# Patent

## Title
Methods for increasing oleic acid content in seeds from transgenic plants containing a mutant delta 12 desaturase

## Abstract

The preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes are described. The invention permits alteration of plant lipid composition. Chimeric genes incorporating mutant delta-12 fatty acid desaturase nucleic acid fragments with suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.

## Background
This is a continuation of application Ser. No. 09/232,948 filed Jan. 19, 1999, now abandoned which is a continuation of application Ser. No. 08/728,025 filed Oct. 9, 1996, now abandoned.


FIELD OF THE INVENTION
The invention relates to the preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes to modify plant lipid composition. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.
BACKGROUND OF THE INVENTION
Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically adapted species is usually required to economically produce large amounts of the desired lipid.
Plant lipids find their major use as edible oils in the form of triacylglycerols. The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly-unsaturated fatty acids.
Many recent research efforts have examined the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, it was believed that mono-unsaturates, in contrast to saturates and poly-unsaturates, had no effect on serum cholesterol and coronary heart disease risk. Several recent human clinical studies suggest that diets high in mono-unsaturated fat and low in saturated fat may reduce the “bad” (low-density lipoprotein) cholesterol while maintaining the “good” (high-density lipoprotein) cholesterol (Mattson et al., Journal of Lipid Research(1985) 26:194–202).
A vegetable oil low in total saturates and high in mono-unsaturates would provide significant health benefits to consumers as well as economic benefits to oil processors. For specialized uses, high levels of poly-unsaturates can be desirable. Linoleate and linolenate are essential fatty acids in human diets, and an edible oil high in these fatty acids can be used for nutritional supplements, for example in baby foods.
The biosynthesis of the major plant lipids has been the focus of much research (Browse et al., Ann. Rev. Plant Physiol. Mol. Biol. (1991) 42:467–506). These studies show that, with the notable exception of the soluble stearoyl-acyl carrier protein desaturase, the controlling steps in the production of unsaturated fatty acids are largely catalyzed by membrane-associated fatty acid desaturases. Desaturation reactions occur in plastids and in the endoplasmic reticulum using a variety of substrates including galactolipids, sulfolipids, and phospholipids Genetic and physiological analyses of Arabidopsis thaliana nuclear mutants defective in various fatty acid desaturation reactions indicates that most of these reactions are catalyzed by enzymes encoded at single genetic loci in the plant. These investigations have demonstrated the role of delta-12 desaturase and delta-15 desaturase activities in the production of linoleate and linolenate from 2-oleoyl-phosphatidylcholine and 2-linoleoyl-phosphatidylcholine, respectively (Wang et al., Plant Physiol. Biochem. (1988) 26:777–792). Thus, modification of the activities of these enzymes represents an attractive target for altering the levels of lipid unsaturation by genetic engineering.
The cloning and characterization of wild-type delta-12 fatty acid desaturases has been reported (Okuley, et al., Plant Cell (1994) 6:147–158). However, there are no teachings concerning plants having seed-specific expression of mutant delta-12 or delta-15 fatty acid desaturase gene products. Furthermore, no methods have been described for altering the fatty acid composition of plants using nucleic aced constructs expressing a mutant delta-12 or a mutant delta-15 fatty acid desaturase.
SUMMARY OF THE INVENTION
Applicants have discovered a means to control the nature and levels of unsaturated fatty acids in plants. Nucleic acid fragments from cDNAs or genes encoding mutant fatty acid desaturases are used to create chimeric genes. The chimeric genes may be used to transform various plants to modify the fatty acid composition of the plant or the oil produced by the plant. The invention comprises nucleic acid constructs containing mutant microsomal delta-12 or mutant microsomal delta-15 fatty acid desaturase coding sequences, which are operably linked in sense orientation to at least one regulatory sequence. Such a construct is effective for altering fatty acid composition of seeds when the construct is introduced into a plant. In one embodiment, a mutant coding sequence for a delta-12 fatty acid desaturase comprises the mutation in the sequence of SEQ ID NO:3.
The invention further comprises seeds, plants and plant lines having a recombinant nucleic acid construct containing at least one regulatory sequence linked in sense orientation to a mutant delta-12 or mutant delta-15 fatty acid desaturase. The mutant chimeric gene preferentially is expressed in seeds and results in an altered fatty acid composition in seeds of such plants. A plant expressing a mutant delta-12 desaturase gene preferably has a reduced level of linoleic acid in seeds. A plant expressing a mutant delta-15 desaturase gene preferably has a reduced level of α-linolenic acid in seeds. If desired, a plant of the invention may express both a mutant delta-12 and a mutant delta-15 fatty acid desaturase, resulting in the reduction of both linoleic acid and α-linolenic acid in seeds.
Yet another embodiment of the invention involves a method of producing seed oil containing altered levels of unsaturated fatty acids comprising: (a) transforming a plant cell with a chimeric gene described above; (b) growing sexually mature plants from the transformed plant cells of step (a); (c) screening progeny seeds from the sexually mature plants of step (b) for the desired levels of unsaturated fatty acids, and (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of the unsaturated fatty acids. Preferred plant cells and oils are derived from soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax, oil palm, and corn. Preferred methods of transforming such plant cells would include the use of Ti and Ri plasmids of Agrobacterium, electroporation, and high-velocity ballistic bombardment.
Yet another aspect of the invention involves a method of producing seeds having altered fatty acid composition. The method comprises the step of introducing a recombinant nucleic acid construct into a plant (i.e., transforming a plant). The construct comprises one or more seed-specific regulatory sequences operably linked in sense orientation to a mutant delta-12 fatty acid desaturase gene or a mutant delta-15 fatty acid desaturase gene. After obtaining transgenic progeny, those transformed plants that produce seeds having an altered fatty acid composition are identified. Suitable plants for transformation include, for example, soybean, rapeseed, sunflower, safflower, castor bean and corn. Suitable methods of transforming such plants include, for example, Agrobacterium—mediated methods, electroporation, and microprojectile bombardment.
The invention also is embodied in a method of RFLP breeding to obtain altered levels of oleic acids in the seed oil of oil producing plant species. This method involves (a) making a cross between two varieties of oil producing plant species differing in the oleic acid trait; (b) making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross; and (c) hybridizing the Southern blot with the radiolabelled nucleic acid fragments encoding the mutant fatty acid desaturases or desaturase-related enzymes.
The invention is also embodied in a method of RFLP mapping that uses the isolated mutant microsomal delta-12 desaturase cDNA or related genomic fragments described herein.
Another embodiment of the instant invention is a method of genotyping plants containing either a mutant or wild-type form of the delta-12 desaturase gene by PCR amplification of genomic DNA using gene-specific primers. This method is capable of discriminating genes that differ by only one or a few nucleotides, thus affording a means for detecting plants containing the mutant delta-12 desaturase.
Another aspect of the invention comprises vegetable oil extracted from seeds of plants disclosed herein. Such a vegetable oil contains an altered fatty acid composition, e.g., a decreased level of α-linolenic acid, a decreased level of linoleic acid, or an increased level of oleic acid, based on total fatty acid composition.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description and the Sequence Descriptions which form a part of this application. The Sequence Descriptions contain the three letter codes for amino acids as defined in 37 C.F.R. 1.822 which are incorporated herein by reference.
SEQ ID NO:1 shows the 5′ to 3′ nucleotide sequence of 1464 base pairs of the Brassica napus cDNA which encodes the wild type D form of microsomal delta-12 desaturase in plasmid pCF2–165d.
SEQ ID NO:2 is the 384 amino acid protein sequence deduced from the open reading frame in SEQ ID NO:1.
SEQ ID NO:3 shows the 5′ to 3′ cDNA nucleotide sequence of a mutant D form of microsomal delta-12 fatty acid desaturase from Brassica napus IMC129 Nucleotides 1–3 are the initiation codon and nucleotides 1153–1155 are the termination codon.
SEQ ID NO:4 is the 384 amino acid protein sequence deduced from the open reading frame of SEQ ID NO 3.
SEQ ID NO:5 shows the 5′ to 3′ cDNA nucleotide sequence of the wild-type F form of microsomal delta-12 fatty acid desaturase in Brassica napus. Nucleotides 1–3 are the initiation codon and nucleotides 1153–1155 are the termination codon.
SEQ ID NO:6 is the 384 amino acid protein sequence deduced from the open reading frame of SEQ ID NO:5.
SEQ ID NO:7 shows the 5′ to 3′ cDNA nucleotide sequence of a mutant F form of microsomal delta-12 fatty acid desaturase from Brassica napus IMC Q508. Nucleotides 1–3 are the initiation codon and nucleotides 1153–1155 are the termination codon.
SEQ ID NO:8 is the 384 amino acid protein sequence deduced from the open reading frame of SEQ ID NO:7.
SEQ ID NO:9 is the upstream (5′) primer used for isolation of the D form of microsomal delta-12 fatty acid desaturase gene from Brassica napus. 
SEQ ID NO:10 is the downstream (3′) primer used for isolation of the D form of microsomal delta-12 fatty acid desaturase gene from Brassica napus. 
SEQ ID NO:11 is the upstream (5′) primer used for isolation of the F form of microsomal delta-12 fatty acid desaturas gene in Brassica napus. 
SEQ ID NO:12 is the downstream (3′) primer used for isolation of the F form of microsomal delta-12 fatty acid desaturase gene in Brassica napus. 
SEQ ID NO:13 is the upstream (5′) primer used for gene-specific detection of the wild type D form of microsomal delta-12 fatty acid desaturase gene in Brassica napus. 
SEQ ID NO:14 is the upstream (5′) primer used for gene-specific detection of the mutant D form of microsomal delta-12 fatty acid desaturase gene in Brassica napus. 
SEQ ID NO:15 is the modified upstream (5′) primer used for gene-specific detection of the wild type D form of microsomal delta-12 fatty acid desaturase gene in Brassica napus. 
SEQ ID NO:16 is the modified upstream (5′) primer used for gene-specific detection of the mutant D form of microsomal delta-12 fatty acid desaturase gene in Brassica napus. 
SEQ ID NO:17 is a 5 amino acid sequence in which at least one mutation in the conserved three amino acids of this motif renders the delta-12 desaturase gene product non-functional.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic drawing of plasmid pZPhMcFd2, showing restriction sites and relative position and orientation of the bean phaseolin promoter (5′ Phas), the IMC129 mutant microsomal delta-12 fatty acid desaturase D form coding sequence (MCFd2) and the bean phaseolin 3′ untranslated region (3′ Phas).
FIG. 2 is a schematic drawing of plasmid pIMC127, showing restriction sites and the relative positions and orientation of the napin promoter (5′ nap), the wild-type microsomal delta-12 fatty acid desaturase D form coding sequence (CanFd2) and the napin 3′ untranslated region (3′ Nap).
FIG. 3 shows the frequency distribution of seed oil linoleic acid (C18:2) content in transgenic Brassica T2 populations transformed with either a napin promoter linked in sense orientation to a wild-type microsomal delta-12 fatty acid desaturase D form coding sequence (WS127) or a phaseolin promoter linked to a mutant delta-12 fatty acid desaturase D form (WS201)
FIG. 4 shows the frequency distribution of seed oil linoleic acid content in transgenic Brassica T2 populations transformed with either a napin promoter linked in sense orientation to a mutant F form (WS140) delta-12 fatty acid desaturase coding sequence or a cruciferin promoter linked to a wild-type delta-12 fatty acid desaturase D form (WS135).



DETAILED DESCRIPTION OF THE INVENTION
Applicants have isolated nucleic acid fragments that encode mutant plant fatty acid desaturases and that are useful in modifying fatty acid composition in oil-producing species by genetic transformation.
Transfer of the nucleic acid fragments of the invention or a part thereof, along with suitable regulatory sequences that direct the transcription of their mRNA, into plants may result in production of decreased levels of unsaturated fatty acids in cellular lipids, including triacylglycerols.
The nucleic acid fragments of the invention can also be used as DNA diagnostic markers in plant genetic mapping and plant breeding programs.
The nucleic acid fragments of the invention or oligomers derived therefrom can also be used to isolate other related fatty acid desaturase genes using DNA, RNA, or a library of cloned nucleotide sequences from the same or different species by well known sequence-dependent protocols, including, for example, methods of nucleic acid hybridization and amplification by the polymerase chain reaction.
Definitions
In the context of this disclosure, a number of terms shall be used. Fatty acids are specified by the number of carbon atoms and the number and position of the double bond: the numbers before and after the colon refer to the chain length and the number of double bonds, respectively. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the “c” affix for the cis-configuration of the double bond. For example, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1, 6c), linoleic acid (18:2, 9c, 12c), γ-linolenic acid (18:3, 6c, 9c, 12c) and α-linolenic acid (18:3, 9c, 12c, 15c). Unless otherwise specified 18:1, 18:2 and 18:3 refer to oleic, linoleic and linolenic fatty acids. The term “fatty acid desaturase” used herein refers to an enzyme which catalyzes the breakage of a carbon-hydrogen bond and the introduction of a carbon—carbon double bond into a fatty acid molecule. The fatty acid may be free or esterified to another molecule including, but not limited to, acyl-carrier protein, coenzyme A, sterols and the glycerol moiety of glycerolipids. The term “glycerolipid desaturases” used here refers to a subset of the fatty acid desaturases that act on fatty acyl moieties esterified to a glycerol backbone. “Delta-12 desaturase” refers to a fatty acid desaturase that catalyzes the formation of a double bond between carbon positions 6 and 7 (numbered from the methyl end), (i.e., those that correspond to carbon positions 12 and 13 (numbered from the carbonyl carbon) of an 18 carbon-long fatty acyl chain. “Delta-15 desaturase”refers to a fatty acid desaturase that catalyzes the formation of a double bond between carbon positions 3 and 4 (numbered from the methyl end), (i.e., those that correspond to carbon positions 15 and 16 (numbered from the carbonyl carbon) of an 18 carbon-long fatty acyl chain. Examples of fatty acid desaturases include, but are not limited to, the microsomal delta-12 and delta-15 desaturases that act on phosphatidylcholine lipid substrates; the chloroplastic or plastid delta-12 and delta-15 desaturases that act on phosphatidyl glycerol and galactolipids; and other desaturases that act on such fatty acid substrates such as phospholipids, galactolipids, and sulfolipids. “Microsomal desaturase” refers to the cytoplasmic location of the enzyme, while “chloroplast desaturase” and “plastid desaturase” refer to the plastid location of the enzyme. These fatty acid desaturases may be found in a variety of organisms including, but not limited to, higher plants, diatoms, and various eukaryotic and prokaryotic microorganisms such as fungi and photosynthetic bacteria and algae. The term “homologous fatty acid desaturases” refers to fatty acid desaturases that catalyze the same desaturation on the same lipid substrate. Thus, microsomal delta-15desaturases, even from different plant species, are homologous fatty acid desaturases. The term “heterologous fatty acid desaturases” refers to fatty acid desaturases that catalyze desaturations at different positions and/or on different lipid substrates. Thus, for example, microsomal delta-12 and delta-15 desaturases, which act on phosphatidylcholine lipids, are heterologous fatty acid desaturases, even when from the same plant. Similarly, microsomal delta-15 desaturase, which acts on phosphatidylcholine lipids, and chloroplast delta-15 desaturase, which acts on galacto-lipids, are heterologous fatty acid desaturases, even when from the same plant. It should be noted that these fatty acid desaturases have never been isolated and characterized as proteins. Accordingly, the terms such as “delta-12 desaturase” and “delta-15 desaturase” are used as a convenience to describe the proteins encoded by nucleic acid fragments that have been isolated based on the phenotypic effects caused by their disruption. They do not imply any catalytic mechanism. For example, delta-12 desaturase refers to the enzyme that catalyzes the formation of a double bond between carbons 12 and 13 of an 18 carbon fatty acid irrespective of whether it “counts” the carbons from the methyl, carboxyl end, or the first double bond.
The term “nucleic acid” refers to a large molecule which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, a phosphate and either a purine or pyrimidine. A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of the information in DNA into proteins. A “genome” is the entire body of genetic material contained in each cell of an organism. The term “nucleotide sequence” refers to the sequence of DNA or RNA polymers, which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The term “oligomer” refers to short nucleotide sequences, usually up to 100 bases long.
From time to time, the term “FAD2” may be used herein as a shorthand notation for a nucleotide sequence encoding a wild type microsomal delta-12 fatty acid desaturase enzyme, and the term “fad2” may be used herein as a shorthand notation for a nucleotide sequence encoding a mutant form of a microsomal delta-12 fatty acid desaturase enzyme.
As used herein, the term “homologous to” refers to the relatedness between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA—DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.); or by the comparison of sequence similarity between two nucleic acids or proteins, such as by the method of Needleman et al. (J. Mol. Biol. (1970) 48:443–453). As used herein, “essentially similar” refers to DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alteration in the gene sequence which reflect the degeneracy of the genetic code, or which results in the production of a chemically equivalent amino acid at a given site, are contemplated; thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another hydrophobic amino acid residue such as glycine, valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding) and following (3′ non-coding) the coding region. “Fatty acid desaturase gene” refers to a nucleic acid fragment that expresses a protein with fatty acid desaturase activity. “Native” gene refers to an isolated gene with its own regulatory sequences as found in nature. “Chimeric gene” refers to a gene that comprises heterogeneous regulatory and coding sequences not found in nature. “Endogenous” gene refers to the native gene normally found in its natural location in the genome and is not isolated. A “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer. “Pseudo-gene” refers to a genomic nucleotide sequence that does not encode a functional enzyme. “Mutant gene” refers to a gene comprising one or more nucleotides that have been altered when compared to the wild-type nucleotide sequence, resulting in a change to the amino acid sequence and functional properti s of the encoded protein.
“Coding sequence” refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences. It may constitute an “uninterrupted coding sequence”, i.e., lacking an intron or it may include one or more introns bounded by appropriate splice junctions. An “intron” is a nucleotide sequence that is transcribed in the primary transcript but that is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
“Initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). “Open reading frame” refers to the coding sequence uninterrup-ted by introns between initiation and termination codons that encodes an amino acid sequence.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. “Ribozyme” refers to a catalytic RNA and includes sequence-specific endoribonucleases.
As used herein, “suitable regulatory sequences” refer to nucleotide sequences in native or chimeric genes that are located upstream (5′), within, and/or downstream (3′) to the nucleic acid fragments of the invention, which control the expression of the nucleic acid fragments of the invention. The term “expression”, as used herein, refers to the transcription and stable accumulation of the sense (mRNA) derived from the nucleic acid fragment(s) of the invention that, in conjunction with the protein apparatus of the cell, results in altered levels of the fatty acid desaturase(s). Expression or overexpression of the gene involves transcription of the gene and translation of the mRNA into precursor or mature fatty acid desaturase proteins. “Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
“Promoter” refers to a DNA sequence in a gene, usually upstream (5′) to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. Promoters may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. It may also contain enhancer elements. An “enhancer” is a DNA sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter. “Constitutive promoters” refers to those that direct gene expression in all tissues and at all times. “Tissue-specific” or “development-specific” promoters as referred to herein are those that direct gene expression almost exclusively in specific tissues, such as leaves or seeds, or at specific development stages in a tissue, such as in early or late embryogenesis, respectively.
The “3′ non-coding sequences” refers to the DNA sequence portion of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
“Transformation” herein refers to the transfer of a foreign gene into the genome of a host organism and its genetically stable inheritance. “Restriction fragment length polymorphism” (RFLP) refers to different sized restriction fragment lengths due to altered nucleotide sequences in or around variant forms of genes. “Molecular breeding” refers to the use of DNA-based diagnostics, such as RFLP, RAPDs, and PCR in breeding. “Fertile” refers to plants that are able to propagate sexually.
“Plants” refer to photosynthetic organisms, both eukaryotic and prokaryotic, whereas the term “Higher plants” refers to eukaryotic plants. “Oil-producing species” herein refers to plant species which produce and store triacylglycerol in specific organs, primarily in seeds. Such species include soybean (Glycine max), rapeseed and canola (including Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), (castor (Ricinus communis)) and peanut (Arachis hypogaea). The group also includes non-agronomic species which are useful in developing appropriate expression vectors such as tobacco, rapid cycling Brassica species, and Arabidopsis thaliana, and wild species which may be a source of unique fatty acids.
“Progeny” includes descendants of a particular plant or plant line, e.g., seeds and plants of F1, F2, F3, and subsequent generations, or seeds and plants of backcrossed populations BC1, BC2, BC3 and subsequent generations. “Sequence-dependent protocols” refer to techniques that rely on a nucleotide sequence for their utility. Examples of sequence-dependent protocols include, but are not limited to, the methods of nucleic acid and oligomer hybridization and methods of DNA and RNA amplification such as are exemplified in various uses of the polymerase chain reaction (PCR).
Various solutions used in the experimental manipulations are referred to by their common names such as “SSC”, “SSPE”, “Denhardt's solution”, etc. The composition of these solutions may be found by reference to Appendix B of Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).
Availability and Relatedness of Wild-Type Microsomal Delta-12 and Delta-15 Fatty Acid Desaturases
U.S. patent application Ser. No. 08/262,401, incorporated herein by reference, describes the isolation and characterization of cDNAs encoding wild-type microsomal delta-12 fatty acid desaturases from a number of plant species, including Arabidopsis thaliana, Brassica napus, Glycine max, Zea mays and Castor bean. Moreover, that application demonstrates successful alteration of fatty acid content of oils from seeds obtained from transgenic plants expressing sense or antisense mRNAs encoding microsomal delta-12 fatty acid desaturases.
Alignments of protein sequences of plant microsomal fatty acid delta-12 desaturases and plant delta-15 desaturases [microsomal and plastid delta-15 desaturases from Arabidopsis and Brassica napus, WO 9311245] allows identification of amino acid sequences conserved between the different desaturases (Table 1).







TABLE 1







Amino Acid Sequence Conserved Between


Plant Microsomal Delta-12 Desaturases and Microsomal and


Plastid Delta-15 Desaturases












Conserved AA






Positions in
Consensus
Consensus



SEQ ID NO:2
Conserved AA
Conserved AA



of USSN
Sequence in
Sequence in
Consensus


Region
08/262,401
Δ12 Desaturases
Δ15 Desaturases
AA Sequence





A
39–44
AIPPHC
AIPKHC
AIP(P/K)HC




(SEQ ID NO:18)
(SEQ ID NO:19)
(SEQ ID NO:20)


B
86–90
WP(L/I)YW
WPLYW
WP(L/I)YW




(SEQ ID NO:21)
(SEQ ID NO:22)
(SEQ ID NO:21)


C
104–109
AHECGH
GHDCGH
(A/G)H(D/E)CGH




(SEQ ID NO:23)
(SEQ ID NO:24)
(SEQ ID NO:25)


D
130–134
LLVPY
ILVPY
(L/I)LVPY




(SEQ ID NO:26)
(SEQ ID NO:27)
(SEQ ID NO:28)


E
137–142
WKYSHR
WRISHR
W(K/R)(Y/I)SHR




(SEQ ID NO:29)
(SEQ ID NO:30)
(SEQ ID NO:31)


F
140–145
SHRRHH
SHRTHH
SHR(R/T)HH




(SEQ ID NO:32)
(SEQ ID NO:33)
(SEQ ID NO:34)


G
269–274
ITYLQ
VTYLH
(I/V)TYL(Q/H)




(SEQ ID NO:35)
(SEQ ID NO:36)
(SEQ ID NO:37)


H
279–282
LPHY
LPWY
LP(H/W)Y




(SEQ ID NO:38)
(SEQ ID NO:39)
(SEQ ID NO:40)


I
289–294
WL(R/K)GAL
YLRGGL
(W/Y)L(R/K)G(A/G)L




(SEQ ID NO:41)
(SEQ ID NO:42)
(SEQ ID NO:43)


J
296–302
TVDRDYG
TLDRDYG
T(V/L)DRDYG




(SEQ ID NO:44)
(SEQ ID NO:45)
(SEQ ID NO:46)


K
314–321
THVAHHLF
THVIHHLF
THV(A/I)HHLF




(SEQ ID NO:47)
(SEQ ID NO:48)
(SEQ ID NO:49)


L
318–327
HHLFSTMPHY
HHLFPQIPHY
HHLF(S/P)(T/Q)(I/M)PHY




(SEQ ID NO:50)
(SEQ ID NO:51)
(SEQ ID NO:52)









Table 1 shows twelve regions of conserved amino acid sequences, designated A–L (column 1), whose positions in SEQ ID NO:2 of U.S. Ser. No. 08/262,401 are shown in column 2. The consensus sequences for these regions in plant delta-12 fatty acid desaturases and plant delta-15 fatty acid desaturases are shown in columns 3 and 4, respectively; amino acids are shown by standard abbreviations, the underlined amino acids are conserved between the delta-12 and the delta-15 desaturases, and amino acids in brackets represent substitutions found at that position. The consensus sequence of these regions are shown in column 5. These short conserved amino acids and their relative positions further confirm that the isolated isolated cDNAs encode a fatty acid desaturase.
Inhibition of Plant Target Genes By Dominant Negative Suppression
In one embodiment, transgenic plants according to the invention contain an introduced nucleic acid construct that comprises at least a portion of a mutant delta-12 or delta-15 desaturase coding sequence. Surprisingly, a construct comprising a mutant delta-12 desaturase or delta-15 desaturase coding sequence, operably linked in sense orientation to one or more regulatory sequences, has been found to inhibit the corresponding endogenous fatty acid desaturase activity in plants which contain such a construct. This phenomenon has been termed dominant negative suppression.
The basis for the phenomenon of dominant negative suppression is not understood. One possible explanation is that the delta-12 desaturase gene product exists as a dimer in vivo. If so, a dimer consisting of the mutant gene product and the wild-type gene product may be non-functional. Regardless of the actual mechanism by which dominant negative suppression operates, the observation that transformation of plants with a mutant delta-12 desaturase gene results in a large proportion of the transgenic progeny having endogenous wild-type enzyme activity inhibited by expression of the mutant gene can be used to advantage. For example, the phenomenon of dominant negative suppression can be used to alter plant desaturase enzyme activity in a tissue-specific manner. The phenomenon may also allow transformation experiments to be carried out in which a higher proportion of the resulting transgenic plants have a desired altered fatty acid profile and allow transgenic plants having desired fatty acid profiles to be more readily obtained.
Preferred constructs contain, in addition, at least one regulatory sequence operably linked in the sense orientation to the mutant coding sequence. Regulatory sequences typically do not themselves code for a gene product. Instead, regulatory sequences affect the expression level of the mutant coding sequence.
In preferred embodiments, regulatory sequences for dominant negative suppression are tissue-specific, i.e., the mutant desaturase gene product is preferentially expr ssed in certain plant tissues and expressed at low levels or not at all in the remaining tissues of the plant. Suitable tissue-specific regulatory sequences include those that permit expression preferentially in developing seeds. Seed-specific regulatory sequences preferably stimulate or induce levels of mutant desaturase gene product expression that coincide with the levels of wild-type desaturase gene product expression.
Dominant negative suppression plants according to the invention preferably yield seeds containing an altered fatty acid profile. For example, oil obtained from seeds of such plants may have from about 69% to about 90% oleic acid, based on the total fatty acid composition of the seed. Such oil preferably has from about 74% to about 90% oleic acid, more preferably from about 80% to about 90% oleic acid. In some embodiments, oil extracted from seeds produced by plants of the invention may have from about 3% to about 5% saturated fatty acids, based on total fatty acid composition of the seeds. In some embodiments, oil extracted from seeds of the invention may have from about 1% to about 10% linoleic acid, or from about 1% to about 10% α-linolenic acid.
After a recombinant nucleic acid construct, comprising a mutant microsomal delta-12 fatty acid desaturase coding sequence operably linked in the sense orientation to one or more regulatory sequences, is introduced into a plant, seeds of transgenic plants are grown and either selfed or outcrossed. Progeny are analyzed to identify those individuals having endogenous wild-type delta-12 fatty acid desaturase activity inhibited by dominant negative suppression as discussed above.
Method similar to those described above are used to make delta-15 desaturase dominant negative suppression constructs, comprising a mutant delta-15 desaturase gene operably linked to at least one regulatory sequence. Transformation of a plant with such a construct will result in dominant negative suppression of endogenous delta-15 desaturase activity in transgenic progeny and in a decreased level of α-linolenic acid in homozygous dominant suppression lines. Such lines will have from about <1% to about 10% α-linolenic acid, preferably from about <1% to about 5%, based on total seed fatty acid composition.
In one embodiment of the invention, a plant contains a mutant delta-12 fatty acid desaturase and a mutant delta-15 fatty acid desaturase, both of which are expressed preferentially in seeds. Such a plant can be produced from the cross of single mutant plants, followed by outcrossing or selfing in order to obtain progeny seeds carrying both mutant chimeric genes. Progeny seeds are screened in order to identify those seeds carrying both mutant genes. Alternatively, seed-specific defects in delta-12 desaturase and delta-15 desaturase may be introduced into a wild-type plant by transformation, simultaneously or sequentially, with one or more dominant negative suppression constructs for delta-12 desaturase and delta-15 desaturase, each driven by suitable regulatory sequences. Other similar methods to construct double mutant plants will be recognized by those of skill in the art.
Double mutant plants can have more extreme fatty acid profiles in seeds than the single mutant plants, e.g., the double mutant phenotype can result in significantly lower levels of α-linolenic acid in seeds than the single mutant delta-15 desaturase plant phenotype. Thus, by combining seed-specific inhibition of microsomal delta-12 desaturase with seed-specific inhibition of microsomal delta-15 desaturase, one can obtain levels of seed α-linolenic acid that are as low or lower than those in a single mutant without adversely affecting desirable properties. The decreased levels of α-linolenic acid in the double homozygotes may be associated with increased levels of oleic acid and decreased levels of saturates and linoleic acid.
Selection of Hosts, Promoters and Enhancers
A preferred class of heterologous hosts for the expression of the nucleic acid fragments of the invention are eukaryotic hosts, particularly the cells of higher plants. Particularly preferred among the higher plants are the oil-producing species, such as soybean (Glycine max), rapeseed (including Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), and peanut (Arachis hypogaea).
Expression in plants will use regulatory sequences functional in such plants. The expression of foreign genes in plants is well-established (De Blaere et al., Meth. Enzymol. (1987) 153:277–291). The source of the promoter chosen to drive the expression of the fragments of the invention is not critical provided it has sufficient transcriptional activity to accomplish the invention by increasing or decreasing, respectively, the level of translatable mRNA for the fatty acid desaturases in the desired host tissue. Preferred promoters include (a) strong constitutive plant promoters, such as those directing the 19S and 35S transcripts in cauliflower mosaic virus (bdell et al., Nature (1985) 313:810–812; Hull et al., Virology (1987) 86:482–493),(b) tissue- or developmentally-specific promoters, and (c) other transcriptional promoter systems engineered in plants, such as those using bacteriophage T7 RNA polymerase promoter sequences to express foreign genes. Examples of tissue-specific promoters are the light-inducible promoter of the small subunit of ribulose 1,5-bis-phosphate carboxylase (if expression is desired in photosynthetic tissues), the maize zein protein promoter (Matzke et al., EMBO J. (1984) 3:1525–1532), and the chlorophyll a/b binding protein promoter (Lampa et al., Nature (1986) 316:750–752).
Particularly preferred promoters are those that allow seed-specific expression. This may be especially useful since seeds are the primary source of vegetable oils and also since seed-specific expression will avoid any potential deleterious effect in non-seed tissues. Examples of seed-specific promoters include, but are not limited to, the promoters of seed storage proteins, which can represent up to 90% of total seed protein in many plants. The seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly tissue-specific and stage-specific manner (Higgins et al., Ann. Rev. Plant Physiol. (1984) 35:191–221; Goldberg et al., Cell (1989) 56:149–160). Moreover, different seed storage proteins may be expressed at different stages of seed development.
Expression of seed-specific genes has been studied in great detail (See reviews by Goldberg et al., Cell (1989) 56:149–160 and Higgins et al., Ann. Rev. Plant Physiol. (1984) 35:191–221). There are currently numerous examples of seed-specific expression of seed storage protein genes in transgenic dicotyledonous plants. These include genes from dicotyledonous plants for bean β-phaseolin (Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA (1985) 82:3320–3324; Hoffman et al., Plant Mol. Biol. (1988) 11:717–729), bean lectin (Voelker et al., EMBO J. (1987) 6:3571–3577), soybean lectin (Okamuro et al., Proc. Natl. Acad. Sci. USA (1986) 83:8240–8244), soybean Kunitz trypsin inhibitor (Perez-Grau et al., Plant Cell (1989) 1:095–1109), soybean β-conglycinin (Beachy et al., EMBO J. (1985) 4:3047–3053; pea vicilin (Higgins et al., Plant Mol. Biol. (1988) 11:683–695), pea convicilin (Newbigin et al., Planta (1990) 180:461–470), pea legumin (Shirsat et al., Mol. Gen. Genetics (1989) 215:326–331); rapeseed napin (Radke et al., Theor. Appl. Genet. (1988) 75:685–694) as well as genes from monocotyledonous plants such as for maize 15 kD zein (Hoffman et al., EMBO J. (1987) 6:3213–3221), maize 18 kD oleosin (Lee et al., Proc. Natl. Acad. Sci. USA (1991) 888:6181–6185), barley β-hordein (Marris et al., Plant Mol. Biol. (1988) 10:359–366) and wheat glutenin (Colot et al., EMBO J. (1987) 6:3559–3564). Moreover, promoters of seed-specific genes operably linked to heterologous coding sequences in chimeric gene constructs also maintain their temporal and spatial expression pattern in transgenic plants. Such examples include use of Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and B. napus seeds (Vandekerckhove et al., Bio/Technology(1989) 7:929–932), bean lectin and bean β-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. (1989) 63:47–57), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J. (1987) 6:3559–3564).
Of particular use in the expression of the nucleic acid fragment of the invention will be the heterologous promoters from several soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor (Jofuku et al., Plant Cell (1989) 1:1079–1093; glycinin (Nielson et al., Plant Cell (1989) 1:313–328), and β-conglycinin (Harada et al., Plant Cell (1989) 1:415–425). Promoters of genes for α- and β-subunits of soybean β-conglycinin storage protein will be particularly useful in expressing the mRNA or the antisense RNA in the cotyledons at mid- to late-stages of seed development (Beachy et al., EMBO J. (1985) 4:3047–3053) in transgenic plants. This is because there is very little position effect on their expression in transgenic seeds, and the two promoters show different temporal regulation. The promoter for the α-subunit gene is expressed a few days before that for the β-subunit gene. This is important for transforming rapeseed where oil biosynthesis begins about a week before seed storage protein synthesis (Murphy et al., J. Plant Physiol. (1989) 135:63–69).
Also of particular use will be promoters of genes expressed during early embryogenesis and oil biosynthesis. The native regulatory sequences, including the native promoters, of the fatty acid desaturase genes expressing the nucleic acid fragments of the invention can be used following their isolation by those skilled in the art. Heterologous promoters from other genes involved in seed oil biosynthesis, such as those for B. napus isocitrate lyase and malate synthase (Comai et al., Plant Cell (1989) 1:293–300), delta-9 desaturase from safflower (Thompson et al. Proc. Natl. Acad. Sci. USA (1991) 88:2578–2582) and castor (Shanklin et al., Proc. Natl. Acad. Sci. USA (1991) 88:2510–2514), acyl carrier protein (ACP) from Arabidopsis(Post-Beittenmiller et al., Nucl. Acids Res. (1989) 17:1777), B. napus (Safford et al., Eur. J. Biochem. (1988) 174:287–295), and B. campestris (Rose et al., Nucl. Acids Res. (1987) 15:7197), β-ketoacyl-ACP synthetase from barley (Siggaard-Andersen et al., Proc. Natl. Acad. Sci. USA (1991) 88:4114–4118), and oleosin from Zea mays (Lee et al., Proc. Natl. Acad. Sci. USA (1991) 88:6181–6185), soybean (Genbank Accession No: X60773) and B. napus (Lee et al., Plant Physiol. (1991) 96:1395–1397) will be of use. If the sequence of the corresponding genes is not disclosed or their promoter region is not identified, one skilled in the art can use the published sequence to isolate the corresponding gene and a fragment thereof containing the promoter. The partial protein sequences for the relatively-abundant enoyl-ACP reductase and acetyl-CoA carboxylase are also published (Slabas et al., Biochim. Biophys. Acta (1987) 877:271–280; Cottingham et al., Biochim. Biophys. Acta (1988) 954:201–207) and one skilled in the art can use these sequences to isolate the corresponding seed genes with their promoters. Similarly, the fragments of the present invention encoding fatty acid desaturases can be used to obtain promoter regions of the corresponding genes for use in expressing chimeric genes.
Attaining the proper level of expression of the nucleic acid fragments of the invention may require the use of different chimeric genes utilizing different promoters. Such chimeric genes can be transferred into host plants either together in a single expression vector or sequentially using more than one vector.
It is envisioned that the introduction of enhancers or enhancer-like elements into the promoter regions of either the native or chimeric nucleic acid fragments of the invention will result in increased expression to accomplish the invention. This would include viral enhancers such as that found in the 35S promoter (Odell et al., Plant Mol. Biol. (1988) 10:263–272), enhancers from the opine genes (Fromm et al., Plant Cell (1989) 1:977–984), or enhancers from any other source that result in increased transcription when placed into a promoter operably linked to a nucleic acid fragment of the invention.
Of particular importance is the DNA sequence element isolated from the gene for the α-subunit of β-conglycinin that can confer 40-fold seed-specific enhancement to a constitutive promoter (Chen et al., Dev. Genet. (1989) 10:112–122). One skilled in the art can readily isolate this element and insert it within the promoter region of any gene in order to obtain seed-specific enhanced expression with the promoter in transgenic plants. Insertion of such an element in any seed-specific gene that is expressed at different times than the β-conglycinin gene will result in expression in transgenic plants for a longer period during seed development.
The invention can also be accomplished by a variety of other methods to obtain the desired end. In one form, the invention is based on modifying plants to produce increased levels of mutant fatty acid desaturases by virtue of introducing more than one copy of the foreign gene containing the nucleic acid fragments of the invention. In some cases, the desired level of polyunsaturated fatty acids may require introduction of foreign genes for more than one kind of mutant fatty acid desaturase.
Any 3′ non-coding region capable of providing a polyadenylation signal and other regulatory sequences that may be required for the proper expression of the nucleic acid fragments of the invention can be used to accomplish the invention. This would include 3′ ends of the native fatty acid desaturase(s), viral genes such as from the 35S or the 19S cauliflower mosaic virus transcripts, from the opine synthesis genes, ribulose 1,5-bisphosphate carboxylase, or chlorophyll a/b binding protein. There are numerous examples in the art that teach the usefulness of different 3′ non-coding regions.
Transformation Methods
Various methods of transforming cells of higher plants according to the present invention are available to those skilled in the art (see EPO Pub. 0 295 959 A2 and 0 318 341 A1). Such methods include those based on transformation vectors utilizing the Ti and Ri plasmids of Agrobacterium spp. It is particularly preferred to use the binary type of these vectors. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants (Sukhapinda et al., Plant Mol. Biol. (1987) 8:209–216; Potrykus, Mol. Gen. Genet. (1985) 199:183). Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EPO Pub. 0 295 959 A2), techniques of electroporation (Fromm et al., Nature (1986) (London) 319:791) or high-velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline et al., Nature (1987) (London) 327:70). Once transformed, the cells can be regenerated by those skilled in the art.
Of particular relevance are the recently described methods to transform foreign genes into commercially important crops, such as rapeseed (De Block et al., Plant Physiol. (1989) 91:694–701), sunflower (Everett et al., Bio/Technology (1987) 5:1201), and soybean (Christou et al., Proc. Natl. Acad. Sci USA (1989) 86:7500–7504.
Application to Plant Breeding
The use of restriction fragment length polymorphism (RFLP) markers in plant breeding has been well-documented in the art (Tanksley et al., Bio/Technology (1989) 7:257–264). Thus, the nucleic acid fragments of the invention can be used as molecular markers for traits associated with mutant fatty acid desaturases. These traits will include altered levels of unsaturated fatty acids. The nucleic acid fragment of the invention can also be used to isolate the fatty acid desaturase gene from other mutant plants with altered levels of unsaturated fatty acids. Sequencing of these genes will reveal nucleotide differences that cause the alteration in levels of unsaturated fatty acids. Oligonucleotides designed around these differences may also be used in plant breeding as diagnostic markers to follow fatty acid variation. In one embodiment, oligonucleotides based on differences betwen wt and mutant Δ12 des may be used as molecular markers in breeding canola lines with variant oil profiles.
EXAMPLES
The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference herein.
Example 1
Sequences of Mutant Delta-12 Fatty Acid Desaturases from B. Napus 
Primers specific for the FAD2 structural gene were used to clone the entire open reading frame (ORF) of the D and F forms of the gene by reverse transcription-polymerase chain reaction (RT-PCR). The sequences of the primers used for isolation of the D form ORF of B. napus FAD2 gene are








5′-CATGGGTGCAGGTGGAAGAATGC-3′
(SEQ ID NO: 9); and


 


5′-GTTTCTTCTTTGCTTCATAAC-3′
(SEQ ID NO: 10).






The sequences of the primers used to clone the F form ORF of B. napus FAD2 gene are









5′-CATGGGTGCAGGTGGAAGAATGC3′
(SEQ ID NO: 11); and


 


5′-TCTTTCACCATCATCATATC-3′
(SEQ ID NO: 12).






RNA from seeds of three lines, IMC129, Q508 and Westar, was isolated by an acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987; Analytical Biochemistry 162, 156–159, 1987). The total RNA was used as a template for reverse transcription and PCR amplification by RNA PCR kit (Perkin Elm r). The RT-PCR amplified fragments were cloned into pGEM-T vector (Promega), and then used for nucleotide sequence determination. The DNA sequence of each gene from each line was determined from both strands by dideoxy sequencing by Sanger et al. (Proc Natl Acad Sci USA 74, 5463–5467).
The D gene of IMC129 contained a G to A transversion at nucleotide 316 (from the translation initiation codon) of the D gene in IMC 129, compared to the sequence of Westar. The transversion changes the codon at this position from GAG to AAG and results in a substitution of lysine for glutamic acid. The same base change was also detected in IMC129 when RNA from leaf tissue was used as template. The G to A mutation at nucleotide 316 was confirmed by sequencing several independent clones containing fragments amplified directly from genomic DNA of IMC129. These results eliminated the possibility of a rare mutation introduced during reverse transcription and PCR in the RT-PCR protocol. The mutation in the D form of delta-12 desaturase in IMC129 mapped to a conserved region of cloned delta-12 and delta-15 membrane bound desaturases (Table 5).
The sequence of the F form of delta-12 desaturase in IMC129 was the same as the F form of delta-12 desaturase in Westar.
For Q508, the sequence of the D form of delta-12 desaturase was the same as the D form of the IMC129 gene. This was expected, as Q508 was derived by mutagenesis of IMC129.
Sequence analysis of the Q508 F form of delta-12 desaturase revealed a T to A transition at nucleotide 515, compared to the wild-type Westar sequence. This mutation results in a change from a CTC codon to a CAC codon, substituting a histidine residue for the wild-type leucine residue.







TABLE 2







Alignment of Amino Acid Sequences of Cloned Canola


Membrane Bound-Desaturases











Desaturase Gene
Sequencea
Positionb















Canola-FAD2-D
HECGH (SEQ ID NO:53)
110



Canola-FAD2-F
HECGH (SEQ ID NO:53)
110



Canola-FAD6c
HDCAH (SEQ ID NO:54)
171



Canola-FAD3d
HDCGH (SEQ ID NO:55)
97



Canola-FAD7e
HDCGH (SEQ ID NO:55)
126







aOne letter amino acid code; conservative substitutions are underlined



bPosition in gene product of first amino acid



cFAD6 = Plastid delta-12



dFAD3 = Microsomal delta-15



eFAD7 = Plastid delta-15






Example 2
Gene-Specific Oligonucleotide Markers for the Mutant and Wild Type Delta-12 Fatty Acid Desaturase Genes
The D form of IMC129 fad2 gene contains a G to A transversion at nucleotide 316 from the translation initiation codon. Two short oligonucleotide upstream (5′) primers, based on the single base change (G to A) between the D form of the IMC129 and wild type FAD2 genes, were designed. The sequences of the upstream (5′) primers are as follows:







5′ gene-specific primer for wild type FAD2-D:








5′-GTCTGGGTCATAGCCCACG-3′
(SEQ ID NO: 13); and


 







5′ gene specific primer for IMC129 fad2-d:








5′-GTCTGGGTCATAGCCCACA-3′
(SEQ ID NO: 14).






A common downstream (3′) primer (SEQ ID NO:10) specific for the D form of the FAD2 gene was used for both IMC129 and wild type FAD2 genes. These gene-specific primers were used in a DNA based PCR diagnostic assay to genotype plants carrying the mutant and/or wild type FAD2 genes.

Genomic DNA was isolated from leaf tissue of IMC129 and Westar plants, and used as PCR templates. The PCR amplification assays were carried out by using a gene amplification kit (Perkin Elmer). The PCR conditions are as follows: denaturing temperature, 95° C. for 1 min; annealing temperature, 52° C. for 1 min; amplification temperature 72° C. for 1 min. Total 20 PCR cycles were extended. Under these conditions primers SEQ ID NO:13 and SEQ ID NO:14 only amplified wild type FAD2-D and IMC 129 mutant fad2-d gene fragments, respectively.
The specificity of the gene-specific primers could be further improved by shortening the length of the primers and by replacing the base C with a T at the second position from the 3′ end of the oligonucleotide PCR primer for FAD2-D (SEQ ID NO:13). The sequences of the modified upstream (5′) oligonucleotide PCR primers are as follows:









5′ modified gene-specific primer for wild



type FAD2-D:










5′-CTGGGTCATAGCCCATG-3′
(SEQ ID NO: 15); and



 









5′ modified gene-specific primer for IMC129



fad2-d:










5′-CTGGGTCATAGCCCACA-3′
(SEQ ID NO: 16).






The same common downstream (3′) oligonucleotide primer (SEQ ID NO:10) was used for amplifying FAD2-D and fad2-d. With the modified primers, the genotype for FAD2-D and fad2-d could be consistently distinguished after extended 30 cycle of PCR amplification. Therefore, the DNA based PCR assay provided a simple and reliable method of genotypping B. Napus germplasms containing mutant and/or wild type FAD2 genes.

Example 3
Constructs for Dominant Negative Suppression of Delta-12 Fatty Acid Desaturase
The vector pZS212 was used to construct plasmids for dominant negative suppression experiments. One construct was prepared by inserting the full-length mutant D gene coding sequence (nucleotides 1 to 1155 of SEQ ID NO:3) in sense orientation between the phaseolin promoter and phaseolin 3′ poly A region of plasmid pCW108. The pCW108 vector contains the bean phaseolin promoter and 3′ untranslated region and was derived from the commercially available pUC18 plasmid (Gibco-BRL) via plasmids AS3 and pCW104. Plasmid AS3 contains 495 base pairs of the bean (Phaseolus vulgaris) phaseolin (7S seed storage protein) promoter starting with 5′-TGGTCTTTTGGT-3′ (SEQ ID NO:56) followed by the entire 1175 base pairs of the 3′ untranslated region of the same gene (see sequence descriptions in Doyle et al., (1986) J. Biol. Chem. 261:9228–9238 and Slightom et al., (1983) Proc. Natl. Acad. Sci. USA, 80:1897–1901. Further sequence description may be found in WO 9113993) cloned into the Hind III site of pUC18. The additional cloning sites of the pUC18 multiple cloning region (Eco RI, Sph I, Pst I and Sal I) were removed by digesting with Eco RI and Sal I, filling in the ends with Klenow and religating to yield the plasmid pCW104. A new multiple cloning site was created between the 495 bp of the 5′ phaseolin and the 1175 bp of the 3′ phaseolin by inserting a dimer of complementary synthetic oligonucleotides consisting of the coding sequence for a Nco I site (5′-CCATGG-3′) followed by three filler bases (5′-TAG-3′), the coding sequence for a Sma I site (5′ CCCGGG-3′), the last three bases of a Kpn I site (5′-TAC-3′), a cytosine and the coding sequence for an Xba I site (5′-TCTAGA-3′) to create the plasmid pCW108. This plasmid contains unique Nco I, Sma I, Kpn I and Xba I sites directly behind the phaseolin promoter.
The resulting 5′-phaseolin promoter-mutant fad2-phaseolin poly A-3′ construct was excised and cloned between the EcoRI/SalI sites of pZS212, resulting in the plasmid designated pZPhMCFd2 (FIG. 1). pZS212 is based on a vector which contains: (1) the chimeric gene nopaline synthase/neomycin phosphotransferase as a selectable marker for transformed plant cells (Brevan et al. (1984) Nature 304: 184–186), (2) the left and right borders of the T-DNA of the Ti plasmid (Brevan et al. (1984) Nucl. Acids Res. 12:8711–8720), (3) the E. coli lacZ α-complementing segment (Vieria and Messing (1982) Gene 19:259–267) with unique restriction endonuclease sites for Eco RI, Kpn I, Bam HI, and Sal I, (4) the bacterial replication origin from the Pseudomonas plasmid pVS1 (Itoh et al. (1984) Plasmid 11:206–220), and (5) the bacterial neomycin phosphotransferase gene from Tn5 (Berg et al. (1975) Proc. Natnl. Acad. Sci. U.S.A. 72:3628–3632) as a selectable marker for transformed A. tumefaciens. The nopaline synthase promoter in the plant selectable marker was replaced by the 35S promoter (Odell et al. (1985) Nature, 313:810–813) by a standard restriction endonuclease digestion and ligation strategy.
A second plasmid was constructed by inserting the full-length wild type canola D gene coding sequence (nucleotides 130 to 1281 of SEQ ID NO:1) into the NotI site of the canola napin promoter expression vector pIMC401 which contains a 2.2 kb napin expression cassette.
The canola napin promoter expression cassette in pIMC401 was constructed as follows: ten oligonucleotide primers were synthesized based upon the nucleotide sequence of napin lambda clone CGN1–2 published in European Patent Application EP 255378). The oligonucleotide sequences were:

    
    
        BR42 and BR43 corresponding to bases 1132 to 1156 (BR42) and the complement of bases 2248 to 2271 (BR43) of the sequence listed in FIG. 2 of EP 255378.
        BR45 and BR46 corresponding to bases 1150 to 1170 (BR46) and the complement of bases 2120 to 2155 (BR45) of the sequence listed in FIG. 2 of EP 255378. In addition BR46 had bases corresponding to a Sal I site (5′-GTCGAC-3′) and a few additional bases (5′-TCAGGCCT-3′) at its 5′ end and BR45 had bases corresponding to a Bgl II site (5′-AGATCT-3′) and two (5′-CT-3′) additional bases at the 5′ end of the primer,
        BR47 and BR48 corresponding to bases 2705 to 2723 (BR47) and bases 2643 to 2666 (BR48) of the sequence listed in FIG. 2 of EP 255378. In addition BR47 had two (5′-CT-3′) additional bases at the 5′ end of the primer followed by bases corresponding to a Bgl II site (5′-AGATCT-3′) followed by a few additional bases (5′-TCAGGCCT-3′),
        BR49 and BR50 corresponding to the complement of bases 3877 to 3897 (BR49) and the complement of bases 3985 to 3919 (BR50) of the sequence listed in FIG. 2 of EP 255378. In addition BR49 had bases corresponding to a Sal I site (5′-GTCGAC-3′) and a few additional bases (5′-TCAGGCCT-3′) at its 5′ end,
        BR57 and BR58 corresponding to the complement of bases 3875 to 3888 (BR57) and bases 2700 to 2714 (BR58) of the sequence listed in FIG. 2 of EP 255378. In addition the 5′ end of BR57 had some extra bases (5′-CCATGG-3′) followed by bases corresponding to a
        Sac I site (5′-GAGCTC-3′) followed by more additional bases (5′-GTCGACGAGG-3′) (SEQ ID NO:57. The 5′ end of BR58 had additional bases (5′-GAGCTC-3′) followed by bases corresponding to a Nco I site (5′-CCATGG-3′) followed by additional bases (5′-AGATCTGGTACC-3′) (SEQ ID NO:58).
        BR61 and BR62 corresponding to bases 1846 to 1865 (BR61) and bases 2094 to 2114 (BR62) of the sequence listed in FIG. 2 of EP 255378. In addition the 5′ end of BR 62 had additional bases (5′-GACA-3′) followed by bases corresponding to a Bgl II site (5′-AGATCT-3′) followed by a few additional bases (5′-GCGGCCGC-3′).
    
    


Genomic DNA from the canola variety ‘Hyola401’ (Zeneca Seeds) was used as a template for PCR amplification of the napin promoter and napin terminator regions. The promoter was first amplified using primers BR42 and BR43, and reamplified using primers BR45 and BR46. Plasmid pIMC01 was derived by digestion of the 1.0 kb promoter PCR product with SalI/BglII and ligation into SalI/BamHI digested pBluescript SK+(Stratagene). The napin terminator region was amplified using primers BR48 and BR50, and reamplified using primers BR47 and BR49. Plasmid pIMC06 was derived by digestion of the 1.2 kb terminator PCR product with SalI/BglII and ligation into SalI/BglII digested pSP72 (Promega). Using pIMC06 as a template, the terminator region was reamplified by PCR using primer BR57 and primer BR58. Plasmid pIMC101 containing both the napin promoter and terminator was generated by digestion of the PCR product with SacI/NcoI and ligation into SacI/NcoI digested pIMC01. Plasmid PIMC101 contains a 2.2 kb napin expression cassette including complete napin 5′ and 3′ non-translated sequences and an introduced NcoI site at the translation start ATG. Primer BR61 and primer BR62 were used to PCR amplify an  ̃270 bp fragment from the 3′ end of the napin promoter. Plasmid pIMC401 was obtained by digestion of the resultant PCR product with EcoRI/BglII and ligation into EcoRI/BglII digested pIMC101. Plasmid pIMC401 contains a 2.2 kb napin expression cassette lacking the napin 5′ non-translated sequence and includes a NotI site at the transcription start.
The fragment containing the 5′-napin-fad2D-napin poly A-3′ cassette was then inserted into the SalI site of pZS212, and the resulting 17.2 Kb plasmid was termed pIMC127 (FIG. 2).
A third plasmid, pIMC135, was constructed in a manner similar to that described above for pIMC127. Plasmid pIMC135 contains a 5′ cruciferin promoter fragment operably linked in sense orientation to the full-length wild-type coding sequence of SEQ ID NO:1, followed by a cruciferin 3′ poly A fragment.
A fourth plasmid, pIMC140 was constructed in a manner similar to that described above. Plasmid pIMC140 contains a 5′ napin promoter fragment operably linked in sense orientation to the full-length mutant Q508 F gene coding sequence (SEQ ID NO:7), followed by a 3′ napin poly A fragment.
Example 4
Fatty Acid Profiles in Dominant Negative Suppression Plants
The plasmids pZPhMCFd2, pIMC127, pIMC135 and pIMC140 were transferred by a freeze/thaw method (Holsters et al. (1978) Mol Gen Genet 163:181–187) to the Agrobacterium strain LBA4404/pAL4404 (Hockema et al. (1983), Nature 303:179–180).
Brassica napus cultivar “Westar” was transformed by co-cultivation of seedling pieces with disarmed Agrobacterium tumefaciens strain LBA4404 carrying the appropriate binary vector.
B. napus seeds were sterilized by stirring in 10% Chlorox, 0.1% SDS for thirty min, and then rinsed thoroughly with sterile distilled water. The seeds were germinated on sterile medium containing 30 mM CaCl2 and 1.5% agar, and grown for six days in the dark at 24° C.
Liquid cultures of Agrobacterium for plant transformation were grown overnight at 28° C. in Minimal A medium containing 100 mg/L kanamycin.
Minimal A Bacterial Growth Medium
Dissolve in distilled water:
10.5 grams potassium phosphate, dibasic
4.5 grams potassium phosphate, monobasic
1.0 gram ammonium sulfate
0.5 gram sodium citrate, dihydrate
Make up to 979 mL with distilled water
Autoclave
Add 20 mL filter-sterilized 10% sucrose
Add 1 mL filter-sterilized 1 M MgSO4 
The bacterial cells were pelleted by centrifugation and resuspended at a concentration of 108 cells/mL in liquid Murashige and Skoog Minimal Organic medium (GIBCO; Cat. No. 510–3118) containing 100 μM acetosyringone.
B. napus seedling hypocotyls were cut into 5 mm segments which were immediately placed into the bacterial suspension. After 30 min, the hypocotyl pieces were removed from the bacterial suspension and placed onto BC-35 callus medium containing 100 μM acetosyringone.













Brassica Callus Medium BC-35









Per liter:



Murashige and Skoog Minimal Organic Medium (MS



salts, 100 mg/L i-inositol, 0.4 mg/L thiamine;



GIBCO #510-3118)



30 grams sucrose



18 grams mannitol



0.5 mg/L 2,4-D



0.3 mg/L kinetin



0.6% agarose



pH 5.8










The plant tissue and Agrobacteria were co-cultivated for three days at 24° C. in dim light.

The co-cultivation was terminated by transferring the hypocotyl pieces to BC-35 callus medium containing 200 mg/L carbenicillin to kill the Agrobacteria, and 25 mg/L kanamycin to select for transformed plant cell growth. The seedling pieces were incubated on this medium for three weeks at 28° C. under continuous light.
After four weeks, the segments were transferred to BS-48 regeneration medium containing 200 mg/L carbenicillin and 25 mg/L kanamycin.













Brassica Regeneration Medium BS-48









Murashige and Skoog Minimal Organic Medium



Gamborg B5 Vitamins (SIGMA #1019)



10 grams glucose



250 mg xylose



600 mg MES



0.4% agarose



pH 5.7



Filter-sterilize and add after autoclaving:



2.0 mg/L zeatin



0.1 mg/L IAA










Plant tissue was subcultured every two weeks onto fresh selective regeneration medium, under the same culture conditions described for the callus medium. Putatively transformed calli grew rapidly on regeneration medium; as calli reached a diameter of about 2 mm, they were removed from the hypocotyl pieces and placed on the same medium lacking kanamycin.

Shoots began to appear within several weeks after transfer to BS-48 regeneration medium. As soon as the shoots form d discernable stems, they were xcised from the calli, transferred to MSV-1A elongation medium, and moved to a 16:8 h photoperiod at 24° C.
Brassica Shoot Elongation Medium MSV-1A
Murashige and Skoog Minimal Organic M dium
Gamborg B5 Vitamins
10 grams sucrose
0.6% agarose
pH 5.8
Once shoots had elongated several internodes, they were cut above the agar surface and the cut ends were dipped in Rootone. Treated shoots were planted directly into wet Metro-Mix 350 soiless potting medium. The pots were covered with plastic bags which were removed when the plants were clearly growing—after about ten days.
Plants were grown under a 16:8 h photoperiod, with a daytime temperature of 23° C. and a nightime temperature of 17° C. When the primary flowering stem began to elongate, it was covered with a mesh pollen-containment bag to prevent outcrossing. Self-pollination was facilitated by shaking the plants several times each day.
Tranagenic progeny plants containing pZPhMCFd2 were designated as the WS201 series. Plants transformed with pIMC127 were designated as the WS127 series. Plants transformed with pIMC135 were designated as the WS135 series. Plants transformed with pIMC140 were designated as the WS140 series. Seeds were obtained by selfing the T1 plants. Fatty acid profiles of the T2 seeds were determined as described in WO 93/11245. The results are shown in Tables 3 and 4 and FIGS. 3 and 4.







TABLE 3







T2 Seeds having Decreased


C18:2 (WS2011 Transformants)2.









Line
Plant
Fatty Acid Composition (%)













No.
Series
C16:0
C18:0
C18:1
C18:2
C18:3
















T300236
WS201
4.1
3.2
72.5
10.6
6.5


T300238
WS201
3.9
2.7
74.4
9.2
6.6


T300390
WS201
4.2
2.5
75.4
9.3
5.4


T300273
WS201
3.9
2.8
77.4
7.5
5.3


T300400
WS201
3.9
2.5
78.6
6.6
5.3


T300354
WS201
4.0
2.7
78.6
6.4
4.51





1WS201: phaseolin promoter/IMC129 mutant, sense orientation.


2Population of 168 selfed individuals from greenhouse.






As shown in FIG. 3, a large proportion of the control WS127 plants have elevated levels of linoleic acid. This result is expected, since the delta-12 desaturase gene dosage is higher due to the extra copy of the wild-type delta-12 desaturase gene. A small proportion of WS127 plants show lower levels of linoleic acid, due to cosuppression.
In contrast, no WS201 plants had elevated levels of linoleic acid. This result confirms that the mutant delta-12 desaturase D form gene in WS201 plants is non-functional. Furthermore, a higher proportion of WS201 plants have decreased C18:2 (5–14%) compared to the proportion of WS127 control plants having decreased C18:2. The proportion of WS201 plants having decreased C18:2 is about 25-fold higher than the proportion of WS127 plants having decreased C18:2. The surprising inhibition of endogenous delta-12 desaturase activity by a mutant delta-12 gene product has been termed dominant negative suppression.
The fatty acid composition of seeds produced by representative dominant negative suppression T2 WS201 plants is shown in Table 3. The plants show an altered fatty acid composition, including decreased C18:2, increased C18:1, and decreased saturates.
A T2 generation plant is not homozygous for the introduc d gene. Consequently, T3 and subsequent generations that are homozygous for the mutant delta-12 desaturase gene will have even lower levels of linoleic acid, from about 1% to about 10%, preferably from about 1% to about 6%. Levels of oleic acid in homozygous lines will be from 75% to about 88%, preferably from about 80% to about 88%.
The results observed with WS140 plants (containing a mutant Q508 F form delta-12 desaturase gene) are shown in FIG. 4. None of the WS140 plants have elevated C18:2 levels, similar to the results obtained with WS201 transgenic plants. As expected, a large proportion of the control WS135 plants have elevated C18:2 levels. The proportion of WS140 and WS135 plants having decreased C18:2 levels is similar, indicating that expression of this particular mutant delta-12 desaturase gene product does not inhibit endogenous wild-type delta-12 desaturase gene product to an extent greater than that expected from cosuppression. The fatty acid composition of seeds produced by a representative T2 WS140 plant with decreased C18:2 levels is shown in Table 4.







TABLE 4







T2 Seed Having Decreased C18:2 (WS1401 Transformants)2.









Fatty Acid Composition (%)













Line No.
Vector
C16:0
C18:0
C18:1
C18:2
C18:3





T300435
pIMC140
3.8
3.5
73.7
9.7
6.11





1WS140: napin promoter/Q508 fad2 F mutation, sense orientation


2Population of 61 selfed individuals from greenhouse

## Claims

1. A method for increasing the oleic acid content in plant seeds, comprising the steps of:
a) introducing a recombinant nucleic acid construct into a plant, said construct comprising at least one seed-specific regulatory sequence operably linked in sense orientation to a full length delta-12 fatty acid desaturase coding sequence, wherein said sequence encodes a delta-12 fatty acid desaturase protein having a substitution of a Lys residue for Asp or Glu in an amino acid region selected from the group consisting of His-Glu-Cys-Gly-His (SEQ ID NO:53), His-Asp-Cys-Gly-His (SEQ ID NO;55), and His-Asp-Cys-Ala-His (SEQ ID NO:54),
b) obtaining progeny from said plant, said progeny producing said seeds having an oleic acid content of from about 72.5% to about 78.6%.

2. A recombinant nucleic acid construct effective for increasing oleic acid content when expressed in seeds, said construct comprising at least one seed-specific regulatory sequence operably linked in sense orientation to a delta-12 fatty acid desaturase coding sequence encoding a delta-12 fatty acid desaturase gene product having at least one mutation which renders said desaturase gene product non-functional, said mutation being the substitution of a Lys residue for Asp in an amino acid region selected from the group consisting of His-Asp-Cys-Gly-His (SEQ ID NO:55) and His-Asp-Cys-Ala-His (SEQ ID NO:54).

3. The method of claim 1, wherein said substitution is in a His-Glu-Cys-Gly-His (SEQ ID NO:53) amino acid region.

4. The method of claim 1, wherein said plant is soybean.

5. The method of claim 1, wherein said plant is rapeseed.

6. The method of claim 1, wherein said plant is cotton.

7. The method of claim 1, wherein said plant is corn.

8. The method of claim 1, wherein said plant is safflower.

9. The method of claim 1, wherein said seed-specific regulatory sequence is a bean β-phaseolin promoter.

10. The method of claim 1, wherein said seed-specific regulatory sequence is an αsubunit of soybean β-conglycinin promoter.

11. The method of claim 1, wherein said seed-specific regulatory sequence is maize 18 kd oleosin promoter.

12. The method of claim 1, wherein said seed-specific regulatory sequence is maize 15 kd zein promoter.

13. The method of claim 1, wherein said seed-specific regulatory sequence is a Brassica napin promoter.

14. The method of claim 1, wherein said seeds have a linoleic acid content of about 6.4% to about 10.6%.

15. The method of claim 1, wherein said seeds have an α-linolenic acid content of about 4.5% to about 6.5%.