degrees of stringency of hybridization can be employed in the assay. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and/or the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100 percent; however, minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
Typically, stringent hybridization conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.1×SSC at 60° C. Typically the time of hybridization is from 4 to 16 hours.
An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Often, cDNA libraries will be normalized to increase the representation of relatively rare cDNAs.
Nucleic acids of interest can also be amplified using a variety of known amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify target sequences directly from genomic DNA or cDNA sequences. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for polypeptides to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques of use for nucleic acid amplification are found in Berger, Sambrook, and Ausubel, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987) and, PCR Protocols A Guide to Methods and Applications, Innis, et al., Eds., Academic Press Inc., San Diego, Calif. (1990). The T4 gene 32 polypeptide (Boehringer Mannheim) can be used to improve yield of long PCR products. PCR-based screening methods have been disclosed. (See, e.g., Wilfinger, et al., (1997) BioTechniques 22(3):481-486).
Isolated nucleic acids can be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra. Lett. 22:859-1862; the solid phase phosphoramidite triester method of Beaucage and Caruthers, (1981) Tetra. Letts. 22(20):1859-1862, using an automated synthesizer as in Needham-VanDevanter, et al., (1984) Nucleic Acids Res. 12:6159-6168 or by the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. While chemical synthesis of DNA is best employed for sequences of about 100 bases or less, longer sequences can be obtained by the ligation of shorter sequences.
A variety of cross-linking agents, alkylating agents and radical generating species can be used to bind, label, detect and/or cleave nucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res 14:4065-4076, disclose covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, et al., (1989) J Am Chem Soc 111:8517-8519 disclose covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been disclosed by Webb and Matteucci, (1986) J Am Chem Soc 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz, et al., (1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect, label and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and 5,681,941.
Various embodiments concern cassettes comprising UXS nucleic acids, which cassettes can be transformed into a target host cell. An expression cassette will typically comprise a nucleic acid operably linked to transcriptional regulatory elements which will direct the transcription of the nucleic acid. For example, plant expression cassettes can include a cloned UXS nucleic acid under the transcriptional control of 5′ and 3′ regulatory sequences. Expression cassettes can contain a promoter sequence (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal.
The nucleotide sequences for use in the methods of the present invention are provided in expression cassettes for transcription in the plant of interest. Such expression cassettes are provided with a plurality of restriction sites for insertion of any sequence of the present invention to be placed under the transcriptional regulation of the regulatory regions. The expression cassettes may additionally contain selectable marker genes.
The expression cassette can include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, any seed protein sequence of the invention and optionally, a transcriptional and translational termination region functional in plants. The transcriptional initiation region may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein, a gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Alternatively, a gene comprises fragments of at least two independent transcripts that are linked in a single transcription unit.
While it may be preferable to express the sequences using heterologous promoters, the native promoter sequences may be used. Such constructs would alter expression levels of the proteins in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered. Alternatively, the promoter sequence may be used to alter expression. For example, the promoter (or fragments thereof) of a UXS sequence of the present invention can modulate expression of the native UXS protein or other closely related proteins.
Use of a termination region is not necessary for proper transcription of plant genes but may be used as part of an expression construct. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.
Where appropriate, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
The use of the term “nucleotide constructs” herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
Furthermore, it is recognized that the methods of the invention may employ a nucleotide construct that is capable of directing, in a transformed plant, the expression of at least one protein, or at least one RNA, such as, for example, an antisense RNA that is complementary to at least a portion of an mRNA. Alternatively, it is also recognized that the methods of the invention may employ a nucleotide construct that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
In addition, it is recognized that methods of the present invention do not depend on the incorporation of the entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of the nucleotide construct into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the nucleotide construct into a cell. For example, the nucleotide construct, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions and substitutions of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions or substitutions comprise at least one nucleotide.
The nucleotide constructs of the invention also encompass nucleotide constructs that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary chimeric oligonucleotides and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use, such as, for example, chimeraplasty, are known in the art. Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778, herein incorporated by reference.
A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred or other promoters for expression in plants, more preferably a promoter functional during seed development.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463 and 5,608,142.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
Tissue-preferred promoters can be utilized to target enhanced protein expression within a particular plant tissue. Tissue-preferred promoters include, but are not limited to: Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen. Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kD zein) and milps (myo-inositol-1-phosphate synthase; see, U.S. Pat. No. 6,225,529, herein incorporated by reference). The 27 kD gamma-zein is a preferred endosperm-specific promoter. Glb-1 is a preferred embryo-specific promoter.
For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like.
For monocots, seed-specific promoters include, but are not limited to, maize 15 kD zein, 22 kD zein, 27 kD zein, 10 kD delta-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc.
While not critical to the invention, the methods of the invention comprise either increasing or decreasing the level of a target gene product. Methods for inhibiting gene expression are well known in the art. Although any method know in the art for reducing the level of protein in a plant could be used, possible methods for reducing protein include, but are not limited to, homology-dependent gene silencing, antisense technology, co-suppression including, for example, RNA interference (RNAi), micro RNA and the like, site-specific recombination, site-specific integration, mutagenesis including transposon tagging, and biosynthetic competition, homologous recombination and gene targeting, alone or in combination. Depending upon the intended goal, the level of at least one seed protein may be increased, decreased or eliminated.
Catalytic RNA molecules or ribozymes may also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is disclosed, for example, in Haseloff, et al., (1988) Nature 334:585-591.
A “co-suppression” cassette may include 5′ (but not necessarily 3′) regulatory sequences, operably linked to at least one of the sequences of the invention. Co-supression cassettes used in the methods of the invention can comprise sequences of the invention in so-called “inverted repeat” structures. The cassette may additionally contain a second copy of the fragment in opposite direction to form an inverted repeat structure: Opposing arms of the structure may or may not be interrupted by any nucleotide sequence related or unrelated to the nucleotide sequences of the invention. (See, Fiers, et al., U.S. Pat. No. 6,506,559). The transcriptional units are linked to be co-transformed into the organism. Alternatively, additional transcriptional units can be provided on multiple over-expression and co-suppression cassettes.
The methods of transgenic co-suppression can be used to reduce or eliminate the level of at least one seed protein in grain. One method of transgenic co-suppression comprise transforming a plant cell with at least one transcriptional unit containing an expression cassette comprising a promoter that drives transcription in the plant operably linked to at least one nucleotide sequence transcript in the sense orientation encoding at least a portion of the seed protein of interest. Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives transcription in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity over the entire length of the sequence. Furthermore, portions, rather than the entire nucleotide sequence, of the polynucleotides may be used to disrupt the expression of the target gene product. Generally, sequences of at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200 nucleotides or greater may be used. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by reference.
The endogenous gene targeted for co-suppression may be a gene encoding any seed protein that accumulates as a seed protein in the plant species of interest, including, but not limited to, the seed genes noted above. For example, where the endogenous gene targeted for co-suppression is a UXS gene disclosed herein, co-suppression is achieved using an expression cassette comprising a UXS gene sequence or variant or fragment thereof.
Additional methods of co-suppression are known in the art and can be similarly applied to the instant invention. These methods involve the silencing of a targeted gene by spliced hairpin RNA's and similar methods also called RNA interference and promoter silencing (see, Smith, et al., (2000) Nature 407:319-320, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964; Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Phystiol. 129:1723-1731 and Patent Applications WO 99/53050; WO 99/49029; WO 99/61631; WO 00/49035 and U.S. Pat. No. 6,506,559, each of which is herein incorporated by reference). For the purpose of this invention the term “co-suppression” is used to collectively designate gene silencing methods based on mechanisms involving the expression of sense RNA molecules, aberrant RNA molecules, double-stranded RNA molecules, micro RNA molecules and the like.
The expression cassette for co-suppression may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, International Publication Number WO 02/00904, herein incorporated by reference.
In other embodiments of the invention, inhibition of the expression of a protein of interest may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example, Javier, et al., (2003) Nature 425: 257-263, herein incorporated by reference.
For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). miRNA molecules are highly efficient at inhibiting the expression of endogenous genes and the RNA interference they induce is inherited by subsequent generations of plants
In one embodiment, the polynucleotide to be introduced into the plant comprises an inhibitory sequence that encodes a zinc finger protein that binds to a gene encoding a protein of the invention resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of a UXS gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding a seed protein and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242 and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in US Patent Application Publication Number 2003/0037355; each of which is herein incorporated by reference.
Methods for antisense suppression can be used to reduce or eliminate the level of at least one protein of the invention. The methods of antisense suppression comprise transforming a plant cell with at least one expression cassette comprising a promoter that drives expression in the plant cell operably linked to at least one nucleotide sequence that is antisense to a nucleotide sequence transcript of the target protein. By “antisense suppression” is intended the use of nucleotide sequences that are antisense to nucleotide sequence transcripts of endogenous plant genes to suppress the expression of those genes in the plant.
Methods for suppressing gene expression in plants using nucleotide sequences in the antisense orientation are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that is antisense to the transcript of the endogenous gene. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the corresponding antisense sequences may be used. Furthermore, portions, rather than the entire nucleotide sequence, of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 10 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides or greater may be used.
Methods for transposon tagging can be used to reduce or eliminate the level of at least one seed protein in grain. The methods of transposon tagging comprise insertion of a transposon within an endogenous plant seed gene to reduce or eliminate expression of the seed protein.
Methods for transposon tagging of specific genes in plants are well known in the art (see, for example, Maes, et al., (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000) Nuc. Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics 153:1919-1928). In addition, the TUSC process for selecting Mu insertions in selected genes has been described (Bensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science 274:1537-1540; U.S. Pat. No. 5,962,764, which is herein incorporated by reference).
Other methods for inhibiting or eliminating the expression of endogenous genes are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted (for examples of these methods see Ohshima, et al., (1998) Virology 243:472-481; Okubara, et al., (1994) Genetics 137:867-874; Quesada, et al., (2000) Genetics 154:421-436. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING, (Targeting Induced Local Lesions In Genomes), using a denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention (see, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457).
Mutation breeding is another of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of induced mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased b many different means including: temperature; long-term seed storage; tissue culture conditions; radiation such as X-rays, Gamma rays (e.g., Cobalt 60 or Cesium 137), neutrons, (product of nuclear fission by Uranium 235 in an atomic reactor, Beta radiation (emitted from radioisotopes such as P32, or C14) or ultraviolet radiation (preferably from 2500 to 2900 nm) or chemical mutagens such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid or acridines. Once a desired trait is observed through mutagenesis, the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in “Principals of Cultivar Development” Fehr, 1993 (Macmillan Publishing Company), the disclosures of which are incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of elite lines that comprise such mutations.
Other methods for inhibiting or eliminating the expression of genes include the transgenic application of transcription factors (Pabo, et al., (2001) Annu Rev Biochem 70:313-40 and Reynolds, et al., (2003) Proc Natl Acad Sci USA 100:1615-20) and homologous recombination methods for gene targeting (see, U.S. Pat. No. 6,187,994).
Similarly, it is possible to eliminate the expression of a single gene by replacing its coding sequence with the coding sequence of a second gene using homologous recombination technologies (see, Bolon, (2004) Basic Clin. Pharmacol. Toxicol. 95(4)(12):154-61; Matsuda and Alba, (2004) Methods Mol. Bio. 259:379-90; Forlino, et. al., (1999) J. Biol. Chem. 274(53):37923-30). For example, by using the knock-out/knock-in technology, the coding sequence of a UXS polypeptide can be replaced by the coding sequence of a closely related polypeptide resulting in suppression of UXS protein expression and in over-expression of the closely related protein.
In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of a protein of interest. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of one or more proteins. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.
In some embodiments of the invention, the polynucleotide comprises an inhibitory sequence that encodes an antibody that binds to at least one isoform of a seed protein and reduces the level of the seed protein. In another embodiment, the binding of the antibody results in increased turnover of the antibody-antigen complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporated herein by reference
Plant transformants containing a desired genetic modification as a result of any of the above described methods resulting in increased, decreased or eliminated expression of the seed protein of the invention can be selected by various methods known in the art. These methods include, but are not limited to, methods such as SDS-PAGE analysis, immunoblotting using antibodies which bind to the seed protein of interest, single nucleotide polymorphism (SNP) analysis or assaying for the products of a reporter or marker gene, and the like.
Transformation protocols as well as protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include, but are not limited to: microinjection (Crossway, et al., (1986) Biotechniques 4:320-334); electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606); Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat. No. 5,981,840; Cai, et al., U.S. patent application Ser. No. 09/056,418); direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, Sanford, et al., U.S. Pat. No. 4,945,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, et al., U.S. Pat. No. 5,886,244; Bidney, et al., U.S. Pat. No. 5,932,782; Tomes, et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926). Also see, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising, et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes, et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens), all of which are herein incorporated by reference.
The methods of the invention can involve introducing a nucleotide construct into a plant. By “introducing” is intended presenting to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The claimed methods do not depend on a particular method for introducing a nucleotide construct to a plant, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus-mediated methods.
By “stable transformation” is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By “transient transformation” is intended that a nucleotide construct introduced into a plant does not integrate into the genome of the plant.
The nucleotide constructs of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the protein of interest of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931, herein incorporated by reference.
Other methods of transformation include Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol. 6, Rigby, Ed., London, Academic Press, 1987; and Lichtenstein and Draper, In: DNA Cloning, Vol. II, Glover, Ed., Oxford, IRI Press, 1985), Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) discloses the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16
The introduction of DNA constructs using PEG precipitation is disclosed in Paszkowski, et al., (1984) EMBO J. 3:2717-2722. Electroporation techniques are disclosed in Fromm, et al., (1985) Proc. Natl. Acad. Sci. (USA) 82:5824. Ballistic transformation techniques are disclosed in Klein, et al., (1987) Nature 327:70-73. Alternative transformation methods include liposome-mediated DNA uptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:353) and the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad. Sci. (USA) 87:1228.
DNA can also be introduced into plants by direct DNA transfer into pollen as disclosed by Zhou, et al., (1983) Methods in Enzymology 101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988) Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as disclosed by Pena, et al., (1987) Nature 325:274. DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as disclosed by Neuhaus, et al., (1987) Theor. Appl. Genet. 75:30 and Benbrook, et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus and tobacco mosaic virus. The claimed methods and compositions are not limiting as to the method of transformation and any such plant transformation method known in the art can be utilized.
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and/or phenotype. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84, incorporated herein by reference. These plants can then be grown and either pollinated with the same transformed strain or different strains and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
Plants cells transformed with a plant expression cassette can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. Various cells, tissues, and organs can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is disclosed in Evans, et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176 (1983) and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
The regeneration of plants containing a foreign gene introduced by Agrobacterium from leaf explants can be achieved as disclosed by Horsch, et al., (1985) Science 227:1229-1231. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed. These transformed shoots are then transferred to an appropriate root-inducing medium containing a selective agent and an antibiotic to prevent bacterial growth.
Regeneration can also be obtained from plant callus, explants, organs or parts thereof. The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, Weissbach and Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3.sup.rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).
The desired genetically altered trait can be bred into other plant lines possessing desirable agronomic characteristics using conventional breeding methods and/or top-cross technology. The top-cross method is taught in U.S. Pat. No. 5,704,160 herein incorporated in its entirety by reference.
Methods for cross pollinating plants are well known to those skilled in the art and are generally accomplished by allowing the pollen of one plant, the pollen donor, to pollinate a flower of a second plant, the pollen recipient, and then allowing the fertilized eggs in the pollinated flower to mature into seeds. Progeny containing the entire complement of heterologous coding sequences of the two parental plants can be selected from all of the progeny by standard methods available in the art for selecting transformed plants. If necessary, the selected progeny can be used as either the pollen donor or pollen recipient in a subsequent cross pollination.
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the claimed methods and compositions, provided that these parts comprise cells comprising the disclosed nucleic acids of the invention. Progeny and variants and mutants of the regenerated plants are also included within the scope of the claimed subject matter, provided that these parts comprise the introduced nucleic acid sequences.
Transgenic plants expressing a selectable marker can be screened for transmission of one or more target nucleic acids by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can be analyzed for polypeptide expression by Western immunoblot analysis using specifically or selectively reactive antibodies. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
The claimed methods and/or compositions further provides for modulating (i.e., increasing or decreasing) the concentration or ratio of polypeptides, such as UXS polypeptides, in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the ratio of the polypeptides in a plant.
The following examples are included to illustrate various embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Eight UXS isoforms were identified and named UXS1 through 8. Amino acid sequences of the maize isoforms are provided in the sequence listing as SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16. Nucleic acid sequences encoding the UXS isoforms are provided in the sequence listing as SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15. The eight isoforms can be generally classified into three distinct types. Type A includes ZmUXS1 and 2, orthologous to AtUXS3, 5, 6, which are likely soluble and cytosolic localized. Type B includes ZmUXS3 and 4, orthologous to AtUXS1. Type C includes ZmUXS5, 6, 7, and 8, orthologous to AtUXS2 and 4. Both type B and C contain a trans-membrane domain and are likely Golgi localized.
Expression levels of different UXS isoforms were determined in different maize tissues using standard techniques. Briefly, the eight UXS DNA sequences were used to blast an MPSS tag library and unique tags were identified for each isoform (Massively Parallel Signature Sequencing) technology from LYNX™ (see, Brenner, et al, (2000) Nature Biotechnology 18:630-634). The unique and 3′ most tags for each isoform were used to blast the MPSS profiling database. The average abundance of the tags was calculated from LYNX libraries for each tissue, with the expression levels of the UXS isoforms taken from the relative abundance of the corresponding tags.
UXS expression in maize endosperm showed a different developmental pattern from other seed tissues, with a decrease in total UXS expression observed between 6 DAP (days after pollination) and 40 DAP. The isoform profile shifted from primarily zmUXS1, zmUXS2 and zmUXS6 at DAP6 to an almost equal, but low, mixture of isoforms 1, 2, 3, 4, 6 and 8 at DAP 40.
In contrast, maize pericarp showed an increase in total UXS expression from DAP 5 to DAP 27. However the dominant isoforms in pericarp did not vary much with time after pollination, being primarily zmUXS1 and zmUXS2, with a lesser amount of zmUXS6 at DAP 5. Although the total expression level increases at DAP 27, the same three isoforms predominated. A comparison of various maize tissues showed that zmUXS1, zmUXS2 and zmUXS6 tended to be the predominant isoforms expressed in maize, although the relative levels varied from tissue to tissue. ZmUXS3, zmUXS4 and zmUXS8 appear to be minor alleles in general in maize.
For Agrobacterium-mediated transformation of maize, nucleotide sequences of interest such as UXS sequences were operably linked to a promoter as disclosed below employing the transformation method of Zhao, et al. (U.S. Pat. No. 5,981,840 and PCT Patent Publication WO98/32326, the contents of which are hereby incorporated by reference). Briefly, immature embryos were isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria were capable of transferring the nucleotide sequence of interest to at least one cell of at least one of the immature embryos (infection). In this step the immature embryos were immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos were co-cultured for a time with the Agrobacterium (co-cultivation). The immature embryos were cultured on solid medium following infection. Following this co-cultivation period an optional “resting” stage was performed. In this resting stage, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (resting). The immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos were cultured on medium containing a selective agent and growing transformed callus was recovered (selection). The immature embryos were cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus was then regenerated into plants and calli grown on selective medium were cultured on solid medium to regenerate the plants.
For a given ear, dissected endosperms were pooled into wild type or transgenic based on Western blot results. The pooled endosperm tissue was ground and weighed out into samples in duplicate 2 ml screw top microfuge tubes for hemicellulose preparations.
Small stir bars were placed in each tube and 1 ml 80% ethanol added and vortexed to mix. Tubes were heated in heat block set at 100° C. for 30 seconds and again vortexed to mix.
Tubes were then centrifuged at 14,000 rpm for 10 minutes and the supernatant discarded using vacuum. 1 ml of acetone was added and tubes vortexed to mix.
After another centrifugation at 14,000 rpm for 10 minutes, supernatant was again discarded using vacuum and pellets left in hood to dry (about 4 hours).
0.3 ml of α-amylase solution (300U/assay amylase in MOP buffer) was added to the tubes and mixed by vortexing. Tubes were then placed in racks and immersed in hot water. Heating proceeded at 90-95° C. for 15 minutes with constant stirring on magnetic stir plate. Samples were cooled slightly and briefly spun down to remove moisture by centrifugation (1 minute). At this point, 0.2 ml of 285 mM Na-acetate containing AMG (20 U/assay) mix was added and tubes incubated at 55° C. for 4 hours or overnight followed by a brief spin down by centrifugation (1 minute) to remove moisture.
1.25 ml of absolute ethanol was added to each tube (bringing final ethanol concentration to 70%). Samples were vortexed and cooled on ice or in −20 freezer for 10-15 minutes.
Three rounds of centrifugation at 14,000 rpm for 10 minutes after which the supernatant was discarded using vacuum and 1 ml 80% ethanol added and vortexed between rounds. After the last round of centrifugation, the supernatant was vacuum removed and discarded and 1 ml acetone added, vortexed, and centrifuged at 14,000 rpm for 10 minutes. This supernatant was also removed using vacuum and discarded. The pellets were then allowed to air-dry in fume hood.
1 ml of 1 M H2SO4 was added to air-dried tubes and vortexed to mix. Samples were then heated in 100° C. heat block for 45 minutes and vortexed every few minutes to mix. Samples were then cooled on ice.
Samples were centrifuged at 14,000 rpm for 10 minutes and 0.6 ml of supernatant removed into new tubes for HPLC.
Sugar residues from hemicellulose were quantified by HPLC Dionex CarboPac PA-1, 4×250 mm analytical column, PN 35391 Dionex PAD, with pulsed amperometric detector.
Individual hemicellulosic sugar residue was expressed as percent of endosperm weight. Arabinoxylan is the sum of arabinose and xylose % of control e.g.: arabinoxylan (transgenic)/arabinoxylan (wild type) X 100.
Based on expression profiles, members of Type A (ZmUXS1 and 2) and Type C (ZmUXS5, 6, 7 and 8) showed relatively high levels of expression in all three tissues of grain (pericarp, endosperm and embryo), while members of Type B (ZmUXS3 and 4) had apparently low endogenous expression in grain. Because of the high degree of nucleotide sequence homology between UXS1 and 2 and between UXS5, 6, 7 and 8, RNAi constructs of UXS2 and UXS6 under the control of promoters specific for pericarp, endosperm and embryo are expected to result in silencing expression of both groups of UXS isoforms. In turn, it is expected that a reduction of hemicellulose content in maize kernel will occur, improving grain digestibility and increasing efficiency of ethanol output per unit grain in dry milling.
As demonstrated in the case of UDP-GDH, (U.S. Pat. No. 6,399,859) a practical approach to reduce hemicellulose content can comprise blocking nucleotide sugar biosynthesis. UDP-xylose synthase (UXS) can also be used for this purpose because the reaction UXS catalyzes is downstream of UDP-GDH and additionally bypasses the myo-inositol pathway. Therefore, blocking UXS should provide potent and specific inhibition of hemicellulose production by inhibiting UDP-xylose synthesis.
The following constructs were prepared for co-suppression of UXS expression in maize. Promoters on each end of the expression cassette direct the insert to be expressed in different tissues at different times if desired. The cassettes contain no termination signals. The cassette included a selectable marker gene such as PAT (Wohlleben, et al., (1988) Gene 70:25-37 or BAR for resistance to Basta/phosphinothricin.
Methods of construction of such expression cassettes is well known to those of skill in the art in light of the present disclosure. See, for example, e.g., Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, Laboratory Press, Plainview, N.Y.; Gelvin, et al., (1990) Plant Molecular Biology Manual, each incorporated herein in its entirety by reference.
Nucleic Acid
Res. 18(21): 6426
Journal 4: 849-860
Zea mays-40 promoter
Construct PHP19991 (see, SEQ ID NO: 17 for insert sequence) was prepared to down-regulate the UXS 6 sequence. However, because of the high level of homology with UXS5 and UXS7, it was expected PHP19991 should inhibit expression of UXS 5, 6 and 7, but not UXS8. Maize was transformed with PHP19991 by Agrobacterium-mediated transformation as described herein. The genotypes of T1 kernels from the same event were screened with PCR, and the endosperms pooled into WT or transgenic. Hemicellulose analysis was performed with pooled endosperms as described herein.
PHP19991: GZ PRO:UXS6 IRNT:OLE PRO
Construct and sequence homology:
PHP19991/UXS6F vs UXS1=47.1%
PHP19991/UXS6F vs UXS2=48.2%
PHP19991/UXS6F vs UXS3=55.8%
PHP19991/UXS6F vs UXS4=57.3%
PHP19991/UXS6F vs UXS5=90.5%
PHP19991/UXS6F vs UXS6=100%
PHP19991/UXS6F vs UXS7=97.9%
PHP19991/UXS6F vs UXS8=65.9%
The effects of co-suppression using the PHP19991 cassette were to reduce the arabinoxylan contents in transgenic endosperm by up to about 20% in the T1 generation. The results demonstrate that UXS 5, 6 and/or 7 contribute part of the UDP-xylose substrate for hemicellulose synthesis.
Construct PHP21179 (see, SEQ ID NO: 18 for insert) was prepared to down-regulate the UXS2 sequence. Because of the high level of homology, PHP21179 was expected to inhibit expression of UXS 1 and 2 (Type A), the soluble isoforms of UXS in maize. Maize was transformed with PHP21179 by Agrobacterium-mediated transformation as described herein. The genotypes of T1 kernels from the same event were screened with PCR, and the endosperms pooled into WT or transgenic. Hemicellulose analysis was performed with pooled endosperms.
PHP21179: LEG1A PRO:Uxs2 IRNT ZM-40 PRO:Uxs2 (IRNT)
Construct and sequence homology:
PHP21179/UXS2F vs UXS1=94.2%
PHP21179/UXS2F vs UXS2=100%
PHP21179/UXS2F vs UXS3=65.7%
PHP21179/UXS2F vs UXS4=65.5%
PHP21179/UXS2F vs UXS5=66.2%
PHP21179/UXS2F vs UXS6=65.7%
PHP21179/UXS2F vs UXS7=66.1%
PHP21179/UXS2F vs UXS8=65.5%
The arabinoxylan contents in transgenic endosperm transformed with PHP21179 also showed up to about a 20% reduction in the T1 generation. The results show that UXS1 and 2 also play a role in producing UDP-xylose for hemicellulose synthesis.
Construct PHP21180 (ZM-40 PRO:Uxs2 IRNT::EAP1B PRO:Uxs2 IRNT) was prepared and transformed into maize by Agrobacterium-mediated transformation, as discussed above. T0 events of PHP21180 were selfed. Hence only 25% of the seeds would be expected to be wild-type in a segregating ear and 25% would be homozygous transgenic.
Twenty seeds from 12 events of PHP21180 were dissected to separate endosperm, embryo and pericarp. Twenty mg of ground endosperm was PCR genotyped to identify wild type and transgenic seeds using MOPAT primers. Nine out of the 12 events that segregated as single copy were further used for cell wall analysis. Embryo from seeds that segregated as wild type and transgenic seeds were pooled for each event. Four samples of 15-25 mg were used for cell wall sugar analysis. Reduction of arabinoxylan contents in transgenic embryos of up to 50% of control levels were observed.
Construct PHP21807 (see, SEQ ID NO: 19 for insert sequence) was prepared to inhibit both Type A and Type C isoforms of UXS. Based on sequence comparisons, this construct was expected to down-regulate UXS 1, 2, 5, 6, 7 and possibly UXS 8. PHP21807 was transformed into maize by Agrobacterium-mediated transformation. The genotypes of T1 kernels from the same event were screened with PCR and the endosperms pooled into WT or transgenic. Hemicellulose analysis was performed with pooled endosperms as described herein.
PHP21807:LEG1APRO:[Uxs2::Uxs6] IRNT ZM-40 PRO:[Uxs2::Uxs6] IRNT
Construct and sequence homology:
PHP21807/UXS2F vs UXS1=94.2%
PHP21807/UXS2F vs UXS2=100%
PHP21807/UXS2F vs UXS3=65.7%
PHP21807/UXS2F vs UXS4=65.5%
PHP21807/UXS2F vs UXS5=66.2%
PHP21807/UXS2F vs UXS6=65.7%
PHP21807/UXS2F vs UXS7=66.1%
PHP21807/UXS2F vs UXS8=65.5%
PHP21807/UXS6F vs UXS1=65.3%
PHP21807/UXS6F vs UXS2=66.2%
PHP21807/UXS6F vs UXS3=71.9%
PHP21807/UXS6F vs UXS4=72.0%
PHP21807/UXS6F vs UXS5=95.4%
PHP21807/UXS6F vs UXS6=100%
PHP21807/UXS6F vs UXS7=99.2%
PHP21807/UXS6F vs UXS8=85.6%
Transformation with PHP21807 resulted in a slightly greater reduction in arabinoxylan content in transgenic endosperm in the T1 generation, compared to either PHP19991 or PHP21179 alone, with up to about a 25% decrease observed.
Although the UXS sequences, enzyme activities and cellular localizations have been studied in plant species other than maize, their involvement in hemicellulose biosynthesis has not previously been demonstrated. These results provide the first demonstration that inhibiting expression of UXS causes a reduction in plant hemicellulose accumulation. The results also demonstrate that both the soluble and membrane bound UXS play a role in producing UDP-xylose for hemicellulose synthesis.
T2 generation seeds were produced by back-crossing transgenic T1 plants. The presence of UXS nucleic acid sequences were determined by PCR analysis, using construct specific primers. Arabinoxylan content was determined in pooled samples as described in Example 3. Maize T2 endosperm transformed with PHP19991, PHP21179 and PHP21807 all showed reduced hemicellulose content, with a greater reduction in arabinoxylan content observed in the T2 endosperm compared to T1 endosperm.
A stacked construct containing sequences targeted against Type A and Type C isoforms was prepared and transformed into maize. T1 endosperm containing the construct were analyzed for hemicellulose, cellulose and hemicellulosic galactose content. Inhibition of two types of UXS isoforms resulted in a greater decrease in hemicellulose content than inhibition of Type A alone or Type C alone.
This construct contained a different promoter than PHP21807: LTP1, a pericarp preferred promoter. The insert sequence in PHP23388 was the same as in PHP21807. PHP23388 was transformed into maize by Agrobacterium-mediated transformation as described herein. Hemicellulose content of T1 endosperm was determined as described above.
Construct PHP23388 resulted in a greater inhibition of hemicellulose content than PHP21807 as well as an unexpected reduction in cellulose and hemicellulosic galactose. Hemicellulose was reduced up to 50% in both endosperm and embryo with the majority of events having reductions of 20% or more. Cellulose was reduced up to 25% in both embryo and endosperm with the majority of events having at least a 10% reduction in both tissues. Embryo galactose was reduced by at least 20% in the majority of events and endosperm galactose reduced by at least 10′)/0 in most events.
These constructs are transformed by Agrobacterium transformation into maize as described above. T1 seed are analyzed and compared to wild-type to determine the function of Type B UXS.
These constructs are transformed by Agrobacterium transformation into maize as described above. T1 seed are analyzed and compared to wild-type and other transformants to determine the efficacy and effects of UXS on pericarp to achieve greater reduction of fiber in seed.
For Agrobacterium-mediated transformation of sorghum the method of Cai, et al., is employed (U.S. patent application Ser. No. 09/056,418, the contents of which are hereby incorporated by reference). This method can be employed with any of the nucleotide sequences described above.
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing any of the nucleotide sequences disclosed above, operably linked to a selected promoter, plus a plasmid containing the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows.
The ears are surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.
A plasmid cassette comprising the nucleotide sequence of interest operably linked to a promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows:
Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2.
All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for the desired phenotypic trait.
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H20 following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H20) and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H20 following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H20); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycine brought to volume with polished D-I H20) (Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H20 after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H20) and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H20), 0.1 g/1 myo-inositol and 40.0 g/l sucrose (brought to volume with polished D-I H20 after adjusting pH to 5.6) and 6 g/l bacto-agar (added after bringing to volume with polished D-I H20), sterilized and cooled to 60° C.
Embryogenic callus cultures derived from the scutellum of germinating seeds serve as the source material for transformation experiments. This material is generated by germinating sterile rice seeds on a callus initiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-D and 10 μM AgNO3) in the dark at 27-28° C. Embryogenic callus proliferating from the scutellum of the embryos is then transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/1 2,4-D, Chu, et al., (1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM by routine sub-culture at two week intervals and used for transformation within 10 weeks of initiation.
Callus is prepared for transformation by subculturing 0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular area of about 4 cm in diameter, in the center of a circle of Whatman #541 paper placed on CM media. The plates with callus are incubated in the dark at 27-28 C for 3-5 days. Prior to bombardment, the filters with callus are transferred to CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr. in the dark. The petri dish lids are then left ajar for 20-45 minutes in a sterile hood to allow moisture on tissue to dissipate.
Circular plasmid DNA from two different plasmids, one containing the selectable marker for rice transformation and one containing a nucleic acid of interest, are co-precipitated onto the surface of gold particles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio of trait:selectable marker DNAs is added to a 50 μl aliquot of gold particles resuspended at a concentration of 60 mg/ml. Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a 0.1 M solution) are then added to the gold-DNA suspension as the tube is vortexing for 3 min. The gold particles are centrifuged in a microfuge for 1 sec and the supernatant removed. The gold particles are then washed twice with 1 ml of absolute ethanol and then resuspended in 50 μl of absolute ethanol and sonicated (bath sonicator) for one second to disperse the gold particles. The gold suspension is incubated at −70 C for five minutes and sonicated (bath sonicator) if needed to disperse the particles. Six μl of the DNA-coated gold particles are then loaded onto mylar macrocarrier disks and the ethanol is allowed to evaporate.
At the end of the drying period, a petri dish containing the tissue is placed in the chamber of the PDS-1000/He. The air in the chamber is then evacuated to a vacuum of 28-29 inches Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi. The tissue is placed approximately 8 cm from the stopping screen and the callus is bombarded two times. Five to seven plates of tissue are bombarded in this way with the DNA-coated gold particles. Following bombardment, the callus tissue is transferred to CM media without supplemental sorbitol or mannitol.
Within 3-5 days after bombardment the callus tissue is transferred to SM media (CM medium containing 50 mg/l hygromycin). To accomplish this, callus tissue is transferred from plates to sterile 50 ml conical tubes and weighed. Molten top-agar at 40° C. is added using 2.5 ml of top agar/100 mg of callus. Callus clumps are broken into fragments of less than 2 mm diameter by repeated dispensing through a 10 ml pipet. Three ml aliquots of the callus suspension are plated onto fresh SM media and the plates incubated in the dark for 4 weeks at 27-28° C. After 4 weeks, transgenic callus events are identified, transferred to fresh SM plates and grown for an additional 2 weeks in the dark at 27-28° C.
Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm hyg B) for 2 weeks in the dark at 25° C. After 2 weeks the callus is transferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4% gelrite+50 ppm hyg B) and placed under cool white light (˜40 μEm−2s−1) with a 12 hr photoperiod at 25° C. and 30-40% humidity. After 2-4 weeks in the light, callus generally begins to organize, and form shoots. Shoots are removed from surrounding callus/media and gently transferred to RM3 media (½×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppm hygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) and incubation is continued using the same conditions as described in the previous step.
Plants are transferred from RM3 to 4″ pots containing Metro mix 350 after 2-3 weeks, when sufficient root and shoot growth has occurred. Plants are grown using a 12 hr/12 hr light/dark cycle using ˜30/18° C. day/night temperature regimen.
A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the 13 subunit of the seed storage polypeptide phaseolin from the bean Phaseolus vulgaris (Doyle, et al., (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of nucleic acids in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), SmaI, KpnI and XbaI. The entire cassette is flanked by Hind III sites.
The cDNA fragment of this gene can be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression cassette. Amplification is then performed and the isolated fragment is inserted into a pUC18 cassette carrying the seed expression cassette.
Soybean embryos can then be transformed with the expression cassette comprising nucleic acid sequences as disclosed herein. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
Soybean embryogenic suspension cultures can then be transformed by the method of particle gun bombardment (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene which can be used to facilitate soybean transformation is a nucleic acid composed of the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, a nucleic acid insert of interest and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the cassette carrying the marker gene.
To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media can be exchanged with fresh media and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue can be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line can be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression cassette pBT430. This cassette is a derivative of pET-3a (Rosenberg, et al., (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 is constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites is inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression cassette. Then, the Nde I site at the position of translation initiation is converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, is converted to 5′-CCCATGG in pBT430.
Plasmid DNA containing a cDNA can be appropriately digested to release a nucleic acid fragment encoding the polypeptide. This fragment can then be purified on a 1% NuSieve GTG low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters can be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The cassette pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared cassette pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier, et al., (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25° C. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the polypeptide concentration of the supernatant determined. One microgram of polypeptide from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for polypeptide bands migrating at the expected molecular weight.
Mature sunflower seeds are dehulled and surface sterilized for 30 min in a 20 percent Chlorox® bleach solution with the addition of two drops of Tween 20 per 50 ml of solution. Seeds are rinsed twice with distilled water.
Seeds are imbibed in distilled water for 60 min following the surface sterilization procedure. The cotyledons of each seed are broken off to produce a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed cut surface up on GBA medium consisting of Murashige and Skoog mineral elements, Shepard's vitamin additions, 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine, 0.25 mg/l indole-3-acetic acid, 0.1 mg/l gibberellic acid pH 5.6, and 8 g/l Phytagar.
Thirty to forty explants at a time are placed in a circle at the center of a 60×20 mm plate for microprojectile bombardment. Approximately 4.7 mg of 1.8 um tungsten microprojectiles are re-suspended in 25 ml of sterile TE buffer (10 mM Tris-Cl, 1 mM EDTA pH 8) and 1.5 m aliquots are used per bombardment. Each plate is bombarded twice through a 150 um Nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.
Plasmid is introduced into Agrobacterium tumefaciens via freeze thawing as described by Holsters, et al., (1978) Mol. Gen. Genet. 163:181-7. Bacteria used for transformation are grown overnight (28° C. and 100 RPM continuous agitation) in liquid YEP medium (10 g/l yeast extract, 10 g/l Bactopeptone and 5 g/l NaCl, pH 7.0) in the presence of kanamycin. The suspension is used when it reached an OD600 of 0.5 in an incubation medium comprised of 12.5 mM 2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH4Cl and 0.3 g/l MgSO4 at pH 5.7.
Freshly bombarded explants are placed in an Agrobacterium suspension, mixed and left undisturbed for 30 min. The explants are then transferred to GBA medium with co-cultivated cut surfaces down at 26° C. for 18-h days. After 3 days of co-cultivation, the explants are transferred to 374B media (GBA medium lacking growth regulators and having a reduced sucrose level of 1 percent) supplemented with 250 mg/l cefotaxime and 25, 50, 100 or 200 mg/l kanamycin sulfate. The explants are cultured for 2-5 weeks on the supplemented medium and then transferred to fresh 374B medium lacking kanamycin for 1-2 weeks of continued development. Explants with differentiating, antibiotic resistant areas of growth that had not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment. Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of selectable marker activity. Those shoots that fail to exhibit activity are discarded.
Marker positive shoots are grafted to Pioneer® hybrid in vitro-grown sunflower seedling rootstock. Surface sterilized seeds are germinated in 48-0 medium (half strength Murashige and Skoog salts, 0.5 percent sucrose, 0.3 percent gelrite pH 5.6) and grown under the conditions described for explant culture. The upper portion of each seedling is removed, a 1-cm vertical slice is made in each hypocotyl, and each transformed shoot is inserted into a cut. The entire area of each prepared graft is wrapped with parafilm to secure each shoot. Grafted plants are transferred to soil following 1 week of in vitro culture. Grafts in soil are maintained under high humidity conditions, followed by a slow acclimatization to a greenhouse environment.
Transformed sectors of T0 plants (parental generation) maturing in the greenhouse are identified by marker analysis of leaf extracts while transgenic seeds harvested from marker-positive T0 plants are identified by analysis of small portions of dry seed cotyledon. Leaf and seed assays of confirmed transgenics also are performed for segregation analysis of selfed progeny populations. Transgenic seeds from a number of events are cultivated under field conditions. The transgenics are grown in an isolation cage designed to minimize pollen dissemination by foraging insects.
DNA is isolated from immature leaves of greenhouse grown sunflower plants by a urea buffer extraction protocol. For each sample, leaf tissue (2 g) is frozen in liquid nitrogen, ground with a mortar and pestle and mixed into 6 ml of urea extraction buffer (50 mM TRIS-HCl pH 8.0, 7M urea, 0.31 M NaCl, 1 percent sarcosine). An equal volume of phenol:chloroform (1:1) is added and the mixture is shaken at room temperature for 15 minutes. After centrifugation (12,000×g, 15 min) the clarified supernatant is removed. DNA is precipitated by the addition of 1 ml of 4.4 M ammonium acetate (pH 5.2) and 7 ml isopropanol. The DNA is collected with a Pasteur pipette hook, allowed to dry for 15 minutes, and then resuspended in 0.5 M TE buffer as described in Ausubel, et al., CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, (1989). DNA (about 10 mg) is digested twice with 5-10 units of XbaI/mg DNA at 370 for 3 hours. Samples are subjected to electrophoresis on a one percent agarose gel and transferred onto nylon membranes for hybridization analysis as described by Southern, (1975) J. Mol. Biol. 98: 503-17.
Appropriate enzyme restriction digestion linearizes the binary plasmid. T-DNA junctions with plant genomic DNA at the right border are detected using restriction enzyme digestion followed by hybridization to a radiolabeled probe generated by random prime labeling of the marker fragment as described by Feinberg and Vogelstein, (1984) Anal. Biochem. 137: 266-7.
This application is a divisional of U.S. patent application Ser. No. 13/195,929 filed Aug. 2, 2011 and also claims priority to and the benefit of U.S. patent application Ser. No. 12/486,786 filed Jun. 18, 2009, now U.S. Pat. No. 8,017,831 issued on Sep. 13, 2011, U.S. patent application Ser. No. 11/614,098 filed Dec. 21, 2006, now U.S. Pat. No. 7,592,505 issued Sep. 22, 2009 and U.S. Provisional Patent Application No. 60/755,253, filed Dec. 30, 2005, all of which are herein incorporated in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60755253 | Dec 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13195929 | Aug 2011 | US |
| Child | 13614800 | US | |
| Parent | 12486786 | Jun 2009 | US |
| Child | 13195929 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11614098 | Dec 2006 | US |
| Child | 12486786 | US |
