Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0083—Miscellaneous (1.14.99)

Definitions

• the present invention is directed to nucleic acid sequences and constructs, and methods related thereto.
• Plant oils are used in a variety of applications. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired.
• oils and/or modified fatty acid compositions are through the genetic engineering of plants. However, it is necessary to identify the appropriate nucleic acid sequences which are capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like.
• fatty acids synthetase pathway fatty acid synthetase pathway
• the first committed step is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACP transacylase (ATA).
• acetyl-ACP acyl carrier protein
• Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a ⁇ -ketoacyl-ACP ( ⁇ -ketoacyl-ACP synthase), reduction of the keto-function to an alcohol ( ⁇ -ketoacyl-ACP reductase), dehydration to form an enoyl-ACP ( ⁇ -hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase).
• ⁇ -ketoacyl-ACP synthase I catalyzes elongation up to palmitoyl-ACP (C16:0)
• ⁇ -ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0).
• Common plant unsaturated fatty acids such as oleic, linoleic and a-linolenic acids found in storage triglycerides, originate from the desaturation of stearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastid ⁇ -9 desaturase (also often referred to as “stearoyl-ACP desaturase”).
• Molecular oxygen is required for desaturation in which reduced ferredoxin serves as an electron co-donor. Additional desaturation is effected sequentially by the actions of membrane bound ⁇ -12 desaturase and ⁇ -15 desaturase.
• nucleic acid sequences capable of producing a phenotypic result in FAS, desaturation and/or incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of an enzyme source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.
• nucleic acid targets and methods for modifying fatty acid compositions are needed.
• constructs and methods to produce a variety of ranges of different fatty acid compositions are needed.
• the present invention is generally directed to genomic desaturase polynucleotides, and in particular to genomic desaturase polynucleotides which encode enzymes that catalyze the insertion of a double bond into a fatty acyl moiety at the twelfth ( ⁇ 12 desaturase or fad2) or fifteenth ( ⁇ 5 desaturase or fad3) carbon position in a fatty acyl chain as counted from the carboxyl terminus. Further, the present invention provides isolated non-coding regions of such genomic polynucleotide sequences, particularly including the introns, and promoter regions.
• oligonucleotides include partial or complete sequences which are derived from ⁇ 12 and ⁇ 15 desaturase promoter and intron sequences. Although the sequences disclosed herein are obtained from soybean plants, it is contemplated that additional sequences can be derived from intron and promoter regions of desaturase genomic polynucleotide sequences which are homologous or have identity to the soybean desaturase sequences. Such additional desaturase sequences can be obtained using standard methods described below from a variety of plant sources, in particular oilseed crops.
• the invention is particularly directed to DNA constructs which include sequences which are derived from the intron or promoter regions of a genomic clone wherein said sequences are in a sense or antisense orientation in a DNA construct. These DNA constructs are then used to transform or transfect host cells to produce plants with modified levels of fatty acids, particularly modified levels of oleic, linoleic and linolenic acid.
• modified plant cells, plants, seeds and oils obtained by the expression of the ⁇ 12 and ⁇ 15 desaturase polynucleotides are also considered part of the invention. Further, it is contemplated to produce oil compositions with specific relative levels of each fatty acid.
• One preferred embodiment comprises at least about 80-85% oleic acid, no more than about 1-2% linoleic acid, and no more than about 1-3% linolenic acid; and a second preferred embodiment comprising at least about 50-75% oleic acid, at least about 10-30% linoleic acid, and no more than about 3% linolenic acid.
• the present invention is directed to genomic desaturase sequences, particularly the isolated non-coding sequences from genomic fatty acid desaturase nucleic acid sequences from host cell sources.
• a desaturase sequence of this invention includes any nucleic acid genomic sequence, including all non-coding regions, encoding amino acids from a source, such as a protein, polypeptide or peptide, obtainable from a cell source, which is capable of catalyzing the insertion of a double bond into a fatty acyl moiety in a plant host cell, i.e., in vivo, or in a plant cell-like environment, i.e. in vitro.
• genomic polynucleotide sequences encoding enzymes which add double bonds at the twelfth ( ⁇ 12 desaturase) and fifteenth ( ⁇ 15 desaturase) carbon positions in a fatty acyl chain as counted from the carboxyl terminus are provided.
• specific non-coding regions of such genomic sequences are provided herein.
• non-coding refers to sequences of polynucleotides that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to introns, promoter regions, and 5′ untranslated regions.
• intron refers to the normal sense of the term as meaning a segment of polynucleotides, usually DNA, that does not encode part or all of an expressed protein.
• exon refers to the normal sense of the term as meaning a segment of polynucleotides, usually DNA, that encodes part or all of an expressed protein.
• the term “intron” refers to gene regions that are transcribed into RNA molecules, but which are spliced out of the RNA before the RNA is translated into a protein.
• exon refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
• genomic ⁇ 12 desaturase and ⁇ 15 desaturase sequences and intron and promoter regions obtained from such sequences are provided herein.
• two ⁇ 12 desaturase genomic clones were identified and are set forth in SEQ ID NOs: 1 and 23.
• a single ⁇ 5 desaturase genomic clone was identified and is set forth in SEQ ID NO: 3.
• a single intron region was obtained from each of the ⁇ 12 desaturase genomic clones with the sequences provided in SEQ ID NOs: 2 and 24, respectively.
• the promoter region from each of the ⁇ 12 desaturase genomic clones are respectively included in SEQ ID NO: 1 (base pairs 1-1094) and SEQ ID NO: 23 (base pairs 1-1704).
• the ⁇ 15 desaturase included seven introns in the coding region (set forth as SEQ ID NOs: 4, 5, 6, 7, 8, 25 and 26). In addition, preliminary results suggest that there is an additional intron within the 5′ untranslated region.
• intron and promoter regions can be obtained from desaturase genomic polynucleotide sequences which are homologous or have identity to the soybean desaturase sequences.
• sequences can be obtained from other plant sources and particularly from oilseed crops. Such genomic sequences can be obtained using standard methods, certain of which are described below.
• the sequences of the present invention can be used to modify the fatty acid composition in a plant (see Example 3 and Table I).
• sense and antisense suppression can be used to obtain broad ranges in the levels of oleic, linoleic and linolenic acid.
• levels of oleic acid can range from about 26 to 80%
• levels of linoleic acid can range from about 2.97 to 49.92%
• levels of linolenic acid can range from about 3.38 to 8.81%.
• these are merely representative of the broad range that be can achieved.
• combinations of the sequences could be used to achieve additional fatty acid compositions. Certain compositions are preferred based on the intended use of the oil.
• One preferred composition includes at least about 50-75% oleic acid, at least about 10-30% linoleic acid and no more than about 3% linolenic acid.
• a particularly preferred embodiment includes at least about 60-70% oleic acid, at least about 15-20% linoleic acid and no more than about 3% linolenic acid.
• genes set forth herein utilize sense or antisense suppression to downregulate the gene of interest, it is contemplated that other means of modifying gene expression can be used.
• gene expression can be down regulated using DNA binding proteins which can be designed to specifically bind to the non-coding regions identified herein or that ribozymes can be designed to cleave such non-coding regions.
• DNA binding proteins which can be designed to specifically bind to the non-coding regions identified herein or that ribozymes can be designed to cleave such non-coding regions.
• other methods of downregulation of gene expression which are well known in the art are contemplated and can be used with the sequences of the present invention.
• a first aspect of the present invention relates to isolated desaturase polynucleotides.
• the polynucleotide sequences of the present invention include isolated polynucleotides that are obtainable from genomic nucleic acid sequences.
• the invention provides a polynucleotide sequence identical over its entire length to each sequence as set forth in the Sequence Listing.
• the polynucleotide includes non-coding sequences, including for example, but not limited to, non-coding 5′ and 3′ sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids.
• a marker sequence can be included to facilitate the purification of the fused polypeptide.
• Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.
• the invention also includes polynucleotides of the formula:
• R 1 and R 3 are any nucleic acid residue
• n is an integer between 1 and 3000, preferably between 1 and 1000
• R 2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 1-8, and 23-29.
• R 2 is oriented so that its 5′ end residue is at the left, bound to R 1 , and its 3′ end residue is at the right, bound to R 3 .
• Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
• polynucleotide of the invention that are at least 50%, 60%, or 70% identical over their entire length to a polynucleotide of the invention, and polynucleotides that are complementary to such polynucleotides. More preferable are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide of the invention and polynucleotides that are complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length are particularly preferred, those at least 95% identical are especially preferred. Further, those with at least 97% identity are highly preferred and those with at least 98% and 99% identity are particularly highly preferred, with those at least 99% being the most highly preferred.
• Preferred embodiments are polynucleotides that are obtained from genomic polynucleotide sequences and set forth in the Sequence Listing.
• the invention further relates to polynucleotides that hybridize to the above-described sequences.
• the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides.
• stringent conditions and “stringent hybridization conditions” mean that hybridization will generally occur if there is at least 95% and preferably at least 97% identity between the sequences.
• An example of stringent hybridization conditions is overnight incubation at 42° C.
• the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein.
• polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing.
• Such probes will generally comprise at least 15 bases.
• Preferably such probes will have at least 30 bases and can have at least 50 bases.
• Particularly preferred probes will have between 30 bases and 50 bases, inclusive.
• each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by screening using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe.
• a labeled oligonucleotide having a sequence complementary to that of a polynucleotide of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe.
• synthetic oligonucleotides are prepared which correspond to the desaturase promoter and intron sequences.
• screening of cDNA libraries in phage vectors is useful in such methods due to lower levels of background hybridization.
• a desaturase sequence obtainable from the use of nucleic acid probes will show 60-70% sequence identity between the target desaturase sequence and the encoding sequence used as a probe.
• lengthy sequences with as little as 50-60% sequence identity may also be obtained.
• the nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe.
• longer nucleic acid fragments are employed as probes (greater than about 100 bp)
• one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the sequences used as probe.
• Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding an desaturase enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related desaturase genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86:1934-1938.).
• PCR polymerase chain reactions
• Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.
• Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).
• the BLAST X program is publicly available from NCBI and other sources ( BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol., 215:403-410 (1990)).
• the well known Smith Waterman algorithm can also be used to determine identity.
• Parameters for polypeptide sequence comparison typically include the following:
• a program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis.
• the above parameters along with no penalty for end gap are the default parameters for peptide comparisons.
• Parameters for polynucleotide sequence comparison include the following:
• a program which can be used with these parameters is publicly available as the “gap” program from Genetics Computer Group, Madison Wis. The above parameters are the default parameters for nucleic acid comparisons.
• antibodies to the protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, such methods of preparing antibodies being well known to those in the art. Either monoclonal or polyclonal antibodies can be produced, although typically polyclonal antibodies are more useful for gene isolation.
• Western analysis may be conducted to determine that a related protein is present in a crude extract of the desired plant species, as determined by cross-reaction with the antibodies to the encoded proteins. When cross-reactivity is observed, genes encoding the related proteins are isolated by screening expression libraries representing the desired plant species.
• Expression libraries can be constructed in a variety of commercially available vectors, including lambda gt11, as described in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• the expression constructs generally comprise a promoter functional in a host plant cell operably linked to a nucleic acid sequence of the present invention and a transcriptional termination region functional in a host plant cell.
• One set of promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that yield high levels of expression in most plant organs.
• Enhanced or duplicated versions of the CaMV35S and FMV35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313:810-812; Rogers, U.S. Pat. No. 5,378, 619).
• nucleic acid sequences of the present invention from transcription initiation regions which are preferentially expressed in a plant seed tissue.
• seed preferential transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or from genes involved in fatty acid biosynthesis in oilseeds.
• promoters include the 5′regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean ⁇ ′ subunit of ⁇ -conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad. Sci., 83:8560-8564 (1986))) and oleosin.
• CTP chloroplast transit peptides
• PTP plastid transit peptides
• the expression construct will additionally contain a gene encoding a transit peptide to direct the gene of interest to the plastid.
• the chloroplast transit peptides may be derived from the gene of interest, or may be derived from a heterologous sequence having a CTP.
• Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478-481.
• the constructs may contain the entire genomic nucleic acid sequence or a particular non-coding region of such a sequence or a portion of such sequences. For example, where antisense inhibition of a given desaturase protein is desired, the entire sequence is not required. Furthermore, where desaturase sequences used in constructs are intended for use as probes, it may be advantageous to prepare constructs containing only a particular portion of a desaturase sequence, for example a sequence which encodes a highly conserved desaturase region.
• Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence.
• Transcript termination regions may be provided in plant expression constructs of this invention as well.
• Transcript termination regions may be provided by the DNA sequence encoding the desaturase or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region.
• the transcript termination region which is naturally associated with the transcript initiation region.
• any convenient transcript termination region which is capable of terminating transcription in a plant cell may be employed in the constructs of the present invention.
• constructs may be prepared to direct the expression of the desaturase sequences directly from the host plant cell plastid.
• constructs and methods are known in the art and are generally described, for example, in Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917 and in U.S. Pat. No. 5,693,507.
• a plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected, or transgenic.
• a transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a desaturase nucleic acid sequence.
• Plant expression or transcription constructs having a desaturase polynucleotide of the present invention as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is applicable to dicotyledons and monocotyledons species alike and will be readily applicable to new and/or improved transformation and regulation techniques.
• plant desaturase promoter and/or intron constructs are used in plants to produce plants or plant parts, including, but not limited to leaves, stems, roots, reproductive, and seed, with a modified fatty acid composition.
• polynucleotides of the present invention can be used in the preparation of constructs for use in a variety of host cells.
• Host for use in the present invention include, but are not limited to plant cells, bacterial cells, fungal cells (including yeast), insect cells, and mammalian cells.
• in vitro assays can be performed in insect cell cultures using baculovirus expression systems.
• baculovirus expression systems are known in the art and are described by Lee, et al. U.S. Pat. No. 5,348,886, the entirety of which is herein incorporated by reference.
• the method of transformation in obtaining such transgenic plants is not critical to the instant invention, and various methods of plant transformation are currently available. Furthermore, as newer methods become available to transform crops, they may also be directly applied hereunder. For example, many plant species naturally susceptible to Agrobacterium infection may be successfully transformed via tripartite or binary vector methods of Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation. In addition, techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
• telomere a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells.
• the gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
• a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
• complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
• one or more markers may be employed, where different conditions for selection are used for the different hosts.
• a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host.
• the Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host.
• the armed plasmid can give a mixture of normal plant cells and gall.
• the expression or transcription construct bordered by the T-DNA border region(s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al., ( Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0 120 515, which are incorporated herein by reference.
• a vector containing separate replication sequences one of which stabilizes the vector in E. coli, and the other in Agrobacterium.
• McBride and Summerfelt Plant Mol. Biol. (1990) 14:269-276
• the pRiHRI Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374
• origin of replication is utilized and provides for added stability of the plant expression vectors in host Agrobacterium cells.
• markers which allow for selection of transformed Agrobacterium and transformed plant cells.
• a number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like.
• the particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.
• explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.
• a second expression construct can be used in accordance with the present invention.
• the desaturase expression construct can be introduced into a host cell in conjunction with a second expression construct having a nucleotide sequence for a protein involved in fatty acid biosynthesis.
• the plant cells of this invention which contain multiple expression constructs.
• Any means for producing a plant comprising a construct having a DNA sequence encoding the expression construct of the present invention, and at least one other construct having another DNA sequence encoding an enzyme are encompassed by the present invention.
• the expression construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a single transformation vector or by using separate vectors, each of which express desired genes.
• the second construct can be introduced into a plant which has already been transformed with the desaturase expression construct, or alternatively, transformed plants, one expressing the desaturase construct and one expressing the second construct, can be crossed to bring the constructs together in the same plant.
• soybean fad 2-1A sequence was identified by screening a soybean genomic library using a soybean fad2-1 cDNA probe. Three putative soy fad 2-1 clones were identified and plaque purified. Two of the three soy fad 2-1 clones were ligated into pBluescript II KS+ (Stratagene) and sequenced. Both clones (14-1 and 11-12) were the same and matched the soy fad 2-1 cDNA exactly. The sequence of the entire fad2-1A clone is provided in SEQ ID NO: 1.
• fad2-1A genomic clone Prior to obtaining this full length clone, a portion of the fad2-1A genomic clone was PCR amplified using PCR primers designed from the 5′ untranslated sequence (Primer 12506, 5′-ATACAA GCCACTAGGCAT-3′, SEQ ID NO: 9) and within the cDNA (Primer 11698: 5′-GATTGGCCATGCAATGAGGGAAAAGG-3′, SEQ ID NO: 10. The resulting PCR product, which contained the fad2-1A intron, was cloned into the vector pCR 2.1 (Invitrogen) and sequenced.
• the soy fad 2-1A partial genomic clone (SEQ ID NO: 27) and its intron region (SEQ ID NO: 2) were identified by comparison to the soybean cDNA sequence using the Pustell comparison program in Macvector.
• the intron sequence begins after the ATG start codon, and is 420 bases long.
• a second fad2-1 gene family member was also identified and cloned, and is referred to herein as fad2-1B.
• the soy fad 2-1B partial genomic clone (SEQ ID NO: 23) (contains the promoter (base pairs 1-1704); 5′UTR (base pairs 1705-1782); intron#1 (base pairs 1786-2190); and a portion of the fad2-1B coding region (base pairs 1783-1785 and 2191-2463)) and its intron region (SEQ ID NO: 24) were identified by comparison to the soybean cDNA sequence using the Pustell comparison program in Macvector.
• the intron sequence begins after the ATG start codon and is 405 bases long.
• the partial soybean fad 3 genomic sequence was PCR amplified from soybean DNA using primers 10632, 5′-CUACUACUACUACTCGAGACAAAGCCTMTAGCCTATG-3′ (SEQ ID NO: 11), and 10633: 5′-CAUCAUCAUCAUGGATCCCATGTC TCTCTATGCAAG-3′ (SEQ ID NO: 12).
• the Expand Long Template PCR system (Boehringer Mannheim) was used according to the manufacturers directions. The resulting PCR products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced.
• the soy fad 3 partial genomic clone sequence and the intron regions were confirmed by comparisons to the soybean fad 3 cDNA sequence using the Pustell program in Macvector. From the identified partial genomic soybean fad3 sequence (SEQ ID NO: 3), seven introns were identified (SEQ ID NO: 4 (intron #1), SEQ ID NO: 5 (intron #2), SEQ ID NO: 6 (intron #3A), SEQ ID NO: 7 (intron #4), SEQ ID NO: 8 (intron #5), SEQ ID NO: 25 (intron #3B) and SEQ ID NO: 26 (intron #3C)).
• Intron #1 is 192 base pairs long and is located between positions 294 and 485
• intron #2 is 348 base pairs long and is located between positions 576 and 923
• intron #3A is 142 base pairs long and is located between positions 991 and 1132
• intron #3B is 98 base pairs long and is located between positions 1225 and 1322
• intron #3C is 115 base pairs long and is located between positions 1509 and 1623
• intron #4 is 1231 base pairs long and is located between positions 1705 and 2935
• intron #5 is 626 base pairs long and is located between positions 3074 and 3699.
• the soybean fad2-1A intron sequence was amplified via PCR using the fad2-1A partial genomic clone (SEQ ID NO: 27) as a template and primers 12701 (5′-ACGAATTCCTCGAGGTAAA TTAAATTGTGCCTGC-3′ (SEQ ID NO: 13)) and 12702 (5′-GCGAGATCTATCG ATCTGTGTCAAAGTATAAAC-3′ (SEQ ID NO: 14)).
• the resulting amplification products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced.
• the soyfad2-1A intron was then cloned into the expression cassette, pCGN3892, in sense and antisense orientations.
• the vector pCGN3892 contains the soybean 7S promoter and a pea RBCS 3′. Both gene fusions were then separately ligated into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter.
• the resulting expression constructs (PCGN5469 sense and pCGN5471 antisense) were used for transformation of soybean using biolistic methods described below.
• soybean fad2-1B intron sequence was amplified via PCR using the fad2-1B partial genomic clone (SEQ ID NO: 23) as a template and primers 13883 (5′-GCGATCGATGTATGATGCTAAATTAAATTGTGCCTG-3′ (SEQ ID NO: 30)) and 13876 (5′-GCGGAATTCCTGTGTCAAAGTATAAAGAAG-3′ (SEQ ID NO: 31)).
• the resulting amplification products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced.
• the soyfad2-1B intron was fused to the 3′ end of the soy fad 2-1A intron in plasmids pCGN5468 (contains the soybean 7S promoter fused to the soy fad2-1A intron (sense) and a pea RBCS 3′) or pCGN5470 (contains the soybean 7S promoter fused to the soy fad2-1A intron (antisense) and a pea RBCS 3′) in sense or antisense orientation respectively.
• the resulting intron combo fusions were then ligated separately into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter.
• the resulting expression constructs (pCGN5485, fad2-1A&B intron sense and pCGN5486, fad2-1A&B intron antisense) were used for transformation of soybean using biolistic methods described below.
• pCGN3892 contains the soybean 7S promoter and a pea RBCS 3′. These fusions were ligated into pCGN9372, a vector that contains the CP4 gene regulated by the FMV promoter for transformation into soybean.
• the soy fad3 Intron #3C and #4 were also PCR amplified from a second fad3 gene family member, herein referred to as fad3-1B.
• the soy fad3-1B introns #3C and #4 were PCR amplified from soybean DNA using the following primers, 5′ CATGCTTTCTGTGCTTCTC 3′ (SEQ ID NO: 32) and, 5′ GTTGATCCAACCATAGTCG 3′ (SEQ ID NO: 33).
• the PCR products were cloned into the vector pCR 2.1 (Invitrogen) and sequenced.
• the sequences for the soy fad3-1B introns #3C and #4 are provided in SEQ ID NOs: 28 and 29.
• Fatty acid compositions were analyzed from seed of soybean lines transformed with the intron expression constructs using gas chromatography.
• T2 pooled seed and T2 single seed oil compositions demonstrate that the mono and polyunsaturated fatty acid compositions were altered in the oil of seeds from transgenic soybean lines as compared to that of the seed from non-transformed soybean.
• Table I provides a summary of results which were obtained using the described constructs. These data clearly show that sense and antisense expression of the non-coding regions of the desaturase gene results in the modification of the fatty acid compositions.
• introns can be used to obtain a variety of lines with varying fatty acid compositions. Selections can be made from such lines depending on the desired relative fatty acid composition.

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