BRPI0810973B1 - METHODS FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A SOYBEAN SEED, METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A CORN SEED, RECOMBINANT POLYNUCLEOTIDE, RECOMBINANT DNA CONSTRUCTION, METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF AN OILSEED PLANT SEED, METHOD FOR PRODUCING A SOYBEAN OR CORN SEED AND METHOD FOR PRODUCING A PRODUCT - Google Patents

Abstract

METHODS FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A SOYBEAN SEED, METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A CORN SEED, RECOMBINANT POLYNUCLEOTIDE, CONSTRUCTION OF RECOMBINANT DNA, METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A SEED OF OILSEED PLANT, METHOD FOR PRODUCING A SOYBEAN OR CORN SEED AND METHOD FOR PRODUCING A PRODUCT. The present invention describes transgenic soybean seeds that have an increased total fatty acid content of at least 10% and altered fatty acid profiles when compared to the total fatty acid content of non-transgenic null segregating soybean seeds. The present invention uses DGAT genes from Yarrowia lipolytica to achieve increased seed storage lipids

Description

CROSS REFERENCE TO RELATED PATENT APPLICATION

[001] The present application claims the benefit of US Provisional Application 60/939872, filed on May 24, 2007, and North American Provisional Application No. 61/013406, filed on December 13, 2007, the disclosures of which are in full incorporated into the present.

FIELD OF INVENTION

[002] The present invention is in the field of biotechnology. In particular, it concerns the polynucleotide sequences encoding diacylglycerol acyltransferase genes and the use of these acyltransferases to increase the production of seed storage lipids and altered fatty acid profiles in soybeans.

BACKGROUND OF THE INVENTION

[003] Plant lipids have a series of industrial and nutritional uses and are fundamental for the function of plant membranes and climate adaptation. These lipids represent a broad set of chemical structures and these structures determine the physiological and industrial properties of the lipid. Many of these structures result directly or indirectly from metabolic processes that alter the degree of lipid unsaturation. Different metabolic regimes in different plants produce these altered lipids, and domestication of exotic plant species or modification of agronomically adapted species is typically required to produce economically large quantities of the desired lipid.

[004] There are serious limitations to the use of mutagenesis to alter the composition and content of fatty acids. Selections will rarely discover mutations that (a) result in a dominant (“gain of function”) phenotype; (b) are found in genes that are essential for plant growth; and (c) are found in an enzyme that is not rate limiting and that is encoded by more than one gene. In cases where desired phenotypes are available in mutant maize lines, their introgression into elite lines through traditional breeding methods is expensive and slow, as the desired oil compositions are likely the result of several recessive genes.

[005] Recent cell and molecular biology methods offer the potential to overcome some of the limitations of the mutagenesis approach, including the need for extensive cultivation. Some of the particularly useful technologies are the seed-specific expression of exogenous genes in transgenic plants (see Goldberg et al. (1989), Cell 56: 149-160) and the use of nonsense RNA to inhibit plant target genes in a dominant and tissue-specific (see van der Krol et al. (1988), Gene 72: 4550). Other advances include the transfer of exogenous genes into elite commercial varieties of commercial oilseeds, such as soybeans (Chee et al. (1989), Plant Physiol. 91: 1212-1218; Christou et al. (1989), Proc. Natl. Acad. Sci. U. S. A. 86: 7500-7504; Hinchee et al. (1988), Bio/Technology 6: 915-922; Physiol. 91: 694-701) and sunflower (Everett et al. (1987), Bio/Technology 5: 1201-1204), and the use of genes as restriction fragment length polymorphism (RFLP) markers in a program of breeding, which makes the introgression of recessive traits into elite lines rapid and less expensive (Tanksley et al. (1989), Bio/Technology 7: 257-264). The application of each of these technologies requires, however, the identification and isolation of commercially important genes.

[006] Most fatty acids become esterified to coenzyme A (CoA) to generate acyl-CoAs. These molecules are substrates for the synthesis of glycerolipids in the cell's endoplasmic reticulum, where phosphatidic acid and diacylglycerol (DAG) are produced. Any of these metabolic intermediates can be targeted to membrane phospholipids (such as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) or DAG can be targeted to the formation of triacylglycerols (TAGs), the primary storage reserve of lipids in eukaryotic cells.

[007] Diacylglycerol acyltransferase (“DGAT”) is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi and mammals. This enzyme is responsible for transferring an acyl group from acyl-coenzyme A to the sn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol (“TAG”). DGAT is associated with lipid body and membrane fractions in plants and fungi, particularly in oilseeds, in which it contributes to the storage of carbon used as energy reserves. TAG is believed to be an important substance for energy storage in cells. DGAT is known to regulate TAG structure and direct TAG synthesis. Furthermore, it is known that the TAG reaction is specific for oil synthesis.

[008] TAG is the primary component of vegetable oil in plants. It is used by the seed as a stored form of energy to be used during seed germination.

[009] Two different families of DGAT proteins have been identified. The first family of DGAT proteins (“DGAT1”) refers to acyl coenzyme A:cholesterol acyltransferase (“ACAT”) and was described in US Patents 6,100,077 and US 6,344,548. A second family of DGAT proteins (“DGAT2”) is unrelated to the DGAT1 family and is described in WO 2004/011671, published February 5, 2004. Other references to DGAT genes and their use in plants include the documents WO 2004/011671, WO 1998/055631 and WO 2000/001713, as well as US Patent 20030115632.

[010] The co-pending Published Patent Application of the applicant's assignee No. US 2006-0094088 describes plant and fungal DGATs genes and their use in modifying the levels of polyunsaturated fatty acids (“PUFAs”) in edible oils.

[011] The applicant's assignee Published PCT Application No. WO 2005/003322 describes the cloning of phosphatidylcholine diacylglycerol acyltransferase and DGAT2 to alter the PUFA and oil content in oleaginous yeast.

BRIEF DESCRIPTION OF THE INVENTION

[012] The present invention relates to a transgenic soybean seed that contains an increased total fatty acid content of at least 10% when compared to the total fatty acid content of a seed and non-transgenic zero segregating soybeans.

[013] In a second embodiment, the present invention relates to a method for increasing the total fatty acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct containing at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-GMO zero segregating soybean seed.

[014] In a third embodiment, the present invention relates to a transgenic corn seed that contains a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-null segregating corn seed. transgenic.

[015] In a fourth embodiment, the present invention relates to a method for increasing the total fatty acid content of a corn seed comprising: (c) transforming at least one corn seed with a recombinant construct that contains at least one DGAT sequence; and (d) selecting the processed corn seed(s) from step (a) that contains a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-transgenic null segregating corn seed.

[016] In a fifth embodiment, the present invention relates to a transgenic soybean seed that contains a total fatty acid content of at least 10% and an oleic acid content increased by at least 25% when compared to the total fatty acid content and oleic acid content of a non-transgenic null segregant soybean seed.

[017] In a further embodiment, the present invention relates to transgenic soybeans that contain total fatty acid content increased by at least 10% and at least any one of (i) oleic acid content increased by at least 25%; (ii) linolenic acid content reduced by at least 25%; (iii) linoleic acid content reduced by at least 4%; (iv) palmitic acid content reduced by at least 8%; and (v) stearic acid content increased by at least 14% when compared to the total fatty acid content and the oleic acid, linolenic acid, linoleic acid, palmitic acid or stearic acid content, respectively, of a segregating soybean seed non-transgenic null.

[018] In a sixth embodiment, the present invention relates to a method of increasing the total fatty acid content and oleic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; (b) selecting the transformed soybean cell(s) from step (a) that has a total fatty acid content increased by at least 10% and oleic acid content increased by at least 25% when compared with the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed. (c) 9] In a seventh embodiment, the present invention relates to a method of increasing the total fatty acid content and reducing the linolenic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linolenic acid content reduced by at least 25% % when compared with the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[019] In an eighth embodiment, the present invention relates to a method of increasing the total fatty acid content and reducing the linoleic acid content of a soybean seed comprising: (a) transforming at least one cell of soybean with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linoleic acid content reduced by at least 4% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[020] In an eighth embodiment, the present invention relates to a method of increasing the total fatty acid content and reducing the linoleic acid content of a soybean seed comprising: (a) transforming at least one cell of soybean with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linoleic acid content reduced by at least 4% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[021] In a ninth embodiment, the present invention relates to a method of increasing the total fatty acid content and reducing the palmitic acid content of a soybean seed comprising: (a) transforming at least one cell of soybean with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linolenic acid content reduced by at least 8% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[022] In a tenth embodiment, the present invention relates to a method of increasing the total fatty acid content and stearic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and stearic acid content increased by at least 14% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[023] Any of the transgenic seeds according to the present invention can comprise a recombinant construct that contains at least one DGAT sequence that can be selected from the group consisting of DGAT1, DGAT2 and DGAT1 in combination with DGAT2. Furthermore, the DGAT sequence may be a Yarrowia sequence.

[024] Product(s) and/or by-product(s) obtained from transgenic soybean seeds according to the present invention are also within the scope of the present invention.

[025] Also within the scope of the present invention are mutated versions of Yarrowia DGATs that retain function. Specific examples include, but are not limited to, a recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide that has diacylglycerol acyltransferase activity, wherein the polypeptide is described in SEQ ID NO: 83, 88 or 93. The complement of nucleotide sequence, recombinant DNA constructs comprising the nucleotide sequence, and cells incorporating the recombinant DNA constructs also fall within the scope of the present invention. Oil plants are useful for understanding recombinant constructs.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTING

[026] The present invention can be understood more completely from the following detailed description and the attached figures and Sequence Listing, which form part of the present application.

[027] Figure 1 provides plasmid maps for pFBAIn-YLDGAT1, for pFBAIn-YLDGAT2 and for pFBAIn-MOD1.

[028] Figure 2 provides plasmid maps for KS352 and KS332.

[029] Figure 3 provides plasmid maps for KS349, KS362 and KS364.

[030] Figure 4 provides a strong correlation (R2 > 0.59) between oleic acid content and total esterified fatty acid content for somatic embryos generated with KS349.

[031] Figure 5 provides a strong correlation (R2 > 0.67) between oleic acid content and total esterified fatty acid content for somatic embryos generated with KS362 alone or in combination with KS349 as well as with KS364.

[032] Figure 6 provides a correlation (R2 > 0.45) between oleic acid content and oil content for transgenic soybean seed (T1 generation) generated through cotransformation of plasmids KS349 and KS362.

[033] Figure 7 provides oil content and weight of T1 seed generated through cotransformation of plasmids KS349 and KS362 (A) and KS362 alone (B).

[034] Figure 8 provides hybridization results of genomic DNA spots. Genomic DNA was isolated from transgenic soybeans obtained from events AFS4818.1.2, AFS4818.1.3, AFS4818.1.5, AFS48182.6, AFS4818.1.9 (see Example 6). The DNA was digested with EcoRI and HindIII, run on a gel and blotted onto nylon filters (AFS4818.1.2 lines 1 and 2, AFS4818.1.3 lines 3 and 4, AFS4818.1.5 lines 5 and 6, AFS48182.6 lines 7 and 8, AFS4818.1.9 lines 9 and 10 and lines 11 and 12 are non-transgenic wild-type DNA also digested with EcoRI and HindIII). Hybridization probes were a Yarrowia DGAT1-specific probe for the top blot (A) and the bottom blot was probed with a Yarrowia DGAT2-specific probe.

[035] Figure 9 provides hybridization results of genomic DNA spots.

[036] The stains are similar to those described in Figure 8, except that all DNAs were digested with BstXIand and the stain was probed with a Yarrowia DGAT2-specific probe.

[037] Sequence descriptions reduce the Sequence Listing attached hereto. The Sequence Listing contains one-letter codes for nucleotide sequence characters and the one- and three-letter codes for amino acids as defined in the IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in Biochemical Journal 219 (2): 345-373 (1984). SUMMARY OF PROTEINS AND NUCLEIC ACID SEQ ID NO:

[038] SEQ ID NO. 7 and 8 correspond to the PCR primers oYLDGAT2-1 (SEQ ID NO. 7) and oYLDGAT2-2 (SEQ ID NO. 8), used to amplify the diacylglycerol acyltransferase 2 gene from Yarrowia lipolytica (YL DGAT2) from a yeast lysate (for details, see Example 1).

[039] SEQ ID NO. 12 to 15 corresponds to oligonucleotide primers used to amplify the coding regions of YL DGAT1 (YDGAT1-F and YDGAT1-R; SEQ ID NO. 12 and 13, respectively) and YL DGAT2 (YDGAT2- F and YDGAT2-R, SEQ ID NO. 14 and 15, respectively) from Yarrowia lipolytica genomic DNA.

[040] SEQ ID No. 23 (oCal-15) and SEQ ID No. 24 (oCal-6) correspond to oligonucleotide primers used to amplify cal a24-4 DNA fragment (SEQ ID No. 22) of CalFad2 plasmid model -2 described in WO 02/008269.

[041] SEQ ID No. 28 (oKR85-1) and SEQ ID No. 29 (oKR85-2) correspond to primers used to amplify the betaconglycinin promoter-(NotI cloning site)-phaseolin 3' termination region of plasmid pKR85 (SEQ ID NO. 27).

[042] SEQ ID No. 37 (oGy1-1) and SEQ ID No. 38 (oGy1-2) correspond to primers used to amplify the soy glycinin 1 promoter (SEQ ID No. 36) and incorporate BamHI and NcoI sites onto the 5' and 3' ends, respectively.

[043] SEQ ID No. 54 (oP34-1) and SEQ ID No. 55 (oP34-2) correspond to primers used to amplify the soybean P34 promoter (SEQ ID No. 53) and which incorporate the BamHI and NotI sites at the 5' and 3' ends, respectively.

[044] SEQ ID NO. 69 (oSGly-2) and SEQ ID NO. 70 (oSGly-3) correspond to primers used to amplify the glycinin GY1 promoter.

[045] SEQ ID NO. 76 (oYDG2-1) and SEQ ID NO. 77 (oYDG2-2) correspond to primers used to amplify DGAT2 from Yarrowia (SEQ ID NO. 10) which was then incorporated into pKR1254 (SEQ ID No. 78).

[046] SEQ ID No. 79 (YID2_Y326F-5) and SEQ ID No. 80 (YID2_Y326F-3) correspond to primers used to mutate the amino acid at position 326 of Yarrowia DGAT2 (SEQ ID No. 10) from tyrosine to phenylalanine.

[047] SEQ ID No. 84 (YID2_Y326L-5) and SEQ ID No. 85 (YID2_Y326L-3) correspond to primers used to mutate the amino acid at position 326 of Yarrowia DGAT2 (SEQ ID No. 10) from tyrosine to leucine.

[048] SEQ ID No. 89 (YID2_R327K-5) and SEQ ID No. 90 (YID2_R327K-3) correspond to primers used to mutate the amino acid at position 327 of Yarrowia DGAT2 (SEQ ID No. 10) from arginine to lysine.

[049] SEQ ID NO. 108 to 111 (GmTE2 5-1, GmTE2 3-1, GmTE2 5-2 and GmTE2 3-2, respectively) correspond to primers used to amplify the soybean thioesterase 2 gene.

[050] SEQ ID NO. 115 to 118 (GmFad2-1 5-1, GmFad2-1 3-1, GmFad2-1 5-2 and GmFad2-1 3-2, respectively) correspond to primers used to amplify the desaturase gene of 2-1 soy fatty acid.

DETAILED DESCRIPTION OF THE INVENTION

[051] The description of each reference described herein is fully incorporated herein by reference.

[052] As used herein and in the attached claims, the singular forms “a”, “an”, “the” and “a” include reference to the plural, unless the context clearly indicates otherwise. Thus, for example, reference to “a plant” includes a series of such plants, reference to “a cell” includes one or more cells and their equivalents known to those skilled in the art, and so on.

[053] In the context of this specification, a series of terms and abbreviations are used. The following definitions are provided: “Open reading frame” is abbreviated ORF. “Polymerase chain reaction” is abbreviated PCR. “North American Type Cultivation Collection” is abbreviated ATCC. “Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2. “Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT. “Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT. “Diacylglycerol” is abbreviated DAG. “Triacylglycerols” are abbreviated TAGs. “Coenzyme A” is abbreviated CoA.

[054] The expression “fatty acids” designates long-chain aliphatic acids (alkanoic acids) with variable chain lengths, from about C12 to C22 (although acids with longer and shorter chain lengths are known). The predominant chain lengths are from C16 to C22. The structure of a fatty acid is represented by a simple “X:Y” notation system, where X is the total number of carbon atoms (C) in the specific fatty acid and Y is the number of double bonds.

[055] Generally, fatty acids are classified as saturated or unsaturated. The expression “saturated fatty acids” refers to fatty acids that do not contain “double bonds” between their main carbon chain. On the other hand, “unsaturated fatty acids” contain “double bonds” along their main carbon chains (which are most commonly found in the cis configuration). “Monounsaturated fatty acids” contain only one “double bond” along the main carbon chain (typically, for example, between the ninth and tenth carbon atoms for palmitoleic acid (16:1) and oleic acid (18:1). )), while “polyunsaturated fatty acids” (or “PUFAs”) contain at least two double bonds along the main carbon chain (such as between the ninth and tenth and between the twelfth and thirteenth carbon atoms). carbon for linoleic acid (18:2); and between the ninth and tenth, the twelfth and thirteenth and between the fifteenth and sixteenth for a-linolenic acid (18:3)).

[056] “Microbial oils” or “isolated cell oils” are oils produced naturally by microorganisms (such as algae, oleaginous yeasts and filamentous fungi) during their useful life. The term “oil” designates a lipid substance that is liquid at 25 °C and normally polyunsaturated. On the other hand, the term “fat” designates a lipid substance that is solid at 25 °C and normally saturated.

[057] “Lipid bodies” designate lipid droplets that are normally delimited by specific proteins and a monolayer of phospholipids. These organelles are places where most organisms transport or store neutral lipids. Lipid bodies are believed to arise from microdomains of the endoplasmic reticulum that contain TAG biosynthesis enzymes; and its synthesis and size are apparently controlled by specific protein components.

[058] “Neutral lipids” designate lipids commonly found in cells in lipid bodies such as oils and storage fats and are so called because, under cellular pH, lipids do not contain charged groups. Generally, they are completely nonpolar with no affinity for water. Neutral lipids generally designate mono-, di- and/or triesters of glycerol with fatty acids, also called monoacylglycerol, diacylglycerol or TAG, respectively (or, collectively, acylglycerols). A hydrolysis reaction must occur to release free fatty acids from acylglycerols.

[059] The terms “triacylglycerol”, “oil” and “TAGs” designate neutral lipids composed of three fatty acyl residues esterified in a glycerol molecule (and these terms will be used interchangeably throughout this specification). These oils can contain long-chain PUFAs, as well as shorter saturated and unsaturated fatty acids and longer-chain saturated fatty acids. Therefore, “oil biosynthesis” generally designates the synthesis of TAGs in the cell.

[060] The expression “DAG AT” designates a diacylglycerol acyltransferase (also known as acyl-CoA-diacylglycerol acyltransferase or diacylglycerol O-acyltransferase) (EC 2.3.1.20). This enzyme is responsible for the conversion of acyl-CoA and 1,2-diacylglycerol into TAG and CoA (therefore involved in the final step of TAG biosynthesis). There are two families of DAG AT enzymes: DGAT1 and DGAT2. The former family shares homology with the acyl-CoA:cholesterol acyltransferase (ACAT) gene family, while the later family is unrelated (Lardizabal et al., J. Biol. Chem. 276 (42): 38862-28869 (2001) ).

[061] The term “PDAT” designates a phospholipid enzyme: diacylglycerol acyltransferase (EC 2.3.1.158). This enzyme is responsible for the transfer of an acyl group from the sn-2 position of a phospholipid to the sn-3 position of 1,2-diacylglycerol, resulting in lysophospholipid and TAG (thus involved in the terminal step of TAG biosynthesis ). This enzyme differs from DGAT (EC 2.3.1.20) by synthesizing TAG via an acyl-CoA-independent mechanism.

[062] The term “ARE2” designates an enzyme acyl-CoA: sterol-acyltransferase (EC 2.3.1.26; also known as a sterol ester synthase 2 enzyme), which catalyzes the following reaction: acyl-CoA + cholesterol = CoA + cholesterol ester.

[063] As used herein, “nucleic acid” designates a polypeptide and includes a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include modified fragments and nucleotides. In this way, the expressions “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” or “nucleic acid fragment” are used interchangeably and are an RNA or DNA polymer that has a single or double strand, optionally containing synthetic, unnatural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are named by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deosicytidylate, “G ” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deosythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H ” for A, C or T, “I” for inosine and “N” for any nucleotide.

[064] The expressions “subfragment that is functionally equivalent” and “functionally equivalent subfragment” are used interchangeably herein. These expressions designate a part or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. The fragment or subfragment can be used, for example, in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by ligating a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the nonsense or nonsense orientation with respect to a plant promoter sequence.

[065] The expression “conserved domain” or “motif” designates a set of amino acids conserved in specific positions along an aligned sequence of evolutionarily related proteins. Although amino acids in other positions may vary among homologous proteins, amino acids that are highly conserved in specific positions indicate amino acids that are essential in the structure, stability, or activity of a protein. Because they are identified by the high degree of conservation in aligned sequences of a family of protein homologs, they can be used as identifiers, or “signatures,” to determine whether a protein with a newly determined sequence belongs to a previously identified protein family.

[066] The expressions “homology”, “homologous”, “substantially similar” and “substantially corresponding” are used interchangeably herein. They designate nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These expressions also designate modifications of the nucleic acid fragments according to the present invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment with respect to the initial unmodified fragment. It is understood, therefore, as those skilled in the art will appreciate, that the present invention encompasses more than specific sequence examples.

[067] Furthermore, those skilled in the art recognize that substantially similar nucleic acid sequences encompassed by the present invention are also defined by their ability to hybridize (under moderately stringent conditions, such as 0.5X SSC, 0.1% SDS, 60 °C) with the sequences exemplified herein, or any part of the nucleotide sequences described herein and which are functionally equivalent to any of the nucleic acid sequences described herein. Stringency conditions can be adjusted to select from moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

[068] The expression “selectively hybridizes” includes reference to the hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified target nucleic acid sequence to a detectably greater degree (such as at least twice over the background) that their hybridization to non-target nucleic acid sequences and even the substantial exclusion of non-target nucleic acids. Selective hybridization sequences typically have sequence identity of at least about 80% or sequence identity of 90%, up to 100% sequence identity, inclusive (i.e., fully complementary), to each other.

[069] The expression “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some lack of coincidence in sequences such that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about a thousand nucleotides in length, optionally less than five hundred nucleotides in length.

[070] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ions, typically a concentration of about 0.01 to 1.0 M Na ions (or other salts) under pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (such as ten to fifty nucleotides) and at least about 60°C for long probes (such as longer fifty nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Examples of low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55 °C. Examples of moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 0.5X to 1X SSC at 55 to 60°C. Examples of high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and washing in 0.1X SSC at 60 to 65°C.

[071] Specificity is typically the function of post-hybridization washes, where the critical factors are the ionic strength and temperature of the final wash solution. For DNA and DNA hybrids, Tm can be approximately calculated from the equation of Meinkoth et al., Anal. Biochem. 138: 267-284 (1984): Tm = 81.5 °C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L: where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matching probe. Tm is reduced by about 1°C for every 1% lack of coincidence; in this way, Tm and hybridization and/or washing conditions can be adjusted for hybridization to sequences with the desired identity. If sequences with > 90% identity are desired, for example, the Tm can be reduced by 10 °C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at defined pH and ionic strength. Severely stringent conditions may, however, require hybridization and/or washing at temperatures 1, 2, 3 or 4 °C lower than the thermal melting point (Tm); moderately stringent conditions may utilize hybridization and/or washing at 6, 7, 8, 9 or 10 °C below the thermal melting point (Tm); Low stringency conditions may utilize hybridization and/or washing at 11, 12, 13, 14, 15 or 20 °C below the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill in the art will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of lack of coincidence results in a Tm of less than 45 °C (aqueous solution) or 32 °C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used . An extensive guide to nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Overview of Principles of Hybridization and the Strategy of Nucleic Acid Probe Assays, Elsevier, Nova York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or washing conditions may be applied for at least ten, thirty, sixty, ninety, 120 or 240 minutes.

[072] “Sequence identity” or “identity”, in the context of polypeptide or nucleic acid sequences, designates the nucleic acid bases or amino acid residues in two sequences that are identical when aligned for maximum correspondence over a window of specified comparison.

[073] Thus, “percent sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., spaces) when compared to the reference sequence (which comprises neither additions nor deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue or nucleic acid base occurs in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the results by one hundred to generate the percent sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.

[074] Sequence alignments and percentage identity or similarity calculations can be determined using a series of comparison methods designed to detect homologous sequences that include, but are not limited to, the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc. , Madison WI). Within the context of the present application, it will be understood that when using sequence analysis software for analysis, the results of the analysis will be based on the “default values” of the indicated program, unless otherwise specified. As used herein, “default values” shall indicate any set of values or parameters originally loaded with the software when first initialized.

[075] The “Clustal V alignment method” corresponds to the Clustal V marked alignment method (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, D. G. et al. (1992), Comput. Appl . Biosci. 8: 189-191) and found in the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison WI). For multiple alignments, the default values correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are: WORD LENGTH = 1, GAP PENALTY = 3, BEST MATCH DISPLAY = 5, and DIAGONAL SPACING = 5. For nucleic acids, these parameters are WORD LENGTH = 2, GAP PENALTY = 5, BEST MATCH DISPLAY = 4, and DIAGONAL SPACING = 4. After aligning the sequences using the Clustal V program, it is possible to obtain a “percentage of identity” by observing the “sequence distances” table in the same program.

[076] “BLASTN alignment method” is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using standard parameters.

[077] Those skilled in the art well understand that many levels of sequence identity are useful in identifying polypeptides, from other species, in which these polypeptides have identical or similar function or activity. Useful examples of identity percentages include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any integer percentage from 50% to 100 %. In fact, any entire amino acid identity from 50% to 100% may be useful in describing the present invention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99%. Furthermore, any partial or full-length complement of this isolated nucleotide fragment is of interest.

[078] “Gene” indicates a nucleic acid fragment that expresses a specific protein, which includes regulatory sequences preceding (5' non-coding sequences) and subsequent (3' non-coding sequences) the coding sequence. “Native gene” indicates a gene found in nature with its own regulatory sequences. “Chimeric” gene designates any gene, other than a native gene, that comprises regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature. “Exogenous” gene indicates a gene not normally found in the host organism, but which is introduced into the host organism through genetic transfer. Exogenous genes may comprise native genes inserted into a non-native organism or chimeric genes. “Transgene” is a gene that has been introduced into the genome through a transformation procedure.

[079] The term “genome”, as it applies to a plant cell, encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found in subcellular components (such as mitochondria, plastids) of the cell.

[080] “Codon-optimized gene” is a gene that has its codon usage frequency designed to mimic the preferred codon usage frequency of the host cell.

[081] “Allele” is one of several alternative forms of a gene that occupies a given location on a chromosome. When all the alleles present at a given location on a chromosome are identical, that plant is homozygous at that location. If the alleles present at a given location on a chromosome are different, that plant is heterozygous at that location.

[082] “Coding sequence” indicates a DNA sequence that encodes a specific amino acid sequence. “Regulatory sequences” indicate nucleotide sequences located above (5' non-coding sequences), within or below (3' non-coding sequences) the coding sequence and which influence the transcription, stability or processing of RNA or translation of the coding sequence associated. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and loop and stem structures.

[083] “Promoter” designates a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of elements near and far in the flow, with the latter elements often called amplifiers. Accordingly, an “amplifier” is a DNA sequence that can stimulate promoter activity and can be an innate promoter element or a heterologous element inserted to amplify the level or tissue specificity of a promoter. Promoters can be derived entirely from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Those skilled in the art understand that different promoters can direct the expression of a gene in different tissues or cell types, at different stages of development, or in response to different environmental conditions. It is also recognized that, as in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments with some variation may have identical promoter activity. The promoters that cause a gene to be expressed in most cell types most of the time are commonly called “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; Numerous examples can be found in the compilation of Okamuro, J. K. and Goldberg, R. B., Biochemistry of Plants 15: 182 (1989).

[084] “Translation leader sequence” designates a sequence of polynucleotides located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence can affect the processing of the primary transcript into mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Mol. Biotechnol. 3: 225-236 (1995)).

[085] “3' non-coding sequences”, “transcription termination” or “termination sequences” designate DNA sequences at a location subsequent to a coding sequence and include polyadenylation recognition sequences and other regulatory signal coding sequences capable of affecting mRNA processing or gene expression. The polyadenylation signal is typically characterized by affecting the addition of traces of polyadenylic acid to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L. et al., Plant Cell 1: 671-680 (1989).

[086] “RNA transcript” means the product resulting from the RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is called a primary transcript. An RNA transcript is called mature RNA when it is an RNA sequence derived from post-transcriptional processing of the primary transcript. “Messenger RNA” or “mRNA” designates RNA that does not contain introns and that can be translated into protein by the cell. “cDNA” designates a DNA that is complementary to an mRNA template and synthesized from it using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted to double-stranded form using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to an RNA transcript that includes mRNA and can be translated into protein in a cell or in vitro. “Nonsense RNA” means an RNA transcript that is complementary to mRNA or a target primary transcript, in whole or in part, and that blocks expression of a target gene (US Patent 5,107,065). The complementarity of a nonsense RNA can occur with any part of the specific genetic transcript, that is, in the 5' non-coding sequence, 3' non-coding sequence, introns or in the coding sequence. “Functional RNA” designates nonsense RNA, ribozyme RNA, or other RNA that may not be translated but still has an effect on cellular processes. The terms “complement” and “reverse complement” are used interchangeably herein with respect to mRNA transcripts and are intended to define the nonsense RNA of the message.

[087] The expression “operationally linked” designates the association of nucleic acid sequences on an isolated nucleic acid fragment, in such a way that the function of one is regulated by the other. A promoter is operably linked to a coding sequence, for example, when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequences can be operatively linked to regulatory sequences in either sense or nonsense orientation. In another example, the complementary RNA regions according to the present invention can be operatively linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or in the target mRNA, or a first complementary region is 5'. ' and its complement is 3' to the target mRNA.

[088] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are more fully described in Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described above.

[089] “PCR” or “polymerase chain reaction” is a technique for synthesizing large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk CT). Typically, double-stranded DNA is denatured by heat, the two primers complementary to the 3’ boundaries of the desired segment are annealed at low temperature and then extended at intermediate temperature. A set of these three consecutive steps is called a “cycle”.

[090] The term “recombinant” designates an artificial combination of two other sequence segments separated in another way, such as through chemical synthesis or manipulation of isolated segments of nucleic acids through genetic engineering methods.

[091] The terms “plasmid”, “vector” and “set” designate an additional chromosomal element that often drives genes that are not part of the cell's central metabolism and usually in the form of circular double-stranded DNA fragments. These elements may be autonomously reproducing sequences, genome integration sequences, phage or nucleotide sequences, linear or circular, from a single- or double-stranded RNA or DNA derived from any source, into which a series of nucleotide sequences have been joined or recombined into a unique construct that is capable of introducing a promoter fragment and DNA sequence for a selected gene product together with appropriate 3' untranslated sequence into a cell. “Transformation set” means a specific vector that contains an exogenous gene and elements additional to the exogenous gene that facilitate transformation of a specific host cell. “Expression set” means a specific vector that contains an exogenous gene and elements additional to the exogenous gene that permit enhanced expression of that gene in an exogenous host (i.e., in a discrete nucleic acid fragment into which a sequence or nucleic acid fragment).

[092] The expressions “recombinant construction”, “expression construction”, “chimeric construction”, “construction” and “recombinant DNA construction” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, such as regulatory and coding sequences that are not found together in nature. A chimeric construct may comprise, for example, regulatory sequences and coding sequences that are derived from different sources or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature. This construction can be used on its own or can be used in conjunction with a vector. If a vector is used, vector selection depends on the method that will be used to transform host cells, as is well known to those skilled in the art. One can use, for example, a plasmid vector. Those skilled in the art are well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the nucleic acid fragments isolated in accordance with the present invention. Those skilled in the art will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4: 2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218 : 78-86 (1989)) and, therefore, several events must be selected in order to obtain lineages that exhibit the desired pattern and level of expression. This selection can be performed using Southern analysis of DNA, Northern analysis of mRNA expression, immunoblot analysis of protein expression or phenotypic analysis, among others.

[093] The term “expression”, as used herein, designates the generation of a functional terminal product (such as an mRNA or protein (whether precursor or mature)).

[094] The term “introduced” indicates the delivery of a nucleic acid (such as expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid may be incorporated into the cell's genome and includes reference to the transient delivery of a nucleic acid or protein to the cell. Introduced includes reference to methods of stable or transient transformation, as well as sexual crossing. Thus, “introduced” in the context of insertion of a nucleic acid fragment (such as a recombinant DNA construct/expression construct) into a cell, indicates “transfection”, “transformation” or “transduction” and includes reference to incorporation of a nucleic acid fragment in a eukaryotic or prokaryotic cell wherein the nucleic acid fragment can be incorporated into the cell's genome (such as chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replica, or expressed in a transient form (such as transfected mRNA).

[095] “Mature” protein designates a post-translationally processed polypeptide (i.e., one from which any pre- or pro-peptides present in the original translation product have been removed). “Precursor” protein designates the primary product of mRNA translation (i.e., with pre- and pro-peptides still present). Pre- and pro-peptides can be, but are not limited to, intracellular localization signals.

[096] “Stable transformation” means the transfer of a nucleic acid fragment to the genome of a host organism, including the nuclear and organellar genomes, resulting in genetically stable inheritance. On the other hand, “transient transformation” indicates the transfer of a nucleic acid fragment to the nucleus, or DNA-containing organelle, of a host organism, resulting in gene expression without stable integration or inheritance. Host organisms that contain the transformed nucleic acid fragments are called “transgenic” organisms.

[097] As used herein, “transgenic” designates a plant or cell that comprises a heterologous polynucleotide in its genome. Preferably, the heterologous polynucleotide is stably integrated into the genome, such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of an expression construct. Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of heterologous nucleic acid which includes transgenics initially altered in this way as well as those created through crossbreeding sexual or asexual propagation from the initial transgenic. The term “transgenic”, as used herein, does not encompass the alteration of the genome (chromosomal or extrachromosomal) through conventional plant breeding methods or naturally occurring events, such as random cross-fertilization, non-recombinant viral infection, transformation non-recombinant bacterial, non-recombinant transposition or spontaneous mutation.

[098] “Nonsense inhibition” designates the production of nonsense RNA transcripts capable of suppressing the expression of the target protein. “Cosuppression” means the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar endogenous or exogenous genes (US Patent 5,231,020). Cosuppression constructs in plants have previously been designed by focusing on the overexpression of a nucleic acid sequence that has homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA that has homology to the overexpressed sequence (Vaucheret et al., Plant J. 16: 651-659 (1998); Gura, Nature 404: 804-808 (2000)). The overall efficiency of this phenomenon is low and the extent of RNA reduction is widely variable. More recent work has described the use of “hairpin” structures that incorporate, in whole or in part, an mRNA coding sequence in a complementary orientation that results in a potential “stem and loop” structure for the expressed RNA ( document WO 99/53050, published October 21, 1999; WO 02/00904, published January 3, 2002). This increases the frequency of cosuppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression or "silencing" of nearby mRNA coding sequences (WO 98/36083, published August 20, 1998). These two cosuppression phenomena have not been elucidated mechanistically, although genetic evidence has begun to disentangle this complex situation (Elmayan et al., Plant Cell 10: 1747-1757 (1998)).

[099] The term “oilseed” designates organisms that tend to store their energy source in the form of lipid (Weete, in Fungal Lipid Biochemistry, second edition, Plenum, 1980). A class of plants identified as oilseeds is commonly called “oil-producing seed” plants. Examples of plants with oil-producing seeds include, but are not limited to: soybean (Glycine and Soja sp), flax (Linum sp), rapeseed (Brassica sp), corn, cotton, saffron (Carthamus sp) and sunflower (Helianthus sp).

[0100] Within oleaginous microorganisms, the TAG or cellular oil content generally follows a sigmoid curve, in which the lipid concentration increases until it reaches a maximum in the later logarithmic or early stationary phase and is then gradually reduced during the later stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57: 419-25 (1991)).

[0101] The term “oleaginous yeast” designates microorganisms classified as oil-producing yeasts. It is not uncommon for oleaginous microorganisms to accumulate more than about 25% of their dry cell weight in the form of oil. Examples of oleaginous yeasts include, but are not limited in any way to the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

[0102] The term “plant” designates whole plants, plant organs, plant tissues, seeds, plant cells, seeds and their offspring. Plant cells include, without limitation, seed cells, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

[0103] “Offspring” comprises any subsequent generation of a plant.

[0104] “Non-transgenic null segregating soybean seed” means a nearly isogenic seed or plant that does not contain the transgene and/or a parental plant used in the transformation process to obtain the transgenic event. Null segregants may be plants or seeds that do not contain the transgenic trait due to normal genetic segregation during the propagation of heterozygous transgenic plants.

[0105] “Seed” is the corn caryopsis, which consists of a mature embryo and endosperm, which are products of double fertilization. The term “corn” represents any variety, cultivar or population of Zea mays L.

[0106] “Grain” means mature corn seed produced by commercial growers for use on the farm or for sale to customers, in either case for purposes other than cultivation or reproduction of the species. The “seed” is the mature corn seed produced for the purpose of propagating the species and for sale to commercial growers. As used herein, the terms seed and grain may be used interchangeably. The “embryo” or also called “germ” is a young sporophytic plant, prior to the onset of a period of rapid growth (seed germination). The corn embryo (germ) contains the vast majority of the oil found in the seed. The embryo structure in the cereal grain includes the embryonic axis and the scutellum. The “scutellum” is the isolated cotyledon of a cereal grain embryo, specialized for absorption of the endosperm. “Aleurone” is a proteinaceous material, usually in the form of small granules, that occurs in the outermost cell layer of the endosperm of corn and other grains.

[0107] The present invention relates to a transgenic soybean seed that contains a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-transgenic null segregating soybean seed. It is understood that any measurable increase in the total fatty acid content of a transgenic over a non-transgenic null segregant would be useful. Such increases in total fatty acid content would include, but are not limited to, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12% , 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0108] A transgenic oilseed according to the present invention may comprise a recombinant construct that contains at least one DGAT sequence. This DGAT sequence can be selected from the group consisting of DGAT1, DGAT2, and DGAT1 in combination with DGAT2. Furthermore, at least one DGAT sequence may be from Yarrowia. Examples of suitable DGAT sequences that can be used to practice the present invention are discussed in the Examples below. SEQ ID NOs. 1,9, 64, 66, 82, 87 and 92 of the present invention may be mentioned. Those skilled in the art will appreciate that the present invention includes, but is not limited to, the DGAT sequences described herein.

[0109] Such a recombinant construct promoter would comprise different components, such as a promoter which is a DNA sequence that directs the cellular machinery of a plant to produce RNA from the contiguous coding sequence below in the stream (3') of the promoter. The promoter region influences the speed, stage of development and type of cell in which the gene's RNA transcript is produced. The RNA transcript is processed to produce mRNA that serves as a template for translating the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' untranslated leader sequence is a region of the mRNA upstream of the protein-coding region that may play a role in the initiation and translation of the mRNA. The 3' polyadenylation and transcription termination signal is an untranslated region downstream of the protein coding region that functions in the plant cell to cause termination of the mRNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.

[0110] The origin of the promoter selected to direct expression of the DGAT coding sequence is not important, as long as it possesses sufficient transcriptional activity to carry out the present invention through expression of mRNA translatable to the desired nucleic acid fragments in the tissue desired host at the right time. Heterologous or non-heterologous (i.e., endogenous) promoters can be used to practice the present invention. Suitable promoters include, for example, but are not limited to: the beta conglycinin promoter prime alpha subunit, the Kunitz trypsin inhibitor 3 promoter, the annexin promoter, the Gy1 glycinin promoter, the beta conglycinin promoter beta subunit, the P34/Gly Bd m 30K, the albumin promoter, the Leg A1 promoter and the Leg A2 promoter.

[0111] The annexin promoter, or P34, is described in WO 2004/071178 (published August 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol. 37: 275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15: 527-538 (1990); Holtorf et al., Plant Mol. Biol. 29: 637-646 (1995); ); Wilmink et al., Plant Mol. Biol. 28: 949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25: 193205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J. Biol. Chem. 265 (21): 12486-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene 211 (2): 26776 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol. Gen. Genet. 217 (2-3): 246-53 (1989)); and (6) a maize histone H3 gene promoter (Atanassova et al., Plant Mol. Biol. 37 (2): 275-85 (1989)).

[0112] Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before ten days after pollination) and is largely idle at later stages. The expression profile of the annexin promoter is different from several seed-specific promoters, such as seed storage protein promoters, which often provide higher activity at later stages of development (Chen et al., Dev. Genet. 10: 112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32: 10191027 (1996); ; read (above)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed-specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression or suppression of a gene is desired in embryos at an early stage of development. It may be desirable, for example, to overexpress an early embryo development regulatory gene or a gene involved in metabolism before seed ripening.

[0113] After identifying an appropriate promoter for expression of a specific DGAT coding sequence, the promoter is then operably linked in sense orientation using conventional means well known to those skilled in the art.

[0114] The molecular cloning and standard recombinant DNA methods used herein are well known in the art and described more fully in Sambrook, J. et al. in Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, New York, 1989 (hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K ., Eds.; in Current Protocols in Molecular Biology, John Wiley and Sons: New York, 1990 (hereafter, “Ausubel et al., 1990”).

[0115] In another aspect, the present invention relates to a method of increasing the total fatty acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct containing at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-transgenic zero segregating soybean seed.

[0116] After the recombinant construct has been made, it can then be introduced into a selected plant cell by methods well known to those skilled in the art (such as transfection, transformation and electroporation). Oilseed plant cells are the preferred plant cells. The transformed plant cell is then cultured and regenerated under appropriate conditions that allow selection of transformed soybean cell(s) that contain a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-transgenic null segregant soybean seed.

[0117] These recombinant constructs can be introduced into a plant cell; or alternatively, each construct may be introduced into separate plant cells.

[0118] Expression in a plant cell can be carried out in a transient or stable manner as described above.

[0119] Seeds or plant parts obtained from these transformed plants are also within the scope of the present invention.

[0120] Plant parts include differentiated and undifferentiated tissues that include, without limitation, the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and cultures (such as isolated cells, protoplasts, embryos, and callus tissue). Plant tissue may be present in a plant or in a plant organ, tissue, or cell culture.

[0121] The expression “plant organ” designates plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term “genome” designates the following: (1) the complete complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, virus, or organelle; and/or (2) a complete set of chromosomes inherited as a unit (haploid) from a parent.

[0122] Methods for transforming dicotyledons (mainly using Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for: cotton (US Patent 5,004,863; US Patent 5,159,135); soybean (US Patent 5,569,834; US Patent 5,416,011); Brassica (US Patent 5,463,174); peanut (Cheng et al., Plant Cell Rep. 15: 653-657 (1996); McKently et al., Plant Cell Rep. 14: 699-703 (1995)); papaya (Ling, K. et al., Bio/Technology 9: 752-758 (1991)); and peas (Grant et al., Plant Cell Rep. 15: 254-258 (1995)). For a review of other commonly used plant transformation methods, see Newell, C. A. (Mol. Biotechnol. 16: 53-65 (2000)). One such transformation method utilizes Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F., Microbiol. Sci. 4: 24-28 (1987)). Soybean transformation using direct DNA delivery has been published using PEG fusion (WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol. 3: 17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U. S. A. 84: 3962-3966 (1987)), microinjection, and particle bombardment (McCabe, D. E. et al., Bio/Technology 6: 923 (1988); Christou et al., Plant Physiol. 87: 671-674 (1988)).

[0123] There are a number of methods for regenerating plants from plant tissue. The specific regeneration method will depend on the initial plant tissue and the specific plant species to be regenerated. The regeneration, development and cultivation of plants from isolated plant protoplast transformers or from various transformed explants are well known in the art (Weissbach and Weissbach, in: Methods for Plant Molecular Biology (Eds.), Academic: San Diego CA (1988)). This process of regeneration and growth typically includes the steps of selecting transformed cells and cultivating the individualized cells through the usual steps of embryonic development throughout the rooted seedling stage. Transgenic embryos and seeds are regenerated in a similar way. The resulting transgenic rooted roots are then planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from regenerated plants is crossed with plants grown with seeds from agronomically important lines. On the other hand, pollen from plants from these important lineages is used to pollinate regenerated plants. A transgenic plant according to the present invention that contains a desired polypeptide is cultivated using methods well known to those skilled in the art.

[0124] In addition to the procedures discussed above, practitioners are familiar with standard resource materials that describe specific conditions and procedures for: the construction, manipulation and isolation of macromolecules (such as DNA molecules, plasmids, etc.); the generation of recombinant DNA fragments and recombinant expression constructs; and the selection and isolation of clones. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: New York (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: New York (1995); Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold Spring Harbor: New York (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: New York (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: New York (1997).

[0125] Examples of oilseed plants include, but are not limited to: soybeans, Brassica species, sunflower, corn, cotton, flax, and safflower.

[0126] In another aspect, the present invention relates to a transgenic corn seed that contains a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-null segregating corn seed. transgenic. Such transgenic corn seed may comprise a recombinant construct that contains at least one DGAT sequence. This DGAT sequence can be selected from the group consisting of DGAT1, DGAT2, or DGAT1 in combination with DGAT2.

[0127] In yet another aspect, the present invention relates to a method of increasing the total fatty acid content of a corn seed comprising: (a) transforming at least one corn seed with a recombinant construct containing at least one DGAT sequence; and (b) selecting the processed corn seed(s) from step (a) that contains a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-transgenic null segregating corn seed.

[0128] The present invention also relates to a transgenic soybean seed that contains at least 10% increased total fatty acid content and at least 25% increased oleic acid content when compared to the total fatty acid content and oleic acid content of a non-transgenic null segregant soybean seed. Furthermore, the present invention further relates to a transgenic soybean that contains at least 10% increased total fatty acid content and at least any one of (i) an oleic acid content increased by at least 25%; (ii) a linolenic acid content reduced by at least 25%; (iii) a linoleic acid content reduced by at least 4%; (iv) a palmitic acid content reduced by at least 8%; and (v) a stearic acid content increased by at least 14% when compared to the total fatty acid and oleic acid, linolenic acid, linoleic acid, palmitic acid or stearic acid content, respectively, of a non-transgenic null-segregating soybean seed.

[0129] In yet another aspect, the present invention also relates to a method of increasing the total fatty acid content and oleic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and oleic acid content increased by at least 25% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[0130] In yet another aspect, the present invention relates to a method of increasing the total fatty acid content and reducing the linolenic acid content of a soybean seed comprising: (a) transforming at least one cell of soybean with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linolenic acid content reduced by at least 25% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[0131] Still again in a further aspect, the present invention relates to a method of increasing the total fatty acid content and reducing the linoleic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and linoleic acid content reduced by at least 4% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[0132] Still again in a further aspect, the present invention relates to a method of increasing the total fatty acid content and reducing the palmitic acid content of a soybean seed comprising: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and palmitic acid content reduced by at least 8% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[0133] In yet another additional aspect, the present invention relates to a method of increasing the total fatty acid content and stearic acid content of a soybean seed comprising: (a) transforming at least one cell of soybean with a recombinant construct that contains at least one DGAT sequence; and (b) selecting the transformed soybean cell(s) from step (a) that contain a total fatty acid content increased by at least 10% and stearic acid content increased by at least 14% when compared to the total fatty acid content and oleic acid content of a non-GMO null segregating soybean seed.

[0134] As discussed above, any of the transgenic oilseeds discussed herein may comprise a recombinant construct that contains at least one DGAT sequence. This DGAT sequence can be selected from the group consisting of DGAT1, DGAT2 or DGAT1 in combination with DGAT2. Furthermore, at least one DGAT sequence is from Yarrowia.

[0135] The transformation of monocots using electroporation, particle bombardment and Agrobacterium has been reported. Plant transformation and regeneration have been achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (U. S. A.) 84: 5354 (1987)); barley (Wan and Lemaux, Plant Physiol. 104: 37 (1994)); Zea mays (Rhodes et al., Science 240: 204 (1988), Gordon-Kamm et al., Plant Cell 2: 603-618 (1990), Fromm et al., Bio/Technology 8: 833 (1990), Koziel et al., Bio/Technology 11: 194 (1993), Armstrong et al., Crop Science 35: 550-557 (1995); oats (Somers et al., Bio/Technology 10: 15-89 (1992)); orchid (Horn et al., Plant Cell Rep. 7: 469 (1988)); rice (Toriyama et al., Theor. Appl. Genet. 205: 34 (1986); Part et al., Plant Mol. Biol. 32 : 1135-1148 (1996); , Plant Cell Rep. 7: 379 (1988); Battraw and Hall, Plant Sci. 86: 191-202 (1992); Christou et al., Bio/Technology 9: 957 (1991)); al., Nature 325: 274 (1987)); sugar cane (Bower and Birch, Plant J. 2: 409 (1992)); wheat (Vasil et al., Bio/Technology 10:667 (1992); US Patent 5,631,152).

[0136] Gene expression tests based on the transient expression of cloned nucleic acid constructs have been developed by introducing nucleic acid molecules into plant cells through treatment with polyethylene glycol, electroporation or particle bombardment (Marcotte et al. , Nature 335: 454-457 (1988); Marcotte et al., Plant Cell 1: 523-532 (1989); McCarty et al., Cell 66: 895-905 (1991); 6: 609-618 (1992); Goff et al., EMBO J. 9: 2517-2522 (1990)).

[0137] Transient expression systems can be used to functionally dissect genetic constructs (see, generally, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995)). It is understood that any of the nucleic acid molecules according to the present invention can be introduced into a plant cell permanently or transiently in combination with other genetic elements such as vectors, promoters, amplifiers, etc.

[0138] In addition to the procedures discussed above, practitioners are familiar with standard resource materials that describe specific conditions and procedures for constructing, manipulating, and isolating macromolecules (such as DNA molecules, plasmids, etc.), generating recombinant organisms, and the selection and isolation of clones (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995) ; Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, New York (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New York (1998); : A Laboratory Manual, Eds. Clark, Springer, New York (1997)).

[0139] The transgenic soybean seeds according to the present invention can be processed to generate soybean oil, soybean products and/or soybean by-products.

[0140] “Soy products” may include, but are not limited to, the items listed in Table 1A. TABLE 1A SOY PROTEIN PRODUCTS DERIVED FROM SOY SEEDS a See Soy Protein Products: Characteristics, Nutritional Aspects and Utilization (1987), Soy Protein Council.

[0141] “Processing” means any physicochemical method used to obtain the products listed in Table 1A and includes, but is not limited to, hot conditioning, flaking and grinding, extrusion, solvent extraction or aqueous soaking and extraction of whole or partial seeds.

[0142] Furthermore, “processing” includes the methods used to concentrate and isolate soy protein from whole or partial seeds, as well as the various traditional Eastern methods of preparing fermented soy food products.

[0143] Commercial Standards and Specifications have been established for many of these products (see National Oilseed Processors Association Yearbook and Trading Rules 1991-1992).

[0144] Products designated as being “high protein” or “low protein” are those described by the Standard Specifications. “NSI” designates the Nitrogen Solubility Index as defined using North American Society of Oil Chemists Method Ac4 41. “KOH Nitrogen Solubility” is an indicator of soybean mass quality and designates the amount of soluble nitrogen in 0.036 M KOH under the conditions described by Araba and Dale ((1990) Poult. Sci. 69: 76-83).

[0145] “White” flakes designate cotyledons peeled and processed into flakes that have been skimmed and heat treated with controlled humidity to have an NSI of about 85 to 90. This expression can also designate a flour with a similar NSI that has been milled until passed through a US Standard Sieve size #100. “Cooked” refers to a soy protein product, typically a flour, with an NSI of about 20 to 60.

[0146] “Roasted” designates a soy protein product, typically a flour, with an NSI below 20. “Brans” designates peeled and skimmed cotyledons that have a North American standard sieve size No. 10 to 80.

[0147] “Soy protein concentrates” designate products made from skimmed and peeled soybeans through three basic processes: acid leaching (about pH 4.5), alcohol extraction (about 55 to 80 %) and denaturation of the protein with moist heat before extraction with water.

[0148] Conditions typically used to prepare soy protein concentrates were described by Pass ((1975) US Patent 3,897,574; Campbell et al. (1985) in New Protein Foods, Ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10, Seed Storage Proteins, pp. 302-338).

[0149] “Extrusion” designates processes whereby materials (brans, flour or concentrate) pass through a jacketed auger using high pressures and temperatures as a means of altering the texture of the material. “Texturing” and “structuring” designate extrusion processes used to modify the physical characteristics of the material.

[0150] The characteristics of these processes, which include thermoplastic extrusion, have been described previously [Atkinson (1970), US Patent 3,488,770, Horan (1985) in New Protein Foods, Ed. by Altschul and Wilcke, Academic Press, Vol. 1A , Chapter 8, pp. 367-414].

[0151] Furthermore, conditions used during extrusion processing of complex food mixtures that include soy protein products have been described previously (Rokey (1983), Feed Manufacturing Technology III, 222-237; McCullouch, US Patent 4,454,804 ).

[0152] More specifically, soybean seeds are cleaned, seasoned, peeled and transformed into flakes, in order to increase the efficiency of oil extraction. Oil extraction is typically accomplished through solvent extraction (such as hexane), but can also be achieved through a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. Crude oil can have fat removed through the hydration of phospholipids and other polar and neutral lipid complexes that facilitate its separation from the non-hydrating triglyceride fraction (soybean oil).

[0153] The resulting lecithin gums can be further processed to produce commercially important lecithin products used in a number of food and industrial products as emulsifying and releasing agents (i.e., anti-caking agents). Oil with fat removed can be further refined to remove impurities (mainly free fatty acids, pigments and residual gums). Refining is accomplished by adding a caustic agent that reacts with free fatty acid to form soap and hydrates phosphatides and proteins in the crude oil. Water is used to wash traces of soap formed during refining. The soap by-product can be used directly in animal or acidified foods to recover free fatty acids. The coloring is removed through adsorption with a bleaching earth that removes most of the chlorophyll and carotenoid compounds. Refined oil can be hydrogenated to result in fats with different textures and melting properties. Winterization (fractionation) can be used to remove stearin from hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization (primarily through vacuum vapor distillation) is the last step and is designed to remove compounds that impart odor or aroma to the oil. Other valuable by-products such as tocopherols and sterols can be removed during the deodorization process. Deodorized distillate containing these byproducts can be sold for the production of natural vitamin E and other high-value pharmaceutical products. Refined, leached (hydrogenated, fractionated) and deodorized fats and oils can be packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production, and utilization of byproducts can be found in Erickson, Practical Handbook of Soybean Processing and Utilization, The American Oil Chemists’ Society and United Soybean Board (1995). Soybean oil is liquid at room temperature because it contains relatively low saturated fatty acids when compared to oils such as coconut, palm, palm kernel and cocoa butter.

[0154] Vegetable and microbial oils containing PUFAs that have been refined and/or purified can be hydrogenated to result in fats with various textures and melting properties. Many processed fats (including spreaders, confectionery fats, hard butters, margarines, baking toppings, etc.) require varying degrees of solidity at room temperature and can only be produced by altering the physical properties of the source oil. This is most commonly achieved through catalytic hydrogenation.

[0155] Hydrogenation is a chemical reaction in which hydrogen is added to the double bonds of unsaturated fatty acids with the help of a catalyst such as nickel. High-oleic soybean oil, for example, contains unsaturated oleic, linoleic, and linolenic fatty acids, each of which can be hydrogenated. Hydrogenation has two primary effects. Firstly, the oxidative stability of the oil increases as a result of the reduction in the unsaturated fatty acid content. Second, the physical properties of the oil are changed because fatty acid modifications increase the melting point, resulting in a semi-liquid or solid fat at room temperature.

[0156] There are several variables that affect the hydrogenation reaction which, in turn, change the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation, and reactor design are among the most important parameters that can be controlled. Selective hydrogenation conditions can be used to hydrogenate more unsaturated fatty acids in preference over less unsaturated ones. Very light or rapid hydrogenation is often employed to increase the stability of liquid oils. Further hydrogenation converts a liquid oil into a physically solid fat. The degree of hydrogenation depends on the desired melting and performance characteristics designed for the specific end product. Liquid coatings (used in the manufacture of baked goods, solid fats and coatings used for commercial frying and roasting operations) and base stocks for the manufacture of margarine are among the range of possible fat and oil products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice, North American Society of Oil Chemists (1994).

[0157] Hydrogenated oils have become somewhat controversial due to the presence of trans fatty acid isomers resulting from the hydrogenation process. Ingestion of large amounts of trans isomers has been linked to detrimental health effects that include higher ratios of low-density to high-density lipoproteins in blood plasma and increased risk of coronary heart disease.

[0158] In yet another aspect, the present invention relates to an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having diacylglycerol acyltransferase activity, wherein the polypeptide is described in SEQ ID NO 83, 88 or 93; or (b) a complement of the nucleotide sequence of (a), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

[0159] Furthermore, a recombinant DNA construct comprising the isolated nucleic acid fragment operatively linked to at least one regulatory sequence. Likewise, a cell that comprises the construction of recombinant DNA in its genome. The cell may be part of an oilseed plant, such as, but not limited to, soybeans, corn, canola, sunflower, flax, cotton and safflower.

[0160] Finally, the present invention relates to a method for increasing the total fatty acid content of an oilseed comprising: (a) transforming at least one oilseed plant cell with the aforementioned recombinant construct; and (b) selecting the transformed oilseed plant cell(s) from step (a) that contain an increased total fatty acid content when compared to the total fatty acid content of a non-transgenic null segregant oilseed.

EXAMPLES

[0161] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise indicated. It should be understood that these Examples, while indicating preferred embodiments of the present invention, are provided solely by way of illustration. From the above discussion and these Examples, those skilled in the art can determine the essential features of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the present invention to adapt it to various uses and conditions. Accordingly, various modifications of the present invention in addition to those shown and described herein will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0162] The meaning of abbreviations is as follows: “mon” indicates second(s), “min” indicates minute(s), “h” indicates hour(s), “d” indicates day(s), “μl” indicates microliter(s), “ml” indicates milliliter(s), “l” indicates liter(s), “μM” indicates micromolar, “mM” indicates millimolar, “M” indicates molar, “mmol” indicates millimol(s) , “μmol” indicates micromol(s), “g” indicates gram(s), “μg” indicates microgram(s), “ng” indicates nanogram(s), “U” indicates unit(s), “bp” indicates base pair(s) and “kB” Petition 870170085071, dated 11/06/2017, p. 71/206 61/185 indicates kilobase(s). EXAMPLE

EXAMPLE 1 EXPRESSION OF DGAT GENES FROM YARROWIA LIPOLYTICA IN SACCHAROMYCES CEREVISIAE:

[0163] The DGAT1 gene (SEQ ID NO. 1) from Yarrowia lipolytica was excised from the plasmid vector pYDA1 (SEQ ID NO. 2) through restriction digestion with NcoI and NotI. The DNA fragment ends were completely filled using T4 DNA polymerase (Promega, Madison WI, United States) and ligated to the unique Not I site of pY75 (SEQ ID NO: 3). Before its use for cloning, the pY75 vector had been linearized with NotI, filled in with T4 DNA polymerase and dephosphorylated with shrimp alkaline phosphatase (NEB, Beverly MA, United States). Plasmid DNA was isolated using standard methods and EcoRI restriction digests were conducted to identify plasmid clones in which the start codon was located near the 3' end of the GPD promoter in pY75 (sense orientation of the DGAT1 gene). This plasmid is hereinafter called pY75 YL DGAT1 (SEQ ID NO. 4). The construction of pYDAI is described in WO 2006/052914, which is incorporated herein by reference.

[0164] The yeast episomal plasmid (YEp)-type vector pRS425 (SEQ ID NO. 5) (Christianson et al., Gene 110: 119-122 (1992)) contains sequences from the endogenous two-micron plasmid of Saccharomyces cerevisiae, a LEU2 selectable marker and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK(+). The strong constitutive glyceraldehyde dehydrogenase (GPD) 3-phosphate promoter from Saccharomyces cerevisiae was cloned between the SacII and SpeI sites of pRS425 in the same way as described by Jia et al. (Physiol. Genomics 3: 83-92 (2000)) to produce pGPD-425 (SEQ ID NO. 6). A NotI site was introduced into the BamHI site of pGPD-425, in order to generate a NotI site flanked by BamHI sites and this plasmid was named pY75 (SEQ ID NO. 3). The DGAT2 gene was amplified by PCR from the genome of Yarrowia lipolytica (ATCC accession no. 20362) as follows. Yeast cells were cultured on solid YPD medium for 72 hours. Cells were resuspended in 200 μl of DNA extraction buffer (100 mM Tris, pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.1% Triton X-100) and supplemented with two to five glass beads (3 mm diameter) and about 0.1 g of glass beads (0.5 mm diameter). The yeast cell suspension was mixed vigorously using a vortex mixer and incubated at 75 °C for 25 minutes. The lysate was cooled to room temperature and cleared by centrifugation.

[0165] The following two oligonucleotide primers were used to generate a PCR fragment of about 1600 bp: oYLDGAT2-1: GCGGCCGCATGACTATCGACTCAATACTACAAGT (SEQ ID NO. 7); and oYLDGAT2-2: GCGGCCGCTTACTCAATCATTCGGAACTCTGGGGCT (SEQ ID NO. 8).

[0166] Briefly, a PCR reaction mixture (100 μl) was created that contains 2.5 mM MgCl2, 2 mM dNTPs, 10 mM Tris/HCl (pH 8.8), 50 mM KCl, 0 .08% Nonidet P40, 1 μM of oYLDGAT2-1 (SEQ ID NO. 7) and oYLDGAT2-2 (SEQ ID NO. 8), 10 U Taq polymerase (Fermentas, Hanover MD) and 2 μl of yeast lysate. The PCR mixture was divided into four 25-μl portions and amplification was conducted for 35 cycles, each comprising 45 seconds at 94°C, 45 seconds at the corresponding combining temperature, and one minute at 72°C. PCR products were gel purified and cloned into pGEM T-easy (Promega) using the manufacturer's instructions.

[0167] Ten independent plasmid clones were completely sequenced. The consensus sequence of this analysis is defined as SEQ ID NO. 9. This DNA sequence differs from the DGAT2 sequence described in WO 2005/003322 at two different nucleotide positions. The DNA sequence difference affects nt 448 and nt 672 of the DGAT2 open reading frame. The previous nt difference alters the predicted amino acid sequence of the DGAT protein. It replaces a serine found in the DGAT sequence described in WO 2005/003322 with a threonine residue. The second sequence difference does not change the amino acid sequence from that described in WO 2005/003322. The difference between the Yarrowia lipolytica DGAT2 sequence described herein and that of WO 2005/003322 can be attributed to the different strains of Yarrowia lipolytica that were used for isolation of the DGAT2 gene. The predicted amino acid sequence of the DGAT protein from the ATCC Accession No. 20362 strain is described in SEQ ID No. 10.

[0168] The DGAT gene (SEQ ID NO. 9) was excised in the form of a Not I restriction fragment from the pGEM T-easy vector and ligated to dephosphorylated DNA linearized with Not I from pY75 (SEQ ID NO. 3). Plasmid DNA was isolated from recombinant clones and restriction digestion with SacI and PacI allowed the identification of clones in which the start codon of the DGAT2 gene was close to the 3' end of the GPD promoter in pY75 (sense orientation of the gene DGAT2). This plasmid is hereinafter called pY75 YL DGAT2 (SEQ ID NO. 11).

[0169] Plasmid DNA of pY75 YL DGAT1 (SEQ ID NO: 4) and empty pY75 vector were transformed into Saccharomyces cerevisiae strain INVSC1 (Invitrogen, United States) using standard methods (Gletz, R. Daniel; Woods, Robin A., Meth. Enzymol. 350: 87-96 (2002)). Recombinant yeast colonies were selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad CA). Five 50 ml cultures of DOBA media supplemented with CSM-leu were inoculated with five independently generated colonies and grown at 30 °C for 72 h.

[0170] The cells were harvested by centrifugation and resuspended in a medium identical to the DOBA medium described above, with the exception of the omission of ammonium sulfate as a nitrogen source.

[0171] The cultures were grown for an additional sixty hours and the cells were harvested through centrifugation. Cells were grown on dry ice and lyophilized.

[0172] The total fatty acid content of each yeast cell sample was measured in triplicate as follows. Approximately 5 to 15 mg of yeast powder was weighed into the bottom of a 13 x 100 mm glass culture tube with a screw cap and Teflon seal. 5 μL of a 17:0 TAG stock solution (10 mg/ml in toluene) was added, followed by the addition of 500 μL of 5% sulfuric acid in methanol (anhydrous). Samples were incubated at 95 °C for one and a half hours.

[0173] Then, tubes were kept cooled at room temperature, after which 1 ml of 1 M sodium chloride was added, followed by mixing. 1 ml of heptane was added, the contents were mixed and the samples were quickly centrifuged to mediate phase separation.

[0174] About 500 μl of the organic phase was transferred to a GC ampoule. Fatty acid methyl esters were analyzed using gas chromatography. Four microliters of heptane extract was analyzed on a Hewlett-Packard 6890 Gas Chromatograph equipped with an Omegawax 320 fused silica capillary column (Supelco Inc., Catalog No. 24152). The oven temperature was programmed to hold at 220°C for 2.7 minutes, increase to 240°C at 20°C/minute, and then hold for an additional 2.3 minutes. Vehicle gas was supplied by a Whatman hydrogen generator. Retention times were compared to those of commercially available standard methyl esters (Nu-Chek Prep, Inc., catalog no. U-99-A).

[0175] Plasmid DNA of pY75 YL DGAT2 (SEQ ID NO: 11) and the empty pY75 vector were transformed into Saccharomyces cerevisiae strain INVSC1 and the total fatty acid content of yeast cultures was analyzed as previously described. The findings regarding overexpression of the two DGAT genes in yeast are summarized in Table 2. TABLE 2 TOTAL FATTY ACID CONTENT OF SACCHAROMYCES CEREVISIAE CULTURES

[0176] Table 2 demonstrates that there is a significant increase in total fatty acids in yeast cells that harbor pY75 YL DGAT1 (SEQ ID NO. 4) when compared to cells that contain only the empty pY75 plasmid. The average fatty acid dry cell weight (DCW) percentage of five independent cultures is 11.9% when compared to 9.3% for vector controls grown under identical conditions. In short, there is a 28% increase in the total production of fatty acids. Furthermore, there is a slight change in the fatty acid profile associated with the expression of YL DGAT1 characterized by an increase in palmitic acid.

[0177] Constitutive expression of YL DGAT2 under nitrogen starvation increased total fatty acid content by 110% when compared to a vector control grown under identical conditions. The total fatty acid content of the vector-only control was 6.6%, although the average fatty acid content of the YL DGAT2 transformers was 14.2%. The fatty acid profile was altered as a result of YL DGAT2 expression. The palmitic acid content increased significantly, accompanied by a moderate reduction in palmitoleic acid. Furthermore, the fatty acid content increased significantly, accompanied by a clear reduction in the oleic content. Taken together, the results demonstrate that overexpression of YL DGATs in yeast under conditions of higher carbon to nitrogen ratios generates increased accumulation of fatty acids. Overexpressed YL DGAT enzymes are capable of increasing endogenous DGAT activity in Saccharomyces cerevisiae. Similar experiments were repeated with the two DGAT genes twice more. A difference in fatty acid content between the YL DGAT culture and the vector control could always be observed and was at least 8% and 14.3% for YL DGAT1 (SEQ ID NO. 1) and YL DGAT2 (SEQ ID No. 9), respectively.

EXAMPLE 2 CLONING OF DGAT1 AND DGAT2 FROM YARROWIA LIPOLYTICA INTO YARROWIA LIPOLYTICA EXPRESSION VECTORS

[0178] The present Example describes the generation of pFBAIN-YDG1 and pFBAIN-YDG2, which comprise a chimeric FBAINm::YDGAT1::PEX20 gene and a chimeric FBAINm::YDGAT2::PEX20 gene, respectively (Figures 1A and 1B). These were designed to overexpress DGAT1 and DGAT2 in Yarrowia lipolytica.

[0179] Oligonucleotides YDGAT1-F (SEQ ID NO. 12) and YDGAT-R (SEQ ID NO. 13) were designed and synthesized to allow amplification of the DGAT1 ORF from Yarrowia lipolytica genomic DNA (isolated from the strain with ATCC Accession No. 20362, purchased from the North American Type Cultivation Collection (Rockville MD)), while oligonucleotides YDGAT2-F (SEQ ID No. 14) and YDGAT2-R (SEQ ID No. 15) were designed and synthesized to allow amplification of the DGAT2 ORF.

[0180] PCR reactions, with Yarrowia lipolytica genomic DNA as template, were conducted individually in a total volume of 50 μl comprising: 1 μl of genomic DNA (100 ng), 10 μl of 5X PCR buffer, 1 μl of dNTP mixture (10 μM each), 35 μl of water, and 1 μl of Phusion polymerase (New England Biolabs, Inc., Ipswich MA). Amplification was conducted at 98°C for one minute, followed by thirty cycles at 98°C for ten seconds, 55°C for ten seconds, and 72°C for thirty seconds, followed by a final cycle of elongation at 72°C for five minutes. A 1603 bp DNA fragment (SEQ ID NO. 16) and a 1567 bp fragment (SEQ ID NO. 17) were generated that contained the DGAT1 and DGAT2 ORFs, respectively.

[0181] The PCR fragments were purified with Qiagen PCR purification kits following the manufacturer's protocol. Purified DNA samples were digested with NcoI and NotI, purified with a Qiagen reaction cleanup kit, and then directionally ligated with NcoI/NotI-digested pFBAIN-MOD-1 (Figure 1C; SEQ ID NO: 18). Specifically, the ligation reaction contained: 10 μl of 2X binding buffer, 1 μl of T4 DNA ligase (Promega), 4 μl (about 300 ng) of the 1600 bp fragment (i.e., DGAT1; SEQ ID NO: 16 ) or the 1564 bp fragment (i.e., DGAT2; SEQ ID NO: 17) and 1 μl of pFBAIN-MOD-1 (about 150 ng). The reaction mixtures were incubated at room temperature for two hours and used to transform competent E. coli Top10 cells (Invitrogen). Plasmid DNA from transformers was recovered using a Qiagen Miniprep kit. Correct clones were identified through restriction mapping and the final constructs were named “pFBAIN-YDG1” and “pFBAIN-YDG2”, respectively.

[0182] Thus, pFBAIN-YDG1 (Figure 1A; SEQ ID NO. 19) contained the following components: TABLE 3 COMPONENTS OF THE PFBAIN-YDG1 PLASMID (SEQ ID NO. 19)

[0183] The pFBAIN-YDG2 plasmid (Figure 1B; SEQ ID No. 20) contained components identical to those of pFBAIN-YDG1, with the exception of the presence of DGAT2 ORF from Yarrowia lipolytica (SEQ ID No. 17; identified as YDG2 in Figure 1B ) instead of the DGAT1 ORF from Yarrowia lipolytica in pFBAIN-YDG1.

[0184] The term “FBAINm promoter” or “FBAINm promoter region” is a modified version of the FBAIN promoter (below), in which FBAINm has a 52 bp deletion between the ATG translation start codon and the intron of the FBAIN promoter (to include only 22 N-terminal amino acids) and a new translation consensus motif after the intron. Furthermore, although the FBAIN promoter generates a fusion protein when fused with the coding region of a gene to be expressed, the FBAINm promoter does not generate such a fusion protein. The FBAIN promoter designates the untranslated region upstream in the 5' stream in front of the translation start codon “ATG” of the fructose-bisphosphate aldolase enzyme from Yarrowia lipolytica (E.C. 4.1.2.13) encoded by the fba1 gene and which is required for expression, plus a part of the 5' coding region that has an intron from the fba1 gene. These promoters are described in detail in WO 2005/049805 and US Patent 7,202,356, which are incorporated herein in their entirety by reference.

EXAMPLE 3 OVEREXPRESSION OF YARROWIA LIPOLYTICA GENES DGAT1 AND DGAT2 IN YARROWIA LIPOLYTICA LINE Y2224:

[0185] The present Example describes increased fatty acid content and modification to the relative abundance of each fatty acid substance in Yarrowia lipolytica strain Y2224 that was transformed to coexpress Yarrowia lipolytica DGAT1 (SEQ ID NO. 16) or DGAT2 of Yarrowia lipolytica (SEQ ID NO. 17). Strain Y2224 is a FOA-resistant mutant of an autonomous mutation of the Ura3 gene from wild-type Yarrowia strain ATCC Accession No. 20362.

[0186] Generation of strain Y2224: strain Y2224 was isolated as follows: Yarrowia lipolytica cells with ATCC Accession No. 20362 from a YPD Agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% Agar) were streaked onto a plate with minimal media (75 mg/l each of uracil and uridine, 6.7 g/l of YNB with ammonium sulfate, without amino acid, and 20 g/l of glucose) containing 250 mg/l of 5 -FOA (5-fluoroacyl-6-carboxylic acid monohydrate; Zymo Research). Plates were incubated at 28°C and four of the resulting colonies were patched separately onto plates with minimal media (MM) containing 200 mg/ml 5-FOA and plates with MM lacking uracil and uridine to confirm auxotrophy of uracil Ura3.

[0187] Transformation of Y2224 strain: a clone of pFBAIn-YDG1, a clone of pFBAIn-YDG2 and control plasmid pFBAIN-MOD-1 were transformed into Yarrowia lipolytica strain Y2224 as described below.

[0188] The transformation of Yarrowia lipolytica was carried out according to the method of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48 (2): 232-235 (1997)), unless otherwise indicated. Briefly, Yarrowia was streaked onto a YPD Agar plate and grown at 30°C for about eighteen hours. Several large circuits of cells were scraped from the plate and resuspended in 1 ml of transformation buffer containing: 2.25 ml of 50% PEG, average MW 3350; 0.125 ml of 2 M lithium acetate, pH 6.0; 0.125 ml of 2 M DTT; and 50 μg of sheared salmon sperm DNA. Next, about 500 ng of linearized plasmid DNA was incubated in 100 μl of resuspended cells and maintained at 39°C for one hour with mixing by vortexing at fifteen minute intervals.

[0189] Cells from each transformation were plated with minimal media (MM) that does not contain uracil (0.17% yeast nitrogen base (DIFCO Laboratories, Detroit MI) without ammonium sulfate or amino acids, 2% glucose , 0.1% proline, pH 6.1, 20 g/l Agar) and kept at 30 °C for two days. Three transformers from each transformation plate were used to inoculate 25 ml individual cultures in MM medium (0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Each culture was kept growing for two days at 30 °C and then switched to 25 ml of high glucose medium (“HG medium”, which comprises 80 g/l glucose, 27 g/l K2HPO4, 6 .3 g/l of KH2PO4, pH of about 7.5) and kept in growth for five days at 30 °C.

[0190] Lipid analysis: Total lipids were extracted and fatty acid methyl esters (FAMEs) were prepared via transesterification and then analyzed with a Hewlett-Packard 6890 GC.

[0191] More specifically, for fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. and Dyer, W. J. (Can. J. Biochem. Physiol. 37: 911-917 (1959 )). Fatty acid methyl esters were prepared by transesterification of lipid extract with sodium methoxide (Roughan, G. and Nishida, I., Arch. Biochem. Biophys. 276 (1): 38-46 (1990)) and analyzed then with a Hewlett-Packard 6890 GC equipped with a 30 m x 0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170 °C (hold for 25 minutes) to 185 °C at 3.5 °C/minute.

[0192] For direct base transesterification, Yarrowia culture (3 ml) was harvested, washed once in distilled water and vacuum dried in a Speed-Vac for five to ten minutes. Sodium methoxide (100 μl of 1%) was added to the sample and then the sample was vortexed and stirred for twenty minutes. After adding three drops of 1 M NaCl and 400 μl of hexane, the sample was vortexed and centrifuged. The top layer was removed and analyzed using GC as described above.

[0193] Based on the above analyses, lipid content and composition were determined in Y2224 transforming lines, comprising pFBAIn-YDG1, pFBAIn-YDG2 and pFBAIN-MOD-1 (control), respectively, as shown below in the Table 4. Three independent transformers from each strain were analyzed and the average results are shown in the rows highlighted in gray. Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), and 18:2 (LA); and the composition of each of them is presented as a percentage of total fatty acids.

[0194] “% FAME/DCW” represents the percentage by weight of dry fatty acid methyl ester cells. Dry cell weight was determined by collecting cells from 10 ml of culture via centrifugation, washing the cells with water once to remove residual medium, drying the cells in a vacuum oven at 80°C for one overnight and weighing the dry cells. The total amount of fatty acid methyl esters in a sample was determined by comparing the areas of all peaks in the GC profile to the peak area of a known amount spiked with internal standard C15:0 fatty acid. TABLE 4 COMPOSITION AND LIPID CONTENT IN YARROWIA LINE Y2224 THAT OVEREXRESSES YDGAT1 AND YDGAT2

[0195] GC analyzes demonstrated that there was a significant increase in total fatty acids in cells carrying pFBAIn-YDG1, when compared to cells carrying pFBAln-MOD-1. The average fatty acid increased from 21.39% FAME/DCW in control to 23.70% FAME/DCW in YDGAT1-expressing cells (i.e., 10.8% increase). Furthermore, there was also an increase in the amount of C16:1 and C18:1 fatty acids and a reduction in C18:2 fatty acid (Table 4).

[0196] Cells carrying pFBAln-YDG2 also showed a large increase in total fatty acid content compared to the control, which results in an average of 27.32% FAME/DCW (which represents an increase of 27.7%. The distribution of fatty acid species was also significantly changed. Specifically, the amount of C18:0 and C18:1 increased, while the amount of C16:0, C16:1 and C18:2 was reduced.

[0197] Collectively, these results demonstrate that overexpression of DGAT1 or DGAT2 from Yarrowia lipolytica has an impact on the total lipid content and the relative abundance of each fatty acid species.

EXAMPLE 4 EXPRESSION OF YARROWIA LIPOLYTICA DGAT GENES IN ARABIDOPSIS SEEDS:

[0198] A binary vector suitable for Agrobacterium-mediated transformation was generated as follows. Various restriction sites were added through a series of cloning steps to the ends of the Bcon/Not I/Phas3' assembly from KS123 (SEQ ID NO: 21), which was previously described in WO 02/008269 ( the contents of which are incorporated herein by reference). Briefly, a DNA fragment (cal a24-4; SEQ ID NO. 22) was amplified from the CalFad2-2 plasmid (described in WO 01/12800) using the primers oCal-15 (SEQ ID NO. 23) and oCal-6 (SEQ ID NO. 24). The cal a24-4 DNA fragment (SEQ ID NO. 22) was digested with BglII and BamHI and cloned into the BamHI site of pKS123 to generate pKR53B (SEQ ID NO. 25). The XbaI/SbfI fragment of pKR53B, which contains the Bcon/NotI/Phas3' assembly, was cloned into the XbaI/SbfI fragment of pKR72 (ATCC accession no. bacterial, to generate pKR85 (SEQ ID NO. 27). The characteristics of pKR72 are as follows. An initial plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID No. 26), a derivative of pKS123 that was previously described in WO 02/008269 (the contents of which are incorporated herein by reference) contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene 25: 179-188 (1983)), flanked by the T7 promoter and transcription terminus (set T7prom/hpt/T7term) and a bacterial origin of reproduction (ori) for selection and reproduction in bacteria (such as E. coli). In addition, pKR72 also contains the hygromycin B phosphotransferase gene, flanked by the 35S promoter (Odell et al., Nature 313: 810-812 (1985)) and NOS 3' transcription terminus (Depicker et al., J. Mol. Appl . Genet. 1: 561-570 (1982)) (35S/hpt/NOS3' set) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the α' subunit of β-conglycinin (Beachy et al., EMBO J. 4: 3047-3053 (1985)) and the 3' transcription termination region of the gene of phaseolin (Doyle et al., J. Biol. Chem. 261: 9228-9238 (1986)), in order to allow strong tissue-specific expression in soybean seeds of genes cloned at the NotI site.

[0199] The Bcon/NotI/Phas3' set was amplified from the plasmid pKR85 (SEQ ID NO. 27) using the primers oKR85-1 (SEQ ID NO. 28) and oKR85-2 (SEQ ID NO. 29) and the resulting DNA fragment was cloned in PCR-Script® (Stratagene), following the manufacturer's protocol, to generate pCR85 (SEQ ID NO. 30).

[0200] The EcoRI/BglII fragment from pPCR85, which contains the Bcon/NotI/Phas3' set, was cloned into the EcoRI/BamHI fragment of plasmid pZS199 (document WO 93/11245; see also US Patent 5,952,544 , which was published on June 10, 1993; the disclosures of which are incorporated herein by reference), which contains the backbone of the Arabidopsis binary vector for producing pKR91 (SEQ ID NO: 31).

[0201] The Bcon/NotI/Phas3' assembly was removed from pKR91 through digestion with AscI and the religated binary vector was produced that contains a unique Asc I cloning site called pKR92 (SEQ ID NO: 32).

CONSTRUCTION OF PKR92 YL DGAT2:

[0202] The construction of the KS362 expression plasmid is described in Example 5. An expression set that harbors the YL DGAT2 gene, fused to the betaconglycinin promoter and phaseolin terminus and the DsRed gene fused to the Kti promoter and termination sequences, was excised of KS362 as a 6.4 kb AscI fragment. This DNA was ligated to dephosphorylated pKR92 vector DNA linearized by Asc I to generate pKR92 YL DGAT2 (SEQ ID NO: 33).

CONSTRUCTION OF PKR92 YL DGAT1/YL DGAT2:

[0203] Construction of the KS364 expression plasmid is described in Example 5. An expression set in which genes YL DGAT1 (SEQ ID NO. 1) and YL DGAT2 (SEQ ID NO. 9) are fused to the identical terminal sequence of phaseolin and the glycinin 1 and betaconglycinin promoters, respectively, was excised from KS364 as a 7-kb AscI fragment. This DNA was ligated to AscI-linearized dephosphorylated pKR92 vector DNA to generate pKR92 YL DGAT1/YL DGAT2 (SEQ ID NO: 34).

GENERATION AND ANALYSIS OF TRANSGENIC ARABIDOPSIS LINES:

[0204] Plasmid DNA of pKR92 YL DGAT2 and pKR92 YL DGAT1/YL DGAT2 was introduced into Agrobacterium tumefaciens NTL4 (Luo et al., Molecular Plant-Microbe Interactions 14(1):98–103 (2001)) via electroporation. Briefly, 1 μg of plasmid DNA was mixed with 100 μl of electrocompetent cells on ice. The cell suspension was transferred to a 100 μl electroporation cuvette (1 mm gap width) and electroporated using a BIORAD electroporator set at 1 kV, 400 Q, and 25 μF. The cells were transferred to 1 ml of LB medium and incubated for two hours at 30 °C. Cells were plated on LB medium containing 50 μg/ml kanamycin. Plates were incubated at 30 °C for sixty hours. Recombinant agrobacteria cultures (500 ml LB, 50 μg/ml kanamycin) were inoculated from isolated colonies of transformed agrobacteria cells and grown at 30 °C for sixty hours. Cells were harvested by centrifugation (5000 x g, 10 min) and resuspended in one liter of 5% (w/v) sucrose containing 0.05% (v/v) Silwet. Arabidopsis plants were grown in soil at a density of thirty plants per 100 cm2 pot in metromix 360 soil mixture for four weeks (22 °C, permanent light, 100 μE m-2s-1). Plants were repeatedly dipped in the Agrobacterium suspension harboring the binary vectors and maintained in a dark environment with high humidity for 24 h. Plants were grown for four to five weeks under standard plant growth conditions described above, and plant material was harvested and dried for one week at room temperature in paper bags. Seeds were collected using a 0.425 mm mesh brass sieve.

[0205] Clean Arabidopsis seeds (2 g, corresponding to about one hundred thousand seeds) were sterilized by washing in 45 ml of 80% ethanol, 0.01% Triton X-100, followed by 45 ml of 30% (v/v) household bleach in water, 0.01% Triton X-100 and finally through repeated rinsing in sterile water. Plots of 20,000 seeds were transferred to square plates (20 x 20 cm) containing 150 ml of sterile plant growth medium composed of 0.5 x MS salts, 1.0% (w/v) sucrose, 0.05 MES /KOH (pH 5.8), 200 μg/ml of timentin and 50 μg/ml of kanamycin solidified with 10 g/l of Agar. Homogeneous seed dispersion over the medium was facilitated by mixing the aqueous seed suspension with an equal volume of molten plant growth medium. Plates were incubated under standard growth conditions for ten days. Kanamycin-resistant seedlings were transferred to selective agent-free plant growth medium and grown to maturity for eight to ten weeks (22°C, permanent dark light, 100 to 200 μE m-2s-1). Plants were grown in flat plates with 36 inserts. On each flat plate, at least six untransformed wild-type control plants were grown near about thirty T2 plants. Seeds were harvested from individual plants and seed oil content was measured using NMR.

SEED OIL CONTENT ANALYSIS BASED ON NMR:

[0206] Seed oil content was determined using an Ultra Maran NMR analyzer (Resonance Instruments Ltd., Whitney, Oxfordshire, United Kingdom). Samples (individual soybean seeds or batches of Arabidopsis seeds ranging in weight from 5 to 200 mg) were placed in pre-weighed 2 ml polypropylene tubes (Corning Inc., Corning NY, United States; Part No. 430917) marked previously. with unique barcode identifiers. The samples were then placed into 96 plate vehicles and processed through the following series of steps by an Adept Cobra 600 SCARA robotic system: 1. Tube picking (the robotic arm was equipped with a vacuum picking device). 2. Read barcode. 3. Expose the tube to an antistatic device (ensuring that the Arabidopsis seed does not adhere to the tube walls). 4. Weigh the tube (containing the sample) to an accuracy of 0.0001 g. 5. NMR reading; measured as the intensity of the proton spin echo one millisecond after applying a 22.95 MHz signal to the sample (data were collected for 32 NMR scans per sample). 6. Return the tube to the shelf. 7. Repeat the process with the next tube.

[0207] Bar codes, tube weights, and NMR readings were recorded by a computer connected to the system. The weight of the sample was determined by subtracting the weight of the polypropylene tube from the weight of the tube containing the sample.

[0208] The oil content of soybean seeds was calculated as follows: % oil (weight % basis) = (NMR signal/sample weight (g)) - 70.58) 351.45

[0209] Calibration parameters were determined by accurately weighing soybean oil samples (ranging from 0.0050 to 0.0700 g in intervals of about 0.0050 g; weighed to an accuracy of 0.0001 g) in Corning tubes (see above) and subjecting them to NMR analysis. An oil content calibration curve (base % seed weight; considering a standard seed weight of 0.1500 g) per NMR value was established.

[0210] The relationship between seed oil contents measured using NMR and absolute oil contents measured using classical analytical chemistry methods was determined as follows. Fifty soybean seeds, selected for having a range of oil contents, were dried at 40 °C in a forced air oven for 48 hours. Individual seeds were subjected to NMR analysis as described above and were then ground into a fine powder in a Geno grinder (SPEX Centripep (Metuchen, NJ, United States); 1500 oscillations per minute for one minute). Portions of 70 to 100 mg were weighed (up to 0.0001 g accuracy) in 13 100 mm glass tubes equipped with Teflon®-lined screw caps; the remainder of the powder from each grain was used to determine the moisture content, through weight difference after eighteen hours in a forced air oven at 105 °C. Heptane (3 ml) was added to the powders in the tubes and after extracting the mixed samples by vortexing on a shaker between ends for one hour at room temperature. The extracts were centrifuged at 1500 x g for ten minutes, the supernatant was decanted into a clean tube and the pellets were extracted two more times (one hour each) with 1 ml of heptane. The supernatants from the three extractions were combined and 50 μl of internal standard (triheptadecanoic acid; 10 mg/ml toluene) was added before evaporation to dryness at room temperature under a stream of nitrogen gas; Standards containing 0, 0.0050, 0.0100, 0.0150, 0.0200 and 0.0300 g of soybean oil in 5 ml of heptane were prepared in the same way. Fats were converted into fatty acid methyl esters (FAMEs) by adding 1 ml of 5% sulfuric acid (v:v in anhydrous methanol) to the dry pellets and heating them at 80 °C for thirty minutes, with mixing for occasional swirling. The samples were kept cooled at room temperature and 1 ml of 25% aqueous sodium chloride was added, followed by 0.8 ml of heptane. After mixing by vortexing, the phases were kept separate and the upper organic phase was transferred to a sample ampoule and subjected to GC analysis.

[0211] Plotting oil contents determined using NMR against oil contents determined using GC resulted in a linear relationship of 9.66 to 26.27% oil (GC values; base % by weight of seed) with a slope of 1.0225 and R2 of 0.9744; based on an average seed moisture content of 2.6 +/- 0.8%. THE OIL CONTENT OF ARABIDOPSIS SEEDS WAS CALCULATED AS FOLLOWS: mg oil = (NMR signal - 2.1112)/37.514% oil (weight % basis) = (mg oil/1000)/sample weight)*100

[0212] Prior to the establishment of this formula, Arabidopsis seed oil was extracted as follows. About 5 g of mature Arabidopsis seeds (cv Columbia) were ground into a fine powder using a pestle and mortar. The powder was placed in a 33 x 94 mm paper thimble (Ahlstrom no. 7100-3394; Ahlstrom, Mount Holly Springs PA, United States) and the oil extracted during approximately forty cycles of extraction with petroleum ether (point of boiling 39.9 to 51.7 °C) in a Soxhlet apparatus. The extract was kept under cooling and the crude oil was recovered by removing the solvent under vacuum in a rotary evaporator. Calibration parameters were determined by precisely weighing eleven standard samples of partially purified Arabidopsis oil (samples contained 3.6, 6.3, 7.9, 9.6, 12.8, 16.3, 20.3 , 28.2, 32.1, 39.9 and 60 mg of partially purified Arabidopsis oil) weighed to an accuracy of 0.0001 g) in 2 ml polypropylene tubes (Corning Inc., Corning NY, United States; part no. 430917) and subjecting them to NMR analysis. A calibration curve was established between oil content (base% seed weight) and NMR value.

[0213] Seeds of pKR92 YL DGAT2 T2 were grown from a total of 293 independent events alongside 75 wild-type controls. The oil content of YL DGAT2 transgenics ranged from 27.9 to 47.3%. The mean oil content was 43.7%. The oil content of wild-type controls ranged from 39.5 to 49.6%. The mean oil content of controls by weight was 44.8%.

[0214] pKR92 YL DGAT1/YL DGAT2 T2 seeds were grown from a total of 295 independent events over 77 wild-type controls. The oil content of transgenic YL DGAT1/YL DGAT2 ranged from 34.5 to 47.6%. The average oil content was 44%. The oil content of wild-type controls ranged from 41.3 to 46.6%. The average oil content of wild-type controls was 45%. Briefly, these findings suggest that seed-specific expression of the YL DGAT gene does not increase the oil content of Arabidopsis seeds.

ANALYSIS OF THE FATTY ACID PROFILE OF ARABIDOPSIS SEEDS EXPRESSING YL DGAT2 ALONE OR IN COMBINATION WITH YL DGAT1:

[0215] GC analysis of FAME was employed to investigate whether expression of YL DGAT alters the fatty acid profile of Arabidopsis seeds. About one hundred F2 seeds were released into individual wells of 96 well-scored tubes.

[0216] For transesterification, 50 μl of trimethylsulfonium hydroxide (TMSH) and 0.5 ml of hexane were added to each striped tube and incubated for thirty minutes at room temperature with shaking. Fatty acid methyl esters (1 μl injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph equipped with an Omegawax 320 fused silica capillary column (Catalog no. 24152, Supelco Inc.).

[0217] The oven temperature was programmed to maintain at 220 °C for 2.6 minutes, increase to 240 °C at 20 °C/minute and then maintain it for an additional 2.4 minutes. Vehicle gas was supplied by a Whatman hydrogen generator.

[0218] Retention times were compared with those of commercially available standard methyl esters (Nu-Chek Prep, Inc.). The results are summarized in Table 5.

[0219] The results clearly demonstrate that expression of YL DAGT2 and, even more so, co-expression of YL DGAT1 and YL DGAT2 in Arabidopsis seeds generates increased incorporation of oleic acid into seed lipids which provides the first indication that YL proteins Active DGAT1 and YL DGAT2 can be produced in transgenic seeds. EXAMPLE 5 EXPRESSION OF YARROWIA LIPOLYTICA DGAT GENES IN SOYBEAN SOMATIC EMBRYOS:

[0220] Table 6 and Table 7 list promoter and termination sequences that were used in plasmid constructions for seed-specific overexpression of YL DGAT genes. TABLE 6 SEED-SPECIFIC PROMOTERS TABLE 7 TRANSCRIPTION TERMINATIONS

[0221] Elaboration of a plasmid construction for expression of the YL DGAT1 gene under control of the glycinin Gy1 promoter (KS349) (Figure 3).

[0222] Isolation of the soy glycinin Gy1 promoter was carried out as follows. Based on the soybean glycinin Gy1 gene sequence sequences (GenBank accession no. of soy glycinin (SEQ ID NO. 36). The oligonucleotide sequences of these two oligos are the following sequences: SEQ ID NO. 37 (oGy1-1): CGCGGATCCTAGCCTAAGTACGTACTCAAAATGCCA SEQ ID NO. 38 (oGy1-2): GAATTCCCATGGGGTGATGACTGATGAGTGTTTAAGGAC

[0223] Plasmid pKS349 was constructed in several steps from a series of different intermediate vectors. The amplified soy glycinin Gy1 promoter fragment was digested with BamHI and NcoI, purified and cloned into the BamHI and NcoI sites of p24K-G4G-@Sall (PCT Application No. WO 98/59062) to generate pZBL114 (SEQ ID NO ° 39). The GUS-containing NcoI/KpnI fragment was replaced with an NcoI/KpnI fragment containing a fusion product of the soybean GY1 gene (SEQ ID NO: 40) and the synthetic barley high-lysine gene 8 (BHL8 ) (US 6,800,726 B1) (SEQ ID NO. 41) to make pZBL133 (SEQ ID NO. 43). The DNA sequence of the fusion product of soybean GY1 gene and BHL8 gene is defined as SEQ ID NO: 42. The phaseolin terminus was removed from pZBL133 (XbaI/filled) and replaced with the phaseolin terminus found in pKS123 (Request PCT No. WO 02/08269 (obtuse) to generate pKS238 (SEQ ID NO. 44). The GY1-BHL8 fusion was replaced with native GY1 sequence as follows. pKS238 was digested with KpnI/BglI and the remaining vector strip ( 5.1 kb) was ligated to a DNA fragment (BglI/KpnI) of the native GY1 gene (SEQ ID NO. 40) to generate pKS240 (SEQ ID NO. 45). The Bam HI/Sal I fragment containing Gy1. /GM-GY1/Phas3' was excised from pKS 240 and ligated into the BamHI/SalI sites of pKS120 (SEQ ID NO: 46) to generate pKS242 (SEQ ID NO: 47).

[0224] Plasmid pKS120 is identical to pKS123 (above), with the exception of the removal of the HindIII fragment that contains the Bcon/NotI/Phas3' set. The GM-GY1-containing NcoI/NotI fragment was replaced with the YL-DGAT1-containing NcoI/NotI fragment of pYDA1 to generate pKS349 (SEQ ID NO: 48). PREPARATION OF A CONSTRUCTION FOR EXPRESSION OF THE YL DGAT2 GENE UNDER THE CONTROL OF THE BETACONGLYCININ PROMOTER (KS362) (FIGURE 3):

[0225] Plasmid pKS362 was constructed in several steps from a series of different intermediate vectors. The AscI pool containing Kti/NotI/Kti3' from pKS121 (PCT Application No. WO 02/00904) was spiked into the NotI site (filled) in pBluescript II SK+ (Stratagene) to generate pKS121/BS. The NcoI/NotI fragment from pDsRed-Express Vector (Clontech) was inserted into the NotI (filled) site of pKS121/BS to generate pDS-RED in KS121/BS (SEQ ID NO: 49). The Bam HI pool containing Kti/DsRed/Kti3' in PDS-RED in KS121/BS (SEQ ID NO. 50) was ligated into the Bam HI site of pKS123 (PCT Application No. WO 02/08269) to generate pKS332 ( SEQ ID NO. 51). The gene for YL-DGAT2 was synthesized using PCR with primers to introduce NotI sites at both ends of the gene (see Example 1). The resulting PCR product is digested with NotI restriction enzyme and ligated to the NotI site of pKS332 to generate pKS362 (SEQ ID NO: 52). CONSTRUCTION OF A CONTROL PLASMID (KS352) (FIGURE 2):

[0226] Based on the sequences of the cloned soybean P34 promoter (WO 2004/071467) (SEQ ID NO: 53), two oligos with Bam HI or NotI sites at the 5' ends were designed to re-amplify the P34 promoter. The oligonucleotide sequences of these two oligos are shown as follows: SEQ ID NO. 54 (oP34-1): CGCGGATCCAACTAAAAAAAGCTCTCAAATTACATTTTGAG SEQ ID NO. 55 (oP34-2): GAATTCGCGGCCGCAACTTGGTGGAAGAATTTTATGATTTGAAA

[0227] The reamplified P34 promoter fragment was digested with BamHI and NotI, purified and cloned into the BamHI and NotI sites of plasmid pZBL115 (SEQ ID NO. 56) to produce pJS89 (SEQ ID NO. 57). Plasmid pZBL115 contains the origin of reproduction of pRB322, the bacterial HPT hygromycin resistance gene driven by the T7 promoter and T7 terminus, and a 35S-HPT-Nos3' promoter gene to serve as a selection marker for hygromycin-resistant plants. The delta-6 desaturase gene from Morteriella alpina (US Patent 5,968,809) (SEQ ID NO. 58) was cloned into the Not I site of pJS89 (SEQ ID NO. 57) in the sense orientation to construct plant expression sets and pJS93 (SEQ ID NO. 59).

[0228] The P34 promoter was excised from pJS93 (SEQ ID No. 59) using double SalI NotI digestion and ligated into Sal I/Not I linearized pKS127 vector (US Patent Application 11/476,510) (SEQ ID No. 60 ) to generate pKS343 (SEQ ID NO: 61). The Bam HI assembly containing Kti/DsRed/Kti3' in pDS-RED in KS121/BS was moderated and ligated into the HindIII (filled) site of pKS343 to generate pKS352 (SEQ ID NO: 62).

CONSTRUCTION OF A PLASMID FOR COEXPRESSION OF YL DGAT1 AND YL DGAT2 (KS364):

[0229] Plasmid pKS364 (SEQ ID NO. 63) was constructed by ligating the 3.3 kb HindIII assembly containing Bcongl PRO/YL- DGAT2/Phas TER from pKS362 (SEQ ID NO. 52) to the Hind III site exclusive downstream of the Gy1 promoter in pKS349 (SEQ ID NO: 48).

GENERATION OF TRANSGENIC SOMATIC EMBRYOS:

[0230] For coexpression of YL DGAT1 and YL DGAT2 gene in somatic soybean embryos, soybean tissue was cobombarded as described below with a mixture of KS349 and KS362. Briefly, KS349 DNA was digested with PtsI, XhoI restriction enzymes to deactivate the selectable marker gene set (CaMV35S PRO/HPT/CaMV NOS TER). This DNA was mixed in a 10:1 ratio with SalI-linearized plasmid DNA from KS362 and used for soybean transformation as described below. Alternatively, soybean tissue from soybean somatic embryos was bombarded as described below with intact KS364 plasmid DNA that contains functional expression sets for both YL DGAT1 and YL DGAT2. For expression of YL DGAT1 alone, uncut plasmid DNA from KS349 was used for particle bombardment from embryo tissue. Similarly, for expression of YL DGAT2 alone, uncut plasmid DNA from KS362 was used for particle bombardment from embryo tissue. Furthermore, DNA that contained only one selectable marker (KS352) was used to transform soybean tissue identically.

CULTIVATION CONDITIONS:

[0231] Embryogenic suspension cultures of soybean (cv. Jack) were maintained in 35 ml of SB196 liquid medium (below) on a rotary shaker, 150 rpm, 26 °C with cool white fluorescent lights on a 16:8 h photoperiod day/light under light intensity of 60 to 85 μE/m2/s. Cultures were subcultured every seven days to two weeks by inoculating about 35 mg of tissue in 35 ml of fresh liquid SB196 (the preferred subculture interval is every seven days).

[0232] Soybean embryogenic suspension cultures were transformed with the soybean expression plasmids by the particle trigger bombardment method (Klein et al., Nature 327: 70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations.

START OF EMBRYOGENIC SOYBEAN SUSPENSION CULTURE:

[0233] Soybean crops were started twice each month with five to seven days between each start. Pods with immature seeds from soybean plants available 45 to 55 days after planting were chopped, removed from their shells and placed in a sterilized magenta box. Soybean seeds were sterilized by shaking them for fifteen minutes in a 5% Clorox solution with a drop of bar soap (i.e., 95 ml of autoclaved distilled water plus 5 ml of Clorox and a drop of soap , well mixed). Seeds were rinsed using two one-liter bottles of sterile distilled water and those smaller than 4 mm were placed on individual microscope slides.

[0234] The small end of the seed was cut off and the cotyledons were pressed out of the seed coat. The cotyledons were transferred to plates containing SB199 medium (25 to 30 cotyledons per plate) for two weeks and then transferred to SB1 for two to four weeks. The plates were packed with fiber tape. After this period, secondary embryos were cut and placed in SB196 liquid media for seven days.

DNA PREPARATION FOR BOMBING:

[0235] An intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene were used for bombardment.

[0236] A 50 μl portion of sterile distilled water containing 1 mg of gold particles was added to 5 μl of a solution of 1 μg/μl DNA (either intact plasmid or DNA fragment prepared as described above), 50 μl of 2.5 M CaCl2 and 20 μl of 0.1 M spermidine. The mixture was pulsed five times on level 4 of a vortex shaker and centrifuged for five seconds in a benchtop microcentrifuge. After washing with 150 μl of 100% ethanol, the pellet was suspended by sonication in 85 μl of 100% ethanol. Five microliters of DNA suspension was released onto each floating disk of the Biolistic PDS1000/HE instrument. Each 5-μl plot contained about 0.058 mg of gold particles per bombardment (i.e., per disk).

TISSUE PREPARATION AND DNA BOMBARDING:

[0237] About 100 to 150 mg of seven-day-old embryonic suspension cultures were placed in an empty sterile 60 x 15 mm petri dish and the dish was placed inside an empty 150 x 25 mm petri dish . The tissue was bombarded once per plate with membrane burst pressure set at 650 PSI and the chamber was evacuated to a vacuum of 680 to 710 mm of mercury. The tissue was placed approximately 6.3 cm from the holding/stopping screen.

SELECTION OF TRANSFORMED EMBRYOS:

[0238] Transformed embryos were selected using hygromycin as the selectable marker. Specifically, after bombardment, tissue was plated on fresh SB196 medium and cultured as described above. Six to eight days after bombardment, SB196 was replaced with fresh SB196 containing 30 mg/L hygromycin. Selection medium was renewed weekly. Four to six weeks after selection, green transformed tissue was observed to grow from untransformed necrotic embryogenic pools. Isolated green tissue was removed and inoculated into multiwell plates to generate new transformed embryogenic suspension cultures and clonally propagated.

EMBRYO MATURATION:

[0239] Transformed embryogenic pools were cultured for one to three weeks at 26°C in SB196 under cool white fluorescent lamps (Philips Cool White Econowatt F40/CW/RS/EW) and Agro (Philips F40 Agro) (40 watts) in a photoperiod of 16:8 hours with light intensity of 90 to 120 μE/m2s. After this period, the embryo sets were removed to solid agar medium, SB166, for one week. They were then subcultured to SB103 medium for three weeks. Alternatively, embryo pools were removed into SB228 liquid media (SHaM), 35 ml in a 250 ml Erlenmeyer flask, for two to three weeks. Tissue grown in SB228 was maintained on a rotating shaker, 130 rpm, 26 °C with cool white fluorescent lights on a photoperiod of 16:8 day/light hours under light intensity of 60 to 85 μE/m2/s. During this period, individual embryos were removed from the pools and selected to determine changes in their fatty acid compositions as described above.

[0240] Media recipes: SB 196 - FN LITE LIQUID PROLIFERATION MEDIA (PER LITER): FN LITE STANDARD SOLUTIONS:

SOLID MEDIUM SB1 (PER LITER):

[0241 ] 1 package of MS salts (Gibco/BRL - Cat. No. 11117-066)

[0242] 1 ml of vitamins B5 1000X standard

[0243] 31.5 g of glucose

[0244] 2 ml of 2,4-D (20 mg/l final concentration)

[0245] pH 5.7

[0246] 8 g of TC agar

SOLID MEDIUM SB199 (PER LITER):

[0247] 1 package of MS salts (Gibco/BRL - Cat. no. 11117-066)

[0248] 1 ml of vitamins B5 1000X standard

[0249] 30 g of sucrose

[0250] 4 ml of 2,4-D (final concentration of 40 mg/l)

[0251] pH 7.0

[0252] 2 g of gelrite

SOLID MEDIUM SB 166 (PER LITER):

[0253] 1 package of MS salts (Gibco/BRL - Cat. No. 11117-066)

[0254] 1 ml of vitamins B5 1000X standard

[0255] 60 g of maltose

[0256] 750 mg of MgCl2 hexahydrate

[0257] 5 g of activated charcoal

[0258] pH 5.7

[0259] 2 g of gelrite

SOLID MEDIUM SB 103 (PER LITER):

[0260] 1 package of MS salts (Gibco/BRL - Cat. No. 11117-066)

[0261] 1 ml of vitamins B5 1000X standard

[0262] 60 g of maltose

[0263] 750 mg of MgCl2 hexahydrate

[0264] pH 5.7

[0265] 2 g of gelrite

SOLID MEDIUM SB 71-4 (PER LITER):

[0266] 1 bottle of Gamborg B5 salts with sucrose (Gibco/BRL - cat. No. 21153-036)

[0267] pH 5.7

[0268] 5 g of TC Agar STANDARD 2,4-D:

[0269] Obtain pre-made from Phytotech Cat. No. D 295 - concentration of 1 mg/ml.

STANDARD VITAMINS B5 (PER 100 ML):

[0270] Store portions at -20 °C: - 10 mg myo-inositol - 100 mg nicotinic acid - 100 mg pyridoxine HCl - 1 g thiamine

[0271] If the solution does not dissolve quickly enough, apply a low level of heat through the plate under hot stirring. SB 228 - HISTODIFFERENTIATION AND MATURATION OF SOYBEANS (SHAM) (PER LITER):

[0272] Adjust volume to 900 ml.

[0273] pH 5.8

[0274] Autoclave

[0275] Add to cooled media (< 30 °C): *Glutamine (final concentration of 30 mM) 4%: 110 ml. *Note: The final volume will be 1010 ml after adding glutamine.

[0276] As glutamine degrades relatively quickly, immediate addition before using the media may be preferable. The expiration date is two weeks after adding glutamine; base media can be maintained longer without glutamine. MACRO FN-LITE FOR SHAM 10X – STOCK #1 (PER LITER):

[0277] Bring to volume

[0278] Autoclave MS MICRO 1000X - STOCK N° 2 (PER ONE LITER):

[0279] Bring to volume

[0280] FEEDTA 100X Autoclave - STOCK N° 3 (PER LITER): *EDTA must be completely dissolved before adding iron.

[0281] Bring to volume

[0282] The solution is photosensitive. The bottle(s) must be packaged in metal foil to omit light.

[0283] Autoclave CA 100X - STOCK N° 4 (PER LITER):

[0284] Bring to volume

[0285] Autoclave VITAMIN B5 1000X - STOCK N° 5 (PER LITER):

[0286] Bring to volume

[0287] Store frozen 4% GLUTAMINE - STOCK N° 6 (PER LITER):

[0288] Add gradually by stirring and applying low heat.

[0289] Do not exceed 35 °C.

[0290] Bring to volume

[0291] Filter and sterilize

[0292] Store frozen* *Note: heat frozen stock in a bath at 31 °C to completely dissolve the crystals.

OIL ANALYSIS:

[0293] Somatic embryos were harvested after two weeks of cultivation in SB228 liquid maturation medium (SHaM). About thirty events were created in transformations with KS352, KS349/KS362, and KS362 and KS364. All embryos generated for a given event were collected in bulk and processed as follows. Embryos were frozen on dry ice or by incubation in a -80°C freezer for two hours, followed by freeze-drying for 48 hours.

[0294] Dried embryos were ground to a fine powder using a Geno grinder ampoule (1/2”x2” polycarbonate) and a steel ball (SPEX Centriprep (Metuchen NJ, United States). The grinding time was thirty seconds at 1450 oscillations per minute. For each event, triplicates of about 10 mg of tissue were weighed into Eppendorf tubes. The tissue was extracted using 200 μl of heptane at room temperature under continuous stirring for two hours. of centrifugation and 25 μl of extract were derivatized into fatty acid methyl esters as follows. One milliliter of a standard 25% sodium methoxide solution was added to 24 ml of HPLC-grade methanol. inert.

[0295] Five microliters of a standard 17:0 TAG solution (Nu-Chek Prep, Elysian MN, United States) (10 mg/ml) was combined with 25 μl of heptane tissue extract in a glass culture tube and 500 μl of 1% sodium methoxide was added. Sample was derived in a water bath at 50 °C for fifteen minutes. Samples were kept cooled to room temperature and 1 ml of 1 M NaCl was added followed by rapid mixing. FAMEs were extracted in 1 ml of heptane and 4 μl of sample was quantified using GC analysis.

[0296] Data analysis was performed by plotting the oleic content (% total FAME) against the total FAME content (% DW). Table 8 demonstrates that somatic embryos generated with a control vector (KS352) exhibit little fluctuation in oleic acid content and some fluctuation in oil content that can most likely be attributed to biological variations introduced in the regeneration process. Embryos most likely exhibit, for example, variation in their developmental stage at the time of harvest. In embryos generated with the control construct, no correlation (R2 = 0.1142) was observed between oleic acid content and oil content (Table 8). In embryos generated with plasmid constructs expressing YL DGAT1 and YL DGAT2 genes alone, KS349 and KS362, respectively, or both YL DGAT1 and DGAT2 genes (KS349/KS362, KS364) under the control of strong seed-specific promoters, both the content of oleic acid and total esterified fatty acid content exhibited a wide fluctuation variation. Furthermore, as shown in Figures 4 and 5, a strong correlation (R2 > 0.59) was observed between oleic acid content and total esterified fatty acid content for somatic embryos generated with KS349 and KS362 alone or in combinations, as well as with KS364, a transformation plasmid that contains expression sets for YL DGAT1 and YL DGAT2 genes. TABLE 8 CONTENT OF STERIFIED FATTY ACIDS AND OLEIC ACID OF SOMATIC EMBRYOS OF SOYBEAN

[0297] In summary, the data demonstrate that, in soybean somatic embryos, similar to Arabidopsis seeds, gene expression of YL DGAT is associated with increased incorporation of oleic acid into the total esterified fatty acid fraction. Unlike Arabidopsis, however, in soybean somatic embryos, increased oleic acid content is tightly correlated with total esterified fatty acid accumulation. In other words, the expression of YL DGAT2 alone and the coexpression of YL DGAT1 and YL DGAT2 in soybean somatic embryos generates increased biosynthesis and incorporation of fatty acids into the total esterified fatty acid fraction. Taken together, this finding strongly suggests that expression of YL DGAT genes provides an efficient strategy to achieve an increase in total soybean seed oil content.

EXAMPLE 6 EXPRESSION OF YARROWIA LIPOLYTICA DGAT GENES IN SOYBEAN SEED:

[0298] The construction of a control plasmid (KS332) (Fig. 2) that contains only CaMV 35S PRO/HPT/NOS TER and Kti PRO/DsRed/Kti TER expression sets is described in Example 5. Its sequence is described as SEQ ID NO. 51.

[0299] Transgenic soybean lines were generated using the particle trigger bombardment method (Klein et al., Nature (London) 327: 70-73 (1987); US Patent 4,945,050) using a PDS1000/He instrument Biolistic BIORAD and plasmid DNA of KS332, KS362 and a 10:1 mixture of KS349 and KS362 prepared as described in Example 3. The following standard solutions and media were used for transformation and regeneration of soybean plants.

STANDARD SOLUTIONS:

[0300] Standard 100

[0301] Standard 100 X Halides: 30.0 g of CaCl2.2H2O, 0.083 g of KI, 0.0025 g of CoCl2.6H2O.

[0302] Standard 100 X P, B, Mo: 18.5 g KH2PO4, 0.62 g H3BO3, 0.025 g Na2MoO4.2H2O.

[0303] Standard 100 X Fe EDTA: 3.724 g of Na2EDTA, 2.784 g of FeSO4.7H2O.

[0304] Standard 2,4-D: 10 mg/ml of standard 1000X Vitamin B5: 10.0 g of myo-inositol, 0.10 g of nicotinic acid, 0.10 g of pyridoxine HCl, 1 g of thiamine.

[0305] Media (per liter): SB196: 10 ml of each of the above standard solutions, 1 ml of Vitamin B5 standard, 0.463 g of (NH4)2SO4, 2.83 g of KNO3, 1 ml of standard 2.4 -D, 1 g of asparagine, 10 g of sucrose, pH 5.7.

[0306] SB103: 1 packet Murashige & Skoog salt mixture, 1 ml Vitamin B5 standard, 750 mg MgCl2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.

[0307] SB166: SB103 supplemented with 5 g per liter of activated carbon.

[0308] SB71-4: Gamborg B5 salts, 1 ml of standard vitamin B5, 30 g of sucrose, 5 g of TC Agar, pH 5.7.

[0309] To prepare tissue for transformation, soybean embryogenic suspension cultures were maintained in 35 ml of liquid media (SB196) on a rotary shaker (150 rpm) at 28 °C with fluorescent lights providing a 16 hour day cycle. and 8 hours at night. Cultures were subcultured every two weeks by inoculating about 35 mg of tissue into 35 ml of fresh liquid media.

[0310] In particle trigger bombardment procedures, it is possible to use, purified, 1) entire plasmid DNA; or 2) DNA fragments that contain only the recombinant DNA expression set(s) of interest. For every seventeen bombardment transformations, 85 μl of suspension is prepared that contains 1 to 90 picograms (pg) of plasmid DNA per base pair of each plasmid DNA. The two recombinant DNA plasmids were coprecipitated onto gold particles as follows. The suspended DNAs were added to 50 μl of a suspension of 0.6 μm gold particles at 20 to 60 mg/ml and then combined with 50 μl of CaCl2 (2.5 M) and 20 μl of spermidine (0 ,1M). The mixture was vortexed for five seconds, centrifuged in a microcentrifuge for five seconds and the supernatant was removed. The DNA-coated particles were then washed once with 150 μl of 100% ethanol, vortexed, centrifuged again in a microcentrifuge and then resuspended in 85 μl of anhydrous ethanol. Five microliters of the DNA-coated gold particles were then loaded onto each macrovehicle disk.

[0311] About 150 to 250 mg of two-week-old suspension culture was placed in an empty 60 mm x 15 mm petri dish and residual liquid removed from the tissue using a pipette. The tissue was placed approximately 8.9 cm away from the holding screen and each tissue plate was bombarded once. The membrane burst pressure was set at 650 psi and the chamber was evacuated to -711 mm Hg. Three plates were bombarded, and after bombardment, tissue from each plate was divided between two flasks, placed back into liquid media, and cultured as described above.

[0312] Seven days after bombardment, the liquid medium was replaced with new SB196 medium supplemented with 30 to 50 mg/l hygromycin. The selective medium was then renewed weekly or bi-weekly. Seven weeks after bombardment, growth of bright green transformed tissue was observed from untransformed chlorotic or necrotic embryogenic pools. Isolated green tissue was removed and inoculated into individual containers in six-well culture dishes to generate new clonally propagated and transformed embryogenic suspension cultures. In this way, each new lineage was treated as an independent transformation event in an individual cavity. These suspensions can then be maintained in the form of joint embryo suspensions at an immature stage of development through subculture or can be regenerated into whole plants through maturation and germination of individual somatic embryos.

[0313] After two weeks in individual cell wells, transformed embryogenic pools were removed from the liquid culture and placed on solidified medium (SB166) that does not contain hormones or antibiotics for one week. Embryos were cultured at 26°C with mixed fluorescent and incandescent lights on a 16-h day, 8-h night schedule. After one week, the cultures were transferred to SB103 medium and maintained under the same growth conditions for three additional weeks.

[0314] Somatic embryos became suitable for germination after four weeks, were then removed from the maturation medium and dried in empty petri dishes for one to five days. The dried embryos were then planted in SB71-4 medium, where they were maintained in germination under the same light and temperature conditions described above. Germinated embryos were transferred to sterile soil and grown to maturity for seed production.

[0315] A total of 29 seed transgenic lines were generated with intact KS362 plasmid DNA at a concentration of 15 pg per bp of plasmid DNA by gold particle preparation (see above). For each event, twenty seeds were analyzed to determine the presence of the DS marker gene. Briefly, seeds were observed under a stereo microscope (Leica MZ Fluo III) using a UV light source. A custom filter set was used for fluorescence associated with DS Red expression with the following properties: excitation λ = 540-580 nm / emission λ ≥ 570 nm. In cases where fewer than twenty seeds were available, all seeds were evaluated in this way. Next, soybean oil content was measured using NMR as previously described 3. Nineteen events generated with KS362 contained seeds that were positive for Red DS. Of these, eleven events exhibited a detectable difference in oil content between DS Red positive transgenic segregants and DS Red negative null segregants. The data is summarized in Table 9. TABLE 9 T1 SOYBEAN SEED OIL CONTENT GENERATED WITH KS362

[0316] A total of ten transgenic lines with KS332 DNA were generated. For each event, twenty seeds were evaluated to determine the presence of the DS marker gene as described above.

[0317] In cases where fewer than twenty seeds were available, all seeds were evaluated in this way.

[0318] Next, the soybean seed oil content was measured using NMR as described in Example 4.

[0319] Seven events generated with KS 332 contained seeds that were positive for Red DS. The data is summarized in Table 10. TABLE 10 OIL CONTENT OF SOYBEAN SEEDS GENERATED WITH KS332

[0320] Unlike seeds generated with KS362, for transgenic seeds generated with KS332, no consistent oil increase could be associated with the presence of the Red DS marker in T1 segregants.

[0321] Four events generated with KS362 were subjected to DS Red and NMR analysis of oil from all available T1 seeds. The data are summarized in Table 11. TABLE 11 OIL CONTENT OF T1 SOYBEAN SEEDS GENERATED WITH KS362

[0322] Briefly, the data from the tables above and Figure 7 demonstrate that seed-specific expression of YL DAGT2 generates increased oil biosynthesis during maturation of soybean seeds and thus provides an efficient metabolic engineering tool for increase oil accumulation in soybeans.

[0323] A total of sixteen transgenic seed lines were generated by co-bombardment with KS349 and KS362 DNA that had been mixed in a 10:1 ratio (see Example 4). The DNA mixture was fed into soybean transformation at a final concentration of 15 pg per bp of plasmid DNA by gold particle preparation. Briefly, before bombardment, KS349 DNA was digested with PstI, XhoI to deactivate the selectable marker gene on this plasmid. KS362 DNA was linearized with SalI. It is reasonable to consider that, due to DNA pretreatment, the selectable marker gene in all transformations was provided from the plasmid (KS362) that was bombarded at the lowest DNA concentration. Initial inspection of these seeds under the fluorescence stereomicroscope revealed that very few events of this transformation contained T1 seed that was positive for the Red DS marker gene. This result may be due to the proximity between the Red DS expression set and the end of the SalI restriction fragment of KS362 that was used for soybean transformation. This may have resulted in the integration of KS362 DNA fragments that did not contain a functional DS Red expression pool. For this reason, T1 seeds were selected for the absence or presence of transgene-derived YL DGAT by testing seed fatty acid composition. For each event, twenty seeds were analyzed using GC. Fifty unprocessed soybean seeds were processed in the same way. Soybean seed flakes were produced by cutting the seed with a razor blade, avoiding the embryonic axis. Seed flakes weighing approximately 2 mg were placed in an ampoule containing 50 μl of trimethylsulfonium hydroxide and 0.5 ml of hexane. The flakes were incubated for thirty minutes at room temperature with shaking. Five microliters of the hexane layer was injected into a Hewlett Packard 6890 Gas Chromatograph containing an Omegawax 320 fused silica capillary column (Supelco Cat. no. 24152). The oven conditions were as follows: initial temperature of 220°C for 2.7 minutes, raised to 240°C over one minute, and maintained at 240°C for a total conduction time of six minutes. Retention times were compared with commercially available standards (Nu-Chek Prep, Inc., cat. no. U-99-A). Fatty acids were determined by direct transesterification of individual standards in 0.5 ml of methanolic H2SO4 (2.5%). Fatty acid methyl esters were extracted from the methanolic solutions in hexane after adding an equal volume of water.

[0324] Ten events were identified that contained T1 seeds with oleic acid content >25%. Because this oleic acid content was not observed in untransformed soybean seeds (see Fig. 6 and Table 12), and increased oleic acid content has previously been associated with coexpression of YL DGAT2 and YL DGAT1 and YL DGAT2 in Arabidopsis seeds and soybean somatic embryos, the presence of oleic acid at levels >25% is believed to provide an efficient means of identifying YL DGAT-positive T1 seeds. Following GC analysis for YL DGAT genotyping, seeds were subjected to oil measurements by NMR as described previously (Example 3). When oleic acid content was plotted against total oil content, seven of the ten events with T1 seed of >25% demonstrated a correlation of R2 >0.3 between oil and oleic acid content. The properties of these events are described in more detail in Table 12. TABLE 12 OIL CONTENT OF SOYBEAN SEEDS GENERATED WITH KS349/KS362

[0325] Wild-type Jack seeds were grown under conditions similar to those used for the generation of T1 seeds and analyzed using NMR and GC analysis. It was observed that the oleic acid and oil content fluctuated from 7.6 to 20.5% and 18.6 to 25.2%, respectively. No correlation between oleic acid content and oil content could be observed in unprocessed soybean seeds.

[0326] Four events generated with KS349/KS362 were subjected to GC and NMR analysis of all available T1 seeds. The data is summarized in Table 13. TABLE 13 OIL CONTENT OF T1 SOY SEEDS GENERATED WITH KS349/KS362

[0327] Taken together, the data from the preceding tables and Figures 6 and 7 strongly support the conclusion that coexpression of YL DGAT1 and YL DGAT2 genes, like expression of the YL DGAT2 gene alone, provides an efficient strategy for achieving increased of the total soybean seed oil content. Furthermore, it should be noted that a high number of events may be identified with an oil difference of >2% points between null and transgenic segregants among a small set of selected transgenic events.

EXAMPLE 7 EXPRESSION OF YARROWIA LIPOLYTICA DGAT GENES IN CORN:

[0328] Based on the results described in Examples 4, 5 and 6 of the present application, the YL DGAT1 and YL DGAT2 genes can be expressed in the corn seed embryo to increase the oil content of this tissue. As described below, this result can be achieved by transforming maize with expression sets comprising Yarrowia lipolytica DGAT1 and DGAT2 open reading frames operably linked at their 5' ends to embryo-preferred promoters, such as the gene promoter 16 kDa oleosin from maize (Lee, K. and Huang, A. H., Plant Mol. Biol. 26: 1981-1987 (1984)) and maize embryo abundant promoter (EAP1) and terminal (US 2006272058 A1).

[0329] An expression set comprising the corn 16 kDa oleosin gene promoter (OLE PRO), the YL DGAT2 gene coding sequence (SEQ ID NO: 9) and the gene terminal/polyadenylation signal sequence nopaline synthase (NOS) from Agrobacterium tumefaciens is constructed using methods and technologies known in the art. A second expression set comprises the YL DGAT1 gene under the transcriptional control of the maize terminus and abundant protein promoter (EAP1), with the maize ADH1 INTRON1 inserted between the promoter and coding sequence for increased expression. The two expression sets are linked, together with a gene encoding a selectable marker, into a binary vector suitable for Agrobacterium-mediated maize transformation.

[0330] An Agrobacterium-based protocol can be used for corn transformation (see below). The resulting binary vector is introduced into Agrobacterium LBA4404 (PHP10523) cells, preferably via electroporation. An in vivo recombination generates a cointegrated plasmid between the introduced binary vector and the vir plasmid (PHP10523) residing in Agrobacterium cells. The resulting Agrobacterium cells are used to transform corn.

CORN TRANSFORMATION MEDIATED BY AGROBACTERIUM:

[0331] Freshly isolated immature corn embryos, about ten days after pollination (DAP), can be incubated with Agrobacterium. The preferred genotype for transformation is the highly transformable genotype Hi-II (Armstrong, Maize Gen. Coop. Newsletter 65: 92-93 (1991)). An F1 hybrid created by crossing a Hi-II with an elite inbred line can also be used. After treating immature embryos with Agrobacterium, the embryos can be cultured on medium containing toxic levels of herbicide. Only cells that receive the herbicide resistance gene and the linked gene(s) grow on selective media. Transgenic events selected in this way can be propagated and regenerated into whole plants, produce seeds, and transmit transgenes to offspring.

AGROBACTERIUM PREPARATION:

[0332] The engineered Agrobacterium tumefaciens LBA4404 can be constructed to contain plasmids for seed-preferred expression of YL DGAT1 and YL DGAT2 genes, as described in US Patent 5,591,616 (the contents of which are incorporated herein by reference). To use the elaborate construct in plant transformation, a master plate from an isolated bacterial colony transformed with plasmids for seed-preferred expression of YL DGAT1 and YL DGAT2 genes can be prepared by inoculating the bacteria onto minimal AB medium and allowing incubation at 28°C for about three days (the composition and preparation of minimal AB medium have been previously described in WO 02/009040 (the contents of which are incorporated herein by reference). A working plate can then be prepared streaking the transformed Agrobacterium onto YP medium (0.5% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) sodium chloride, 1.5% ( w/v) Agar) containing 50 μg/ml spectinomycin.

[0333] Transformed Agrobacterium for transfection and plant co-cultivation can then be prepared one day prior to maize transformation. In 30 ml of minimal medium A (prepared as described in WO 02/009040) in a flask, 50 μg/ml spectinomycin, 100 μM acetosyringone and about 1/8 full circuit of Agrobacterium from a two-day-old work plate were placed. The Agrobacterium can then be grown at 28 °C with shaking at 200 rpm for about fourteen hours. At mid-log stage, the Agrobacterium can be harvested and resuspended at a density of 3 to 5 x 108 CFU/ml in 561Q medium containing 100 μM acetosyringone using standard microbial methods. The composition and preparation of medium 561Q were described in WO 02/009040.

PREPARATION OF IMMATURE EMBRYOS:

[0334] Nine to ten days after controlled pollination of a corn plant, developing immature embryos are opaque and 1 to 1.5 mm long. This length is the ideal size for infection with Agrobacterium transformed by PHP18749. The shelled ears can be sterilized in 50% commercial bleach and a drop of Tween 20 for thirty minutes and rinsed twice with sterile water. Immature embryos can be removed aseptically following caryopsis and placed in 2 ml of sterile maintenance solution consisting of 561Q medium containing 100 μM acetosyringone.

INFECTION WITH AGROBACTERIUM AND EMBRYO COCULTURE:

[0335] The maintenance solution can be decanted from the excised immature embryos and replaced with transformed Agrobacterium. After gentle mixing and incubation for about five minutes, the Agrobacterium can be decanted from the immature embryos. The immature embryos were then moved to a plate of 562P medium, the composition of which was previously described in WO 02/009040. Immature embryos can be placed on this upward-facing media scutellum surface and then incubated at 20°C for three days in the dark. This step can be followed by incubation at 28 °C for three days in the dark over 562P medium containing 100 μg/ml carbenecillin as described in US Patent 5,981,840.

SELECTION OF TRANSGENIC EVENTS:

[0336] After incubation, immature embryos can be transferred to medium 5630, which can be prepared as described in document WO 02/009040. This medium contains Bialaphos for selection of transgenic plant cells as conferred by the BAR gene which is linked to the barley HGGT expression pool. At ten to fourteen day intervals, embryos were transferred to 5630 medium. Putative transgenic embryogenic tissue can be actively growing after six to eight weeks of incubation on 5630 medium.

PLANT REGENERATION TO:

[0337] Transgenic embryogenic tissue is transferred to 288W medium and incubated at 28 °C in the dark until somatic embryos mature or about ten to eighteen days.

[0338] Individual matured somatic embryos with well-defined scutellum and coleoptile are transferred to embryo germination medium 272 and incubated at 28 ° C in the light. After roots and shoots emerge, individual plants are potted in the ground and hardened off using typical horticultural methods.

[0339] Medium 288W contains the following ingredients: 950 ml of deionized water; 4.3 g of MS salts (Gibco); 0.1 g of myoinositol; 5 ml of standard MS vitamin solution (Gibco); 1 ml of zeatin (5 mg/ml solution); 60 g of sucrose; 8 g of agar (Sigma A-7049, purified), 2 ml of indoloacetic acid (0.5 mg/ml* solution); 1 ml of 0.1 mM ABA*; 3 ml of Bialafos (1 mg/ml solution*); and 2 ml of carbenicillin (50 mg/ml solution). The pH of this solution is adjusted to pH 5.6. The solution is placed in an autoclave and the ingredients marked with an asterisk (*) are added after cooling the media to 60 °C.

[0340] Medium 272 contains the following ingredients: 950 ml deionized water; 4.3 g MS salts (Gibco); 0.1 g myoinositol; 5 ml MS vitamin standard solution (Gibco); 40 g sucrose; and 1.5 g gelrite. This solution is adjusted to pH 5.6 and then autoclaved.

EXAMPLE 8 SEED OIL CONTENT ANALYSIS:

[0341] Nuclear magnetic resonance (NMR) analysis: The seeds are soaked in distilled water for twelve to 24 hours at 4 ° C; The embryo is dissected and stored in a 48-well plate. Samples are freeze-dried overnight in a Virtis 24x48 freeze dryer. The NMR (Process Control Technologies - PCT (Ft. Collins CO) is set up per the manufacturer's instructions. The NMR is calibrated using a series of 5 mm NMR tubes that contain precisely measured amounts of corn oil (Mazola). The standards calibration parameters are 3, 6, 9, 12, 15, 18, 21, 27, 33 and 40 mg of oil.

EXAMPLE 9 SYNTHESIS OF YL DGAT1 AND YL DGAT2 GENES:

[0342] Nucleotide sequences encoding YL DGAT1 and YL DGAT2 were designed for optimized expression in soybean seeds using methods similar to those described in Wu, G. et al., Nucleic Acids Research (2007), 35: D76-D79; Villalobos, A. et al., BMC Bioinformatics (2006), 7, pages not provided; Wu, G. et al., Protein Expression and Purification (2006), 47: 441-445; Richardson, S. M. et al., Genome Research (2006), 16: 550-556; Jayaraj, S. et al., Nucleic Acids Research (2005) 33: 3011-3016. DNA molecules were synthesized using DNA 2.0 (Menlo Park CA, United States). Expression-optimized DNA sequences of YL DGAT1 and YL DGAT2 are described in SEQ ID NO. 64 and SEQ ID NO. 66, respectively. The amino acid sequences of soy-optimized enzymes are described in SEQ ID NO. 65 (YL DGAT1) and SEQ ID NO. 67 (YL DGAT2) and are identical to the translation products of SEQ ID NO. 1 and SEQ ID NO. 9 , respectively.

EXAMPLE 10 COMPOSITION OF FATTY ACIDS OF SOMATIC EMBRYOS OF SOYBEANS EXPRESSING YL DGAT GENES:

[0343] Transgenic somatic embryos were generated using the KS352, KS349, KS362 and KS364 plasmid constructs. The generation of the DNA constructs and the transformation process are described in detail in Example 5. The fatty acid composition was determined via GC analysis of fatty acid methyl esters generated via sodium methoxide derivatization of heptane extracts. . The findings are summarized in Table 14. The table compares the fatty acid composition of one hundred events generated with a control plasmid that does not contain YL DGAT genes with that of events created with plasmids that contain YL DGAT1 (KS349), YL DGAT2 ( KS362) or both genes (KS364). For events generated with YL DGAT that contain DNA constructs, the average fatty acid composition of all events with more than 30% oleic is displayed. TABLE 14 COMPOSITION OF FATTY ACIDS OF SOYBEAN SOMATIC EMBRYOS GENERATED WITH KS 352, 349, 362, 349+362 AND 364

[0344] The table demonstrates that the expression of YL DGAT1 or YL DGAT2, as well as the co-expression of these genes, alters the FA profile of soybean somatic embryos.

[0345] The most pronounced change is an increase in oleic acid and reduction in linolenic acid which are consistently observed with all DNA constructs tested. Expression of YL DGAT genes also generates a reduction in palmitic and linoleic acid and an increase in stearic acid. EXAMPLE 11 FATTY ACID COMPOSITION OF YL DGAT GENES EXPRESSING SOYBEAN SEEDS:

[0346] Event AFS4822.1.13.1 was generated using KS362 plasmid DNA as described in Example 6. Transgenic T1 seeds exhibit an increase in oil content of 24.6% when compared to null segregating seeds from the same T1 plant. This observation strongly supports the conclusion that these events express YL DGAT2. T1 seed of AFS4822.1.13.1 with or without the YL DGAT2 transgene germinated and grew in the growth chamber for three months. Red DS positive T1 seeds from event AFS4703.1.6 germinated and grew alongside event YL DGAT2. AFS4703.1.6 was generated with KS332, a plasmid vector that contains the Red DS marker gene, but does not contain YL DGAT2 (see Example 5). Several T1 segregants could be identified that produced only T2 Red DS positive seeds, which indicates that these lines were homozygous for the corresponding transgene. T2 seeds harvested from null segregating offspring were negative for Red DS, confirming that these lines likely did not contain a functional transgene.

[0347] For each selection, six seeds were chopped and the fatty acid composition of the seed flakes were analyzed via TMSH derivatization followed by gas chromatography as described in Example 6. Table 15A compares the average fatty acid composition of six positive Red DS segregant seed flakes from AFS4822.1.13.1 with that of seed flakes derived from a null segregating plant and seed flakes from event AFS4703.1.6 that contain only the Red DS marker gene. It demonstrates that the expression of YL DGAT2 alters the FA profile of soybean seeds. The most pronounced change is an increase in oleic acid and a reduction in linolenic acid. Expression of YL DGAT genes also generates a reduction in palmitic and linoleic acid and an increase in stearic acid. TABLE 15A COMPOSITION OF FATTY ACIDS OF T2 SOY SEEDS GENERATED WITH KS362 TABLE 15B COMPOSITION OF FATTY ACIDS OF T2 SOYBEAN SEEDS GENERATED THROUGH COTRANSFORMATION WITH KS349 AND KS362

[0348] Events AFS4818.1.9 and AFS4818.1.3 were generated using a mixture of DNA fragments derived from plasmids KS349 (YL DGAT1) and KS362 (YL DGAT2) as described in Example 5. The transgenic T1 seed from these two events exhibits a increase in oil content of 22.9 and 16.2%, respectively, when compared with null segregating seed from the same T1 plant.

[0349] Although this observation strongly supports the conclusion that both events express transgene-derived YL DGAT, it is unclear whether these two events contain intact copies of both or just one DGAT gene present in the DNA mixture used for transformation. T1 seeds with increased oil and oleic acid content (see Example 6) were planted for events AFS4818.1.2, AFS4818.1.3, AFS4818.1.5, AFS4818.2.6 and AFS4818.1.9. DNA was isolated, digested with the two restriction enzymes EcoRI and HindIII and transferred to nylon membranes using standard protocols. Duplicate blots were produced and hybridized independently with probes corresponding to a 1.21 kb restriction fragment of the YL DGAT1 gene (generated through digestion of KS349 with NcoI/EcoRI) and the intact YL DGAT2 genes (generated through digestion of NotI of KS 362). Based on the sequence of KS 349 (SEQ ID NO. 48) and KS 362 (SEQ ID NO. 52), the insertion of an intact copy of YL DGAT1 and YL DGAT2 genes into the soybean genome would be indicated by a strong hybridization signal of restriction fragments with a size of >1,908 and >3,335 kb, respectively. In meeting this, all events exhibited strongly hybridizing bands of >1,908 kb when using a YL DGAT1 probe (Figure 8A). No hybridization signal was observed when using DNA from unmodified soybeans (lines 11 and 12, Figure 8A and B).

[0350] This demonstrates that all tested events have insertions of at least one copy of the intact YL DGAT1 expression pool present on KS 349.

[0351] When using YL DGAT2 DNA in hybridization experiments, however, event 4818.1.9 not only exhibited a very weakly hybridizing band with high MW, but also all other events tested exhibited strongly hybridizing bands of > 3,335 kb (line 9, Figure 8B).

[0352] The next genomic DNA from all five events was digested with BstXI, transferred to nylon membranes and probed with intact YL DGAT2 DNA generated as described above (Figure 9).

[0353] Insertion of an intact copy of the YL DGAT2 expression set would be indicated by strongly hybridizing bands of 0.584 kb (internal fragment) and additional fragments of > 0.2 and > 0.77 kb.

[0354] All events except 4818.1.9 exhibit the hybridization pattern indicative of complete YL DGAT2 insertion. It was concluded that 4818.1.9 contains only one functional expression unit for YL DGAT1.

[0355] T1 plants of events 4818.1.9 and 4818.1.3 that were derived from seed with high oil and oleic content of event 4818.1.9 and 4818.1.3 were grown to maturity and the seeds were harvested.

[0356] The fatty acid composition of T2 seeds was determined through TMSH derivation and GC analysis of seed flakes derived from T2 seeds.

[0357] The table compares the fatty acid composition of null and transgenic segregating seeds from a T2 plant of 4818.1.9 and 4818.1.3 (Table 15B). It demonstrates that the expression of YL DGAT1, as well as the coexpression of YL DGAT1 and YL DGAT2, alters the FA profile of soybean seeds.

[0358] The most pronounced change is an increase in oleic acid content and reduction in linolenic acid.

[0359] Expression of YL DGAT genes also generates a reduction in palmitic and linoleic acid and an increase in stearic acid.

EXAMPLE 12 ANALYSIS OF TRANSGENIC EVENTS - GROWTH CHAMBER:

[0360] The present example describes measurements of the oil content of soybeans derived from T2 plants that were homozygous or heterozygous for transgenes comprising YL DGAT1 or YL DGAT2 or both YL DGAT genes. T2 plants were grown in a controlled environment (growth chamber).

[0361] Analysis of oil from T2 soybean seeds derived from plants grown in a plant growth chamber was performed by means of NMR. Seeds were harvested from individual plants.

[0362] Seed selections from heterozygous plants derived from transformations with the Yarrowia DGAT2 gene exhibited segregation of the DS Red marker. The oil content of DS Red positive seeds (with DGAT2 transgene) and null segregating seeds from the same plant is shown in Table 16.

[0363] In the aforementioned table, the oil content of seeds containing the DGAT2 transgene is compared with that of non-transgenic null segregating seeds of the same plant. TABLE 16 OIL CONTENT OF TRANSGENIC SEEDS AND NULL-SEGREGANT SEEDS DERIVED FROM T2 TRANSGENIC SOYBEAN PLANTS THAT SEGREGATE TRANSGENE WITH THE YARROWIA DGAT2 GENE

[0364] T2 seed selections from events generated through cotransformation of KS349 and KS362 were performed through GC analysis of seed flakes as described above (Example 6). Seeds were collected from individual plants. Heterozygous plant seed selections derived from transformations with Yarrowia DGAT genes segregated by high oleic acid content (>22% total FA). Example 11 describes that event 4818.1.9 contains only an intact expression set for the Yarrowia DGAT1 gene. The oil content of high oleic acid seeds (with DGAT1 transgene) and null segregating seed of the same plant is shown in Table 17. In this table, the oil content of seeds containing the DGAT1 transgene was compared with that of plants non-transgenic null segregants from the same plant. TABLE 17 OIL CONTENT OF TRANSGENIC SEEDS AND NULL-SEGREGANT SEEDS DERIVED FROM T1 TRANSGENIC SOYBEAN PLANTS THAT SEGREGATE TRANSGENE WITH THE YARROWIA DGAT1 GENE

[0365] Example 11 describes that other events generated through cotransformation of KS349 and KS362 contain an intact expression set for the two Yarrowia DGAT genes. The oil content of high oleic acid seeds (with both DGAT transgenes) and null segregating seeds of the same plant is shown in Table 18. In this table, the oil content of seeds containing both DGAT transgenes was compared with that of non-transgenic null segregating plants of the same plant. TABLE 18 OIL CONTENT OF TRANSGENIC SEEDS AND NULL-SEGREGANT SEEDS DERIVED FROM T1 TRANSGENIC SOYBEAN PLANTS THAT SEGREGATE TRANSGENS WITH YARROWIA DGAT1 AND DGAT2 GENES

[0366] Selection of T2 seeds homozygous for the Yarrowia DGAT2 expression pool derived from KS362 no longer segregated the DS Red marker. The oil content of 48 seeds (or all available seeds if fewer than 48 seeds were available) was measured using NMR. For each event, T1 Red DS negative seeds were planted and T2 seeds of null segregants were harvested from plants grown in the same growth chamber used to grow T1 plants homozygous for the DGAT transgene. The oil content of seeds derived from null segregating selections and lines homozygous for the DGAT transgene is shown in Table 19. YARROWIA DGAT2 GENE

[0367] Selection of T2 seeds homozygous for expression sets of Yarrowia DGAT1 derived from KS349 and DGAT2 derived from KS362 no longer segregated with respect to the high oleic acid phenotype (>22% oleic) associated with the expression of DGAT genes from Yarrowia. The oil content of 48 seeds (or all available seeds if fewer than 48 seeds were available) was measured using NMR. For each event, T1 null segregating seeds that did not exhibit oleic acid elevation were planted and T2 seeds of these null segregants were harvested from plants grown in the same growth chamber used to grow T1 plants homozygous for the DGAT transgenes. The oil content of seeds derived from null segregating selections and lines homozygous for the DGAT transgene is shown in Table 20. YARROWIA DGAT1 AND DGAT2 GENES

[0368] Briefly, the growth chamber results exhibit excellent heritability of increased oil traits associated with overexpression of a single Yarrowia DGAT gene or co-expression of Yarrowia DGAT genes in soybean seeds.

[0369] The increase in oil (when compared to null segregating seeds) associated with the expression of a single Yarrowia DGAT gene is at least 10.9% and up to 25.1%.

[0370] The increase in oil (when compared to null segregating seed) associated with the expression of the two Yarrowia DGAT genes is at least 14.4% and up to 24.2%.

EXAMPLE 13A ANALYSIS OF TRANSGENIC EVENTS: FIELD

[0371] The present example describes measurements of soybean oil content derived from T2 plants that were homozygous or heterozygous for transgenes comprising YL DGAT1 or YL DGAT2 or the two YL DGAT genes. T2 plants were grown in an uncontrolled environment (field).

[0372] T1 Red DS positive seed from transgenic events generated with YL DGAT2 (contained in KS 362) and corresponding Red DS negative DS null segregating seed were grown in a field in Iowa in the summer of 2007.

[0373] T1 seeds with high oleic acid content that had been generated through cotransformation with YL DGAT1 and YL DGAT2 and corresponding null segregating seed with normal oleic acid levels were cultivated in a similar way. T2 seeds were harvested from individual plants and subjected to NMR analysis to measure oil content.

[0374] Table 21 displays the oil content of 48 Red DS positive seeds uniformly derived from events that were homozygous for the KS362 transgene and that of Red DS negative seeds from null segregating seeds from the same event grown in the same environment. TABLE 21 OIL CONTENT OF NULL SEGREGANT SEEDS AND TRANSGENIC SEEDS DERIVED FROM FIELD-GROWN T1 TRANSGENIC SOYBEAN PLANTS THAT ARE HOMOZYGOTIC FOR TRANSGENES WITH THE YARROWIA DGAT2 GENE

[0375] Table 22 displays the oil content of 48 seeds from segregants that were homozygous for YL DGAT1 and YL DGAT2 transgenes.

[0376] All seeds harvested from these homozygous T2 seed selections exhibited the high oleic acid content associated with YL DGAT expression.

[0377] In Table 22, the oil content of these lines is compared with that of null segregating seeds from the same events with unchanged levels of oleic acid, derived from plants grown in the same environment. TABLE 22 OIL CONTENT OF NULL SEGREGANT SEEDS AND TRANSGENIC SEEDS DERIVED FROM FIELD-GROWN T1 TRANSGENIC SOYBEAN PLANTS THAT ARE HOMOZYGOTIC FOR TRANSGENES WITH THE YARROWIA DGAT1 AND DGAT2 GENES

[0378] Example 11 describes that event 4818.1.9 contains an intact expression set for the Yarrowia DGAT1 gene. Three T1 plants from this event derived from T1 seeds with high oleic acid content were grown in the field in Iowa along with lateral null segregating plants derived from T1 seeds with unchanged oleic acid content. T2 seeds from all three transgenic segregants still exhibited segregation of the high oleic acid phenotype which indicates that the parental lines were still heterozygous for the DGAT1 transgene. Using GC analysis, all transgene-positive seeds were identified from these lines and subjected to oil analysis using NMR. In Table 23, the oil content of these seeds is compared with that of null segregating seeds derived from T1 plants grown in the same environment. TABLE 23 OIL CONTENT OF NULL SEGREGANT SEEDS AND TRANSGENIC SEEDS DERIVED FROM FIELD-GROWN T1 TRANSGENIC SOYBEAN PLANTS THAT ARE HETEROZYGOTIC FOR TRANSGENES WITH THE YARROWIA DGAT1 GENE

[0379] In summary, the field environment results exhibit excellent heritability of the increased oil trait associated with overexpression of an isolated Yarrowia DGAT gene or co-expression of the two Yarrowia DGAT genes in soybean seed. The increase in oil (when compared to null segregating seed) associated with the expression of a single Yarrowia DGAT gene is at least 11% and up to 14%. The increase in oil (when compared to null segregating seed) associated with the expression of the two Yarrowia DGAT genes is at least 19% and up to 20%.

EXAMPLE13B COMPOSITION ANALYSIS OF SOYBEAN SEEDS:

[0380] The present example describes measurements of seed composition parameters such as protein content and soluble carbohydrate content of soybean seeds derived from transgenic events expressing isolated YL DGAT genes (YL DGAT2) from the two YL DGAT genes.

[0381] Changes in the composition of soybean seeds associated with the expression of YL DGAT genes were measured. For this purpose, the concentrations of protein, soluble carbohydrates and starch were measured as follows.

ANALYSIS OF PROTEINS AND NON-STRUCTURAL CARBOHYDRATES:

[0382] Dried soybean seeds were ground into a fine powder in a Geno grinder and subsamples were weighed (to 0.1 mg accuracy) into thirteen 100 mm glass tubes; The tubes contained Teflon®-lined screw cap closures. Three replicates were prepared for each sample tested. Fabric dry weights were calculated by weighing subsamples before and after drying in a forced air oven for eighteen hours at 105°C. Lipid extraction was performed by adding 2 ml portions of heptane to each tube. The tubes were mixed by vortexing and placed in an ultrasonic bath (VWR Scientific Model 750D) filled with water heated to 60 °C. The samples were sonicated at full power (around 360 W) for fifteen minutes and then centrifuged (5 min x 1700 g). The supernatants were transferred to clean thirteen 100 mm glass tubes and the pellets were extracted twice more with heptane (2 ml, second extraction, 1 ml third extraction) with the supernatants from each extraction pooled. After lipid extraction, 1 ml of acetone was added to the pellets and after mixing by vortexing, to completely disperse the material, they were taken to dry in a Speedvac.

ANALYSIS AND EXTRACTION OF NON-STRUCTURAL CARBOHYDRATES.

[0383] Two milliliters of 80% ethanol were added to the top dry pellets. The samples were thoroughly mixed by vortexing until complete dispersion of the plant material in the solvent before sonication at 60 °C for fifteen minutes. After centrifugation, 5 min x 1700 g, the supernatants were decanted into thirteen clean 100 mm glass tubes. Two other extractions with 80% ethanol were performed and the supernatants from each were pooled. The extracted pellets were suspended in acetone and dried (as above). An internal standard β-phenyl glucopyranoside (100 μl of a 0.5000 +/- 0.0010 g/100 ml standard) was added to each extract before drying in a Speedvac. Extracts were kept in a dissector until further analysis.

[0384] The acetone-dried powders from above were suspended in 0.9 ml of MOPS buffer (3-N[morpholino]propanesulfonic acid; 50 mM, 5 mM CaCl2, pH 7.0) containing 100 U of α- heat-stable amylase (from Bacillus licheniformis; Sigma A-4551). The samples were placed in a heating block (90 °C) for 75 minutes and were mixed by vortexing every fifteen minutes. The samples were then kept cooled at room temperature and 0.6 ml of acetate buffer (285 mM, pH 4.5) containing 5 U of amyloglucosidase (Roche 110 202 367 001) was added to each. Samples were incubated for fifteen to eighteen hours at 55°C in a water bath equipped with a reciprocating shaker; Soluble potato starch standards (Sigma S-2630) were included to ensure that starch digestion proceeded to completion.

[0385] After digestion, the released carbohydrates were extracted before analysis. Absolute ethanol (6 ml) was added to each tube and, after mixing by vortexing, the samples were sonicated for fifteen minutes at 60 °C. Samples were centrifuged (5 min x 1700 g) and supernatants were decanted into thirteen clean 100 mm glass tubes. The pellets were extracted two more times with 3 ml of 80% ethanol and the resulting supernatants were pooled. Internal standard (100 μl of β-phenyl glucopyranoside, as above) was added to each sample before drying in a Speedvac.

SAMPLE PREPARATION AND ANALYSIS:

[0386] The dried samples from the starch and soluble extractions described above were solubilized in anhydrous pyridine (Sigma-Aldrich P57506) containing 30 mg/ml hydroxylamine HCl (Sigma-Aldrich 159417). The samples were placed on an orbital shaker (300 rpm) overnight and then heated for one hour (75 °C) with vigorous vortex mixing applied every fifteen minutes. After cooling to room temperature, 1 ml of hexamethyldisilazane (Sigma-Aldrich H-4875) and 100 μl of trifluoroacetic acid (Sigma-Aldrich T-6508) were added. Samples were mixed by vortexing and precipitates were allowed to settle before transferring the supernatants to GC sample vials.

[0387] Samples were analyzed on an Agilent 6890 gas chromatograph equipped with a DB-17MS capillary column (15 m x 0.32 mm x 0.25 μm film). The inlet and detector temperatures were both 275 °C. After injection (2 μl, 20:1 split), the initial column temperature (150 °C) was increased to 180 °C at a rate of 3 °C/minute and then at 25 °C/minute to a temperature final temperature of 320 °C.

[0388] The final temperature was maintained for ten minutes. The carrier gas was H2 at a linear velocity of 51 cm/sec. Detection was by flame ionization.

[0389] Data analysis was performed using Agilent ChemStation software. Each sugar was quantified relative to the internal standard and detector responses were applied to each individual carbohydrate (calculated from standards run with each sample set). Final carbohydrate concentrations were expressed on a tissue dry weight basis.

PROTEIN ANALYSIS:

[0390] Protein content was estimated through combustion analysis on a Thermo Finnigan Flash 1112EA combustion analyzer. Samples weighing 4 to 8 mg, weighed to an accuracy of 0.001 mg on a Mettler-Toledo MX5 microbalance, were used for analysis. Protein content was calculated by multiplying %N, determined by the analyzer, by 6.25. The final protein content was expressed based on percentage of tissue dry weight. TABLE 24 COMPOSITION ANALYSIS OF SOYBEAN SEEDS DERIVED FROM TWO T1 PLANTS THAT WERE A NULL SEGREGANT OR HOMOZYGOTIC FOR A YL DGAT1 YL DGAT2 TRANSGENE

[0391] The plants were grown in the same growth chamber environment. Unless otherwise noted, values are reported as g/kg DW. TABLE 25 COMPOSITION ANALYSIS OF SOYBEAN SEEDS DERIVED FROM A T1 PLANT THAT WAS HETEROZYGOTIC FOR A YL DGAT2 TRANSGENE

[0392] The plant was grown in a growth chamber. YL DGAT transgenic seeds and null segregating seeds were selected based on expression of Red DS as a visible marker. Eight Red DS positive and Red DS negative seeds were combined and analyzed as described above. Unless otherwise noted, values are reported as g/kg DW. TABLE 26 COMPOSITION ANALYSIS OF SOYBEAN SEEDS DERIVED FROM T1 PLANTS THAT WERE HETEROZYGOTIC FOR A YL DGAT2 OR YL DGAT1 YL DGAT2 TRANSGENE

[0393] The plants were grown in the field. YLD DGAT transgenic seeds and null segregating seeds were selected based on expression of RS Red (4822.2.10) as a visible marker or high oleic acid content determined through GC analysis (4818.1.2). Eight transgene-positive and transgene-negative seeds were combined and analyzed as described above. Unless otherwise noted, values are reported as g/kg DW.

[0394] Tables 24 to 26 illustrate that the expression of one or two YL DGAT genes in different environments (growth chamber, field) is associated with a consistent change in seed composition that is characterized by a reduction in soluble carbohydrates, namely a reduction in sucrose and, to a lesser extent, a reduction in stachyose. Most importantly, there is no reduction in protein content observed when increasing oil accumulation through expression of YL DGAT genes.

EXAMPLE 14 MEASUREMENTS OF DGAT ACTIVITY IN DEVELOPING SEEDS AND SOMATIC EMBRYOS:

[0395] The present example describes the construction of soybean expression vectors comprising DGAT2 isolated from Yarrowia or DGAT2 and DGAT1 from Yarrowia, the expression of these gene(s) in soybean seeds or somatic embryos, and the activity of DGAT enzyme in these tissues.

CONSTRUCTION OF PKR1234 COMPRISING YL DGAT2:

[0396] The NotI fragment of KS362 (SEQ ID NO:52), which contains the YL DGAT2, was cloned into the NotI fragment of pKR72 (SEQ ID NO:26; Example 4) to produce pKR1234 (SEQ ID NO:68).

CONSTRUCTION OF PKR1236 COMPRISING YL DGAT1 AND DGAT2:

[0397] The glycinin Gy1 promoter was amplified by PCR from pZBL119 (which is described in WO 2004/071467, the content of which is incorporated herein by reference) using the oSGly-2 primers (SEQ ID NO. 69 ) and oSGly-3 (SEQ ID NO. 70). The resulting PCR fragment was subcloned into the PCR-Script AMP SK(+) intermediate cloning vector (Stratagene), according to the manufacturer's protocol, to produce the plasmid pSgly32 (SEQ ID NO. 71).

[0398] The Pst I/Not I fragment of plasmid pSGly32 (SEQ ID NO. 71), which contains the Gy1 promoter, was cloned into the PstI/NotI fragment of plasmid pKR142 (which is described in document WO 2004/071467, which contains the transcription termination region of legumin A 2 3', an ampicillin resistance gene of bacterial origin, to produce pKR264 (SEQ ID NO. 72). Thus, the pKR264 vector contains a NotI site flanked by the promoter for the gene. Glycinin Gy1 and the legumin A 2 3' transcription termination region (Gy1/NotI/legA2 set).

[0399] The NcoI/XbaI fragment of KS349 (SEQ ID NO: 48), which contains Yarrowia DGAT1, was cloned into the NcoI/XbaI sites of pKR908 (US Patent 20080095915), which contains NcoI/XbaI sites flanked by NotI sites, to produce pKR1212 (SEQ ID NO: 73).

[0400] The NotI fragment of pKR1212 (SEQ ID NO. 73), which contains the Yarrowia DGAT1 gene, was cloned into the NotI site of pKR264 (SEQ ID NO. 72) to produce pKR1235 (SEQ ID NO. 74).

[0401] The BsiWI fragment of pKR1235 (74), which contains the Yarrowia DGAT1 gene, was cloned into the BsiWI site of pKR1234 (SEQ ID NO. 68) to produce pKR1236 (SEQ ID NO. 75). DGAT TESTS ON MICROSOME EXTRACTS FROM T2 SEEDS UNDER DEVELOPMENT:

[0402] Soybean embryogenic suspension cultures (cv. Jack) were transformed with KS362 as described herein (Example 6), which comprises Yarrowia DGAT2 as described herein and T1 seeds from AFS4822.1.13.1 soybean plants ( seed designated 7GR11-58) and AFS4822.2.10.1 (seed designated 7GR11-66) were planted and the plants grown as described in Example 12. In these two events, the oil concentration of transgene-positive seeds was found to be high when compared with null segregating seed from the same event.

[0403] Similarly, soybean (cv. Jack) embryogenic suspension cultures were cotransformed with KS362 (comprising YL DGAT2) and KS349 (comprising YL DGAT1) as described herein (Example 6). Transgene-positive T1 seeds from event AFS4818.1.2.1 are represented by seed 7GR11-2. In this event, the oil concentration of transgene-positive seeds was found to be elevated when compared to null segregating seeds from the same event (Example 6). Transgene-negative null segregating T1 seeds derived from AFS488.1.2.1 and AFS4818.1.3.1 are represented by 7GR11-7 and 7GR11-15. These seeds were planted and the plants grown as described in Example 12.

[0404] Approximately one gram of T2 seeds was collected from selected plants thirty days after flowering (DAF), snap-frozen in liquid nitrogen, and stored at -80 °C until ready for processing. After grinding one gram of liquid nitrogen-frozen seed tissue in a pestle and mortar, 3 ml of plant homogenization buffer (300 mM sucrose; 1 mM EDTA; 10 mM Tris HCl, pH 8.0; 1 mM DTT; 0.1% polyvinylpolypyrrolidine) was added and the tissue was further homogenized using a polytron homogenizer for one minute. Fragments were collected by vacuum filtration through three layers of cheesecloth followed by filtration through one layer of Miracloth paper. The resulting filtrate was centrifuged for fifteen minutes twice at 1500 x g, and the resulting supernatant was then centrifuged at 100,000 x g for sixty minutes. The resulting pellet was reacted in approximately 0.5 to 1 ml of microsome buffer (100 mM potassium phosphate, pH 7.2) by gentle pipetting, followed by additional resuspension in a 2 ml Teflon-coated Dounce homogenizer. Protein concentrations were determined using Bradford reagent (Sigma-Aldrich), and microsomes were snap-frozen in liquid nitrogen and stored at −80 °C until assayed.

[0405] DGAT tests were conducted for five minutes at 25°C in plant test buffer (500 mM Tricine, pH 7.8; 28 mM sodium chloride; 0.06% CHAPS), with 20 μM of 1-14C-labeled oleoylcoenzyme A (50 mCi/mmol, Perkin Elmer), 1.5 mM dioleoylglyceride (Sigma-Aldrich) and 20 μg of microsomal protein in a total reaction volume of 100 μl. Each reaction was initiated by adding the microsomal membranes to the rest of the reaction components. The tests were terminated by adding 1 ml of hot isopropanol (75 °C) and heating at 75 °C for ten minutes. The tests were cooled to room temperature, 1.5 ml of hexane was added and the samples were mixed. The phases were separated by centrifugation at low speed after the addition of 1.25 ml of 500 mM sodium sulfate and the upper phase was transferred to another glass tube. The upper phase was then dried under nitrogen gas. The lipid from each test was redissolved in 75 μl of hexane blocked with 1 μl of soybean oil. Lipid was applied to a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 μm thickness, 20 cm x 20 cm) and triglycerides were separated from other lipids by development with 80:20:1 (v/ v/v) hexane: diethyl ether: acetic acid. Triacylglycerol was visualized and marked by light spots in iodine vapor in a tank. The plate was removed from the iodine tank and, after the stain had faded, the triacylglycerol was scraped off and radioactivity was determined by liquid scintillation counting and expressed as dpm per minute.

[0406] Total activity was determined as the amount of radiolabeled oleic acid incorporated into triacylglycerol per minute per mg of protein using the following formula: ([dpm] / [2200000 dpm/uCi] / [50 uCi/mol] / [ 5 min] / [0.02 mg protein] x [1000 nmol/μmol]). DGAT activities for each of the samples described are shown in Table 27. TABLE 27 DGAT ACTIVITIES FOR SELECTED T2 SEEDS UNDER DEVELOPMENT DGAT TESTS ON MICROSOMAL EXTRACTS FROM SOYBEAN SOMATIC EMBRYOS:

[0407] Soybean embryogenic suspension cultures (cv. Jack) were transformed with pKR1234 (SEQ ID NO. 68), which comprises Yarrowia DGAT2 and has experiment number MSE2181 or with pKR1236 (SEQ ID NO. 75), which comprises DGAT2 and DGAT1 from Yarrowia and has experiment number MSE2182. Events were selected and somatic embryos matured into SHaM as described in Example 5.

[0408] After two weeks of maturation in SHaM, about 1 g of tissue from each event was frozen in liquid nitrogen and tissue was ground with a pestle and mortar as described for developing soybean seed. A small amount of ground tissue (about 100 mg) was freeze-dried overnight and the remaining tissue was stored at −80 °C.

[0409] Oil concentrations were determined on about 10 mg of lyophilized tissue from each event using the GC method and 17:0 internal standard exactly as described in Example 5 and the results of oil concentrations and oleic acid content (percent of total FAME) are shown in Table 28. Microsomal protein preparations were prepared, protein concentrations were determined, and DGAT tests were conducted on selected events determined to have a range of oil concentrations exactly as previously described for tissue. seeds. DGAT test results are also displayed in Table 28. TABLE 28 OLEIC ACID, ESTERIFIED FATTY ACID CONTENT AND DGAT ACTIVITIES OF SOYBEAN SOMATIC EMBRYOS TRANSFORMED WITH PKR1 234 OR PKR1236 MSE2181-pKR1234 (YL DGAT2)

[0410] Events transformed with pKR1234 and containing some of the highest oil concentrations showed increases in DGAT activity of up to ninefold when compared to events containing wild-type oil levels. Events transformed with pKR1236 and containing some of the highest oil concentrations showed increases in DGAT activity of up to 15.8-fold when compared to events having wild-type oil levels.

[0411] Soybean embryogenic suspension culture (cv. Jack) was also transformed with KS364 (SEQ ID NO. 63), which comprises DGAT2 and DGAT1 from Yarrowia (Experiment No. MSE2134) and individual events were analyzed to determine the profile of fatty acid and oil concentration described in Example 5. Based on these data, an event (Event 54) that contains high oleic acid content (32.83% of total fatty acid) and oil concentrations (12.5% DCWt ) and one event (Event 33) containing wild-type oleic acid levels (15.52% of total fatty acids) and oil concentrations (8.2% DCWt) were selected for DGAT testing. Transformed embryogenic suspension culture from each event was accumulated in SB 196 media and embryos matured in SHaM as described in Example 5.

[0412] After two weeks of maturation in SHaM, approximately 1 g of tissue from each event was frozen in liquid nitrogen, microsomal protein preparations were made, and DGAT tests were conducted on each event exactly as previously described and the results are shown in Table 29. Total lipids were also extracted and oleic acid and oil concentrations were determined as described below and the results are reported in Table 29. SOMATIC SOYBEANS PROCESSED WITH KS364

[0413] The event containing high oil concentration (Event 54) showed increases in DGAT activity of 10.7-fold when compared to the event containing wild-type oil levels (Event 33).

EXAMPLE 15 ANALYSIS OF LIPID FRACTIONS OF TRANSGENIC SEEDS AND SOMATIC EMBRYOS EXPRESSING DGAT GENES:

[0414] Soybean somatic embryos transformed with KS364 from an event with wild-type oil and oleic acid concentrations (Event 33) and an event with high oil and oleic acid concentrations (Event 54) were freeze-dried for 48 hours and the tissue was ground using a genogrinder exactly as described in Example 5.

TOTAL LIPID EXTRACTION:

[0415] Total lipids were extracted from each event using the method of Bligh, E. G. and Dyer, W. J. (Can. J. Biochem. Physiol. 37: 911-917 (1959)) with some modifications. Briefly, approximately 100 mg of ground tissue from each event was added to a 16 mm x 125 mm test tube with a Teflon-lined screw cap. A 2:1 mixture of methanol and chloroform (6 ml) was added and the sample was extracted with gentle mixing for one hour, after which 2 ml of chloroform was added, followed by continuous mixing for thirty minutes. Then, 3.6 ml of water was added, the tube was vortexed vigorously and the phases were separated by centrifugation in a clinical centrifuge. The lower organic layer was gently removed to a second glass test tube and the upper aqueous layers were again extracted with 2 ml of chloroform. Centrifugation was repeated and the lower organic phase was combined with the first organic phase. Samples were dried under a stream of nitrogen at 50°C, total lipid was estimated by weighing, and the lipid was dissolved in 6:1 chloroform:methanol to a concentration of about 10 mg/ml. FAME analysis was conducted on about 50 μg of each sample using the sulfuric acid and methanol procedure described herein (Example 4) and the results are displayed in Table 30.

SEPARATION OF POLAR AND NEUTRAL LIPIDS:

[0416] Seppak aminopropyl solid phase extraction columns (Waters; 6 cc columns; WAT054560) were equilibrated with 5 ml of methanol, followed by 5 ml of 1:1 methanol:chloroform followed by 5 ml of chloroform. About 5 mg of total lipid in 6:1 chloroform:methanol was added to each column, followed by five 1 ml portions of chloroform to elute neutral lipids, and all fractions were collected, combined, and dried under nitrogen flow at 50° W. Polar lipids were eluted from each column using five 1 ml portions of 1:1 methanol:chloroform followed by five 1 ml portions of methanol and all fractions were combined and dried under nitrogen. Neutral lipids were dissolved in about 1 ml of CHCl3:MeOH 6:1 and polar lipids were dissolved in about 200 μl of CHCl3:MeOH 6:1. FAME analysis was conducted on about 50 μg of neutral lipids using the sulfuric acid and methanol procedure described herein (Example 4) and the results are displayed in Table 30.

SEPARATION OF TAG, PC AND PE BY MEANS OF TLC:

[0417] About 100 μl of neutral lipid extract was loaded 2 cm from the bottom of a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 μm thickness, 20 cm x 20 cm). Similarly, about 200 μl of the polar lipid fraction was loaded onto the same TLC plate. Standard solutions (10 mg/ml in chloroform:methanol 6:1) of TAG, PC and PE were also placed on the plates. TLC plates were developed in 65:35:8 CHCl3:MeOH:AcOH until the solvent front was halfway up the plate. The TLC plates were then dried in air for ten minutes and completely developed in 70:30:1 (v/v/v) hexane: diethyl ether: acetic acid. Patterns were visualized by iodine vapor light staining and corresponding bands for TAG, PC, and PE were cut from the TLC plate. Silica gel containing each lipid species was derived directly with sulfuric acid and methanol as described herein (Example 4) and the results are displayed in Table 30.

ANALYSIS OF TAG FATTY ACID POSITION

[0418] The fatty acid profiles of the sn2 position of TAG were determined using porcine pancreatic lipase to remove acyl groups from the sn1 and sn3 position of TAG only, followed by transesterification of the resulting monoacylglyceride (MAG) produced. Approximately 5 mg of neutral lipid extract was suspended in 2 ml of 1M Tris.HCl, pH 8.0 along with 0.2 ml of 2.2% calcium chloride and 0.5 ml of 0.05% bile salts in a glass screw-capped test tube. The lipid was incubated at 37 °C for five minutes, 5 mg of porcine pancreatic lipase was added directly, and the suspension was incubated with shaking at 37 °C for twenty minutes. After incubation, the reaction was terminated by the addition of 1 ml of ethanol followed by 1 ml of 6 M HCl. After mixing, 2.5 ml of diethyl ether was added, the phases were separated by centrifugation, and the upper organic layer was carefully removed. The diethyl ether extraction was repeated, and the upper diethyl ether phase was combined with the first. After drying over anhydrous sodium sulfate, the diethyl ether was evaporated under a nitrogen stream at 50 °C, and the resulting lipid was dissolved in 200 μl of chloroform and methanol 6:1. The lipid was loaded onto a Partisil K6 TLC plate along with trialcylglyceride (TAG), dialcylglyceride (DAG), monoacylglyceride (MAG), and free fatty acid (FFA) standards, and the TLC plate was developed as described herein. Then, the standards were visualized with clear iodine stains, and the MAG band was cut and derivatized with methanol and sulfuric acid as previously described herein. The results for the FAME fatty acid profile of the MAG range, which represents the fatty acid profile of the sn2 position of TAG (i.e., the acyl group on C2 of glycerol), along with the calculated sn1 and sn3 positions, are displayed in Table 30. In Table 30, the percent total fatty acid for each fatty acid (i.e., 16:0, 18:0, 18:1, 18:2, 18:3) at the sn1 and sn3 positions of TAG is calculated with the following formula: = ([TAGx]-[sn2x]/3)*3/2; where the x indicates the fatty acid of interest. TABLE 30 FATTY ACID COMPOSITION OF VARIOUS LIPID SPECIES AND DISTRIBUTION OF POSITIONS IN TAG

[0419] Changes in fatty acid profiles associated with YL DGAT expression observed in TAG are also observed in polar lipids.

EXAMPLE 16 YARROWIA DGAT VARIANTS WITH ALTERED AMINO ACID SEQUENCE:

[0420] The present example describes the creation of mutant forms of Yarrowia DGAT2, cloning them into a yeast expression vector and testing microsomal protein fractions to determine DGAT activity.

CONSTRUCTION OF SACCHAROMYCES EXPRESSION VECTORS CONTAINING MUTANT YARROWIA DGAT2S:

[0421] Yarrowia DGAT2 was amplified from pKR1234 (SEQ ID NO. 68; Example 14) with oligonucleotide primers oYDG2-1 (SEQ ID NO. 76) and oYDG2-2 (SEQ ID NO. 77), using DNA Phusion® High Fidelity Polymerase (Cat. no. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR1254 (SEQ ID NO. 78).

[0422] An isolated codon in the Yarrowia DGAT2 sequence, which encodes the amino acid Y326 in the corresponding amino acid sequence SEQ ID NO. 10, was altered using the Quickchange® Site-Directed Mutagenesis Kit (Cat. No. 200518, Stratagene, La Jolla CA) with oligonucleotides YID2_Y326F-5 (SEQ ID NO. 79) and YID2_Y326F-3 (SEQ ID NO. 80), following the manufacturer's protocol. After extensive sequencing, a clone encoding an amino acid sequence that is identical to Yarrowia DGAT2 (SEQ ID NO. 10), except that Y326 was changed to F326, was selected for further study. This clone was designated pKR1254_Y326F (SEQ ID NO: 81). The nucleotide sequence for altered coding sequence (YIDGAT2_Y326F) is described in SEQ ID NO: 82 and the corresponding amino acid sequence is described in SEQ ID NO: 83.

[0423] A single codon in the Yarrowia DGAT2 sequence, which encodes the amino acid Y326 in the corresponding amino acid sequence SEQ ID NO. 10, was altered using the Quickchange® site-directed mutagenesis kit (Cat. No. 200518, Stratagene, La Jolla CA) with oligonucleotides YID2_Y326L-5 (SEQ ID NO. 84) and YID2_Y326L-3 (SEQ ID NO. 85), following the manufacturer's protocol. After extensive sequencing, a clone encoding an amino acid sequence that is identical to Yarrowia DGAT2 (SEQ ID NO. 10), except that Y326 was changed to L326, was selected for further study. This clone was named pKR1254_Y326L (SEQ ID NO: 86). The nucleotide sequence for altered coding sequence (YIDGAT2_Y326L) is described in SEQ ID NO: 87 and the corresponding amino acid sequence is described in SEQ ID NO: 88.

[0424] A single codon in the Yarrowia DGAT2 sequence, which encodes the amino acid R327 in the corresponding amino acid sequence SEQ ID NO. 10, was altered using the Quickchange® Site-Directed Mutagenesis Kit (Cat. No. 200518, Stratagene, La Jolla CA) with the oligonucleotides YID2_R327K-5 (SEQ ID NO. 89) and YID2_R327K-3 (SEQ ID NO. 90), following the manufacturer's protocol. After extensive sequencing, a clone encoding an amino acid sequence that is identical to Yarrowia DGAT2 (SEQ ID NO. 10), except that R327 was changed to K327, was selected for further study. This clone was named pKR1254_R327K (SEQ ID NO. 91). The nucleotide sequence for altered coding sequence (YIDGAT2_Y326L) is described in SEQ ID NO: 92 and the corresponding amino acid sequence is described in SEQ ID NO: 93.

[0425] NotI fragments from pKR1254, pKR1254_Y326F, pKR1254_Y326L or pKR1254_R327K, each of which contains a mutant or wild-type version of YIDGAT2, were cloned into the NotI site of pY75 (SEQ ID NO: 3; Example 1) to produce pY191 (SEQ ID NO. 94), pY192 (SEQ ID NO. 95), pY193 (SEQ ID NO. 96) or pY194 (SEQ ID NO. 97), respectively.

DGAT ACTIVITY TEST OF DGAT2S FROM YARROWIA MUTANTS:

[0426] A mutant strain of Saccharomyces cerevisiae in which the endogenous DGAT2 gene (DGA1) has been knocked out and has the following genotype (BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was obtained from Open Biosystems (http://www.openbiosystems .with/). It was transformed with pY191, pY192, pY193, or pY194, and transformers were isolated as described herein. Three individual transformants per transformation were inoculated into 2 ml DOBA media cultures supplemented with CSM-leu at 30°C for sixteen hours. Cells (1 ml) were transferred to 50 ml of DOBA medium described above and cultured at 30 °C for an additional sixteen hours. Cells were pelleted via centrifugation, frozen in liquid nitrogen, and stored at −80°C until needed for use.

[0427] Pellets were resuspended in 2 ml of yeast homogenization buffer (20 mM Tris.HCl, pH 8.0; 10 mM MgCl2; 1 mM EDTA; 5% glycerol; 1 mM DTT; 0. 3 M (NH4)2SO4) and the suspension was added to a 2 ml screw cap tube containing about 1 ml of 0.5 mm glass beads. After removing air pockets by vortexing, the new suspension was filled to the top of the tube, the tube was capped and the cells were disrupted with three one-minute pulses in a mini ball beater at 5000 rpm with storage on ice. for five minutes. The yeast homogenate was centrifuged at 1500 x g for fifteen minutes at 4 °C and the resulting supernatant was then centrifuged at 100,000 x g for sixty minutes. The resulting pellet was added to approximately 0.2 to 0.5 ml of microsome buffer (100 mM potassium phosphate, pH 7.2) by gently pipetting, followed by additional resuspending in a sized Teflon-coated Dounce homogenizer. with 2 ml. Protein concentrations were determined using Bradford reagent (Sigma-Aldrich) and microsomes were snap-frozen in liquid nitrogen and stored at −80°C until assayed.

[0428] DGAT tests were conducted for one minute at 25°C in yeast test buffer (50 mM potassium phosphate (pH 7.2)) with 20 μM 1-14C-labeled oleoylcoenzyme A (50 mCi /mmol, Perkin Elmer) and 20 μg of microsomal protein in a total reaction volume of 100 μl. Each reaction was initiated by adding the microsomal membranes to the rest of the reaction components. The tests were terminated and the radioactivity in TAG was determined exactly as described for the plant DGAT tests, except for the formula, which was changed to reflect a one minute test time (i.e., [dpm] / [2200000 dpm/ uCi] / [50 uCi/umol] / [5 min] / [0.02 mg protein] x [1000 nmol/umol]). The DGAT activities for each of the samples, as well as the averages described, are shown in Table 31. TABLE 31 DGAT ACTIVITIES FOR DGA1 TRANSFORMED WITH PY191, PY192, PY193 OR PY194

[0429] From Table 31, it is concluded that the Y326F and Y326L amino acid changes have minimal effect on Yarrowia DGAT2 activity when tested in yeast and these two mutants were selected for expression in soybean somatic embryos.

CONSTRUCTION OF SOY EXPRESSION VECTORS CONTAINING MUTANT YARROWIA DGAT2S:

[0430] The NotI fragment of pKR1254 (SEQ ID NO. 78), pKR1254_Y326F (SEQ ID NO. 81) or pKR1254_Y326L (SEQ ID NO. 86), which contains mutant or wild-type forms of Yarrowia DGAT2, was cloned into NotI fragment of pKR72 (SEQ ID NO: 26; Example 4) to produce pKR1256 (SEQ ID NO: 98), pKR1277 (SEQ ID NO: 99) or pKR1278 (SEQ ID NO: 100), respectively.

DETERMINATION OF OIL CONCENTRATIONS FROM SOYBEAN SOMATIC EMBRYOS EXPRESSING MUTANT YARROWIA DGAT2s:

[0431] Soybean embryogenic suspension culture (cv. Jack) was transformed with pKR1256 (SEQ ID NO. 98) which comprises wild-type Yarrowia DGAT2 and has experiment number MSE2228, pKR1277 (SEQ ID NO. 99), which comprises DGAT2_Y326F from Yarrowia and has experiment number MSE2229 or pKR1278 (SEQ ID NO. 100), which comprises DGAT2_Y326L and has experiment number MSE2230.

[0432] Events were selected and somatic embryos matured in SHaM as described in Example 5.

[0433] Oil concentrations were determined for each event using NMR and described herein, fatty acid profiles were determined using GC exactly as described herein and the results of oil concentrations and oleic acid content (% of total FAME) are shown in Table 32. TABLE 32 OIL CONCENTRATIONS FOR SOMATIC EMBRYOS TRANSFORMED WITH PKR1256, PKR1277 OR PKR1278 MSE2228-pKR1256 (wt DGAT2)

[0434] In somatic soybean embryos, a variant of the YL DGAT2 protein that drives the Y326F mutation increases oil concentrations and alters the fatty acid profile of the oil at least to the same extent as wild-type Yarrowia DGAT2.

EXAMPLE 17 EXPRESSION OF OPTIMIZED DGAT GENES:

[0435] Sequences encoding YL DGAT1 and YL DGAT2 genes that are optimized for expression in soybean plants are described in SEQ ID NO. 64 and SEQ ID NO. 66. The design of these sequences is described in Example 9. DNA molecules with this DNA sequence flanked by Not I restriction sites were synthesized using DNA 2.0 (California, United States). Plasmid DNA with the synthesized genes was digested with Not I. Restriction fragments of Not I with the DGAT genes were ligated to linearized dephosphorylated DNA from KS332, which is described in Example 5. The resulting DNA constructs in which the expression of variants optimized by expression of Yarrowia DGAT1 or Yarrowia DGAT2 genes under the control of the betaconglycinin promoter are therefore indicated as KS392 and KS393. Its sequence is defined as SEQ ID NO. 101 and SEQ ID NO. 102. In addition, plasmid KS391 was constructed. For this purpose, KS349 DNA was digested with NotI and NcoI. The ends of the resulting DGAT1 fragment were completely filled in, ligated to linearized NotI, and filled into DA of KS332. The resulting plasmid construct is therefore called KS391. In this construct, the native YL DGAT1 sequences are under the control of the betaconglycinin promoter. The KS391 sequence is defined as SEQ ID NO: 103. Transgenic soybean somatic embryos were regenerated after particle bombardment with plasmid DNA from KS391, KS392, KS362 and KS393, as described above (Example 5). The oil content of somatic embryos was measured using NMR. Rapidly freeze-dried embryo tissue was sprayed into the genogrinder ampoule as previously described (Example 4). 20 to 200 mg of tissue powder were transferred to NMR tubes. The oil content of the somatic embryo tissue powder was calculated from the NMR signal as described in Example 4. TABLE 33 OIL CONTENT OF SOYBEAN SOMATIC EMBRYOS TRANSFORMED WITH PKS392 AND PKS391

[0436] Table 33 compares the oil content of 30 and 31 events generated with KS 392 and KS391, respectively.

[0437] The average oil content of all events generated with KS392 was 9.1%, while the oil content of all events generated with KS391 was 7.6%.

[0438] Furthermore, the highest oil content observed with KS392 was 15.7%, when compared to 12.8% for KS391. The depositors demonstrated that optimizing YL DGAT1 expression generates increased oil content in developing soybean embryos when compared to the native YL DGAT1 gene. TABLE 34 OIL CONTENT OF SOYBEAN SOMATIC EMBRYOS TRANSFORMED WITH PKS393 AND PKS362

[0439] Table 34 compares the oil content of 31 and 33 events generated with KS393 and KS362. The average oil content of all events generated with KS393 was 8.0%, while the oil content of all events generated with KS393 was 7.8%. Furthermore, the highest oil content observed with KS393 was 12.3% when compared to 11.6% for KS362.

[0440] The applicants demonstrated that optimizing YL DGAT2 expression generates a very small increase in oil content in developing seed embryos when compared to the native YL DGAT2 gene.

EXAMPLE 18 COEXPRESSION OF YL DGAT1 WITH A FAD2/TE2 DOWNREGULATION CONSTRUCTION IN SOYBEAN SOMATIC EMBRYOS:

[0441] The present example describes the construction of soybean expression vectors pKR1274, which comprise Yarrowia DGAT1 (YL DGAT1) and pKR1267 or pKR1269, which comprise a soybean fatty acid desaturase 2 (GM FAD2) down-regulation construct. /thioesterase 2 (GM TE2). Although the GM FAD2-TE2 down-regulation regions of pKR1267 and pKR1269 are identical in each construct and both are driven by the KTi3 promoter, pKR1267 contains only the KTi3 terminus and pKR1269 contains the KTi3 and soy albumin termini.

CONSTRUCTION OF PKR1274 COMPRISING YL DGAT1:

[0442] A starting plasmid pKR85 (SEQ ID NO: 27) that was previously described in Example 4 contains the hygromycin B phosphotransferase (HPT) gene (Gritz, L. and Davies, J., Gene 25: 179-188 (1983)), flanked by the T7 promoter and transcription terminator (T7prom/hpt/T7term cluster) and a bacterial origin of reproduction (ori) for selection and reproduction in bacteria (such as E. coli). In addition, pKR72 also contains the hygromycin B phosphotransferase gene, flanked by the 35S promoter (Odell et al., Nature 313: 810–812 (1985)) and NOS 3′ transcription terminus (Depicker et al., J. Mol. Appl. Genet. 1: 561–570 (1982)) (35S/hpt/NOS3′ assembly) for selection in plants such as soybean. Plasmid pKR85 (SEQ ID NO: 27) also contains a NotI restriction site flanked by the promoter for the α′ subunit of β-conglycinin (Beachy et al., EMBO J. 4: 3047-3053 (1985)) and the 3′ transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem. 261: 9228-9238 (1986)), termed the Bcon/NotI/Phas3′ cluster.

[0443] The Bcon/NotI/Phas3' assembly was removed from pKR85 (SEQ ID NO. 27) through digestion with HindIII and the resulting fragment was ligated back to produce pKR278 (SEQ ID NO. 104).

[0444] The BsiWI fragment of pKR1235 (SEQ ID NO. 74, Example 14), which contains the YL DGAT1, was cloned into the BsiWI site of pKR278 (SEQ ID NO. 104), which was previously described in BB1574; published in US20080095915 (the contents of which are incorporated by reference), to produce pKR1274 (SEQ ID NO: 105).

CONSTRUCTION OF PKR1267 COMPRISING GM DOWN ADJUSTMENT SET FAD2-TE2:

[0445] The 5' end of GM TE2 (SEQ ID NO. 106) was amplified from pTC4 (SEQ ID NO. 107), which was previously described in WO 1996006936A1 (the contents of which are incorporated by reference) with the primers of oligonucleotides GmTE2_5-1 (SEQ ID No. 108) and GmTE2_3-1 (SEQ ID No. 109), using Phusion® high-fidelity DNA polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. The 3' end of GM TE2 (SEQ ID NO: 106) was amplified from pTC4 (SEQ ID NO: 107) with the oligonucleotide primers GmTE2_5-2 (SEQ ID NO: 110) and GmTE2_3-2 (SEQ ID NO ° 111), using Phusion® high-fidelity DNA polymerase (Cat. no. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. The two resulting PCR products were combined and amplified with GmTE2_5-1 (SEQ ID NO. 108) and GmTE2_3-2 (SEQ ID NO. 111) using Phusion® high-fidelity DNA polymerase (Cat. NO. F553S, Finnzymes Oy, Finland), following the manufacturer's protocol.

[0446] The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR1258 (SEQ ID NO. 112).

[0447] The 5’ end of GM FAD2 (SEQ ID NO:113) was amplified from pBS43 (SEQ ID NO:114), which was previously described in WO 1197047731A2 (the contents of which are incorporated by reference), with oligonucleotide primers GmFAD2-1_5-1 (SEQ ID NO:115) and GmFAD2-1_3-1 (SEQ ID NO:116), using Phusion® high fidelity DNA polymerase (Cat. No. F553S, Finnzymes Oy, Finland), following the manufacturer's protocol.

[0448] The 3' end of GM FAD2-1 (SEQ ID NO. 113) was amplified from pBS43 (SEQ ID NO. 114) with the oligonucleotide primers GmFAD2-1_5-2 (SEQ ID NO. 117) and GmFAD2-1_3-2 (SEQ ID NO. 118), using Phusion® high-fidelity DNA polymerase (Cat. No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.

[0449] The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce PCRblunt-Fad-2-1 (SEQ ID No. 119).

[0450] The MluI fragment of pKR1258 (SEQ ID NO. 112), which contains GM TE2, was cloned into the MluI fragment of pCRblunt-Fad2-1 (SEQ ID NO. 119), which contains GM FAD2-1, to produce pKR1259 (SEQ ID NO. 120).

[0451] The EcoRI fragment of pKR1259 (SEQ ID NO. 120) comprised the 5' end of the GM FAD2/TE2 fragment, was cloned into the MfeI site of pKR1259 (SEQ ID NO. 122) to produce pKR1261 (SEQ ID NO. 121 ) and forms a GM FAD2-TE2-TE2loop-TE2-FAD2 hairpin structure flanked by NotI sites.

[0452] The NotI fragment of pKR1261 (SEQ ID NO. 121), which contains GM FAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR123R (SEQ ID NO. 122), which was previously described in WO 2004071467A2 (the contents of which are incorporated by reference), to produce pKR1266 (SEQ ID NO: 123).

[0453] The BsiWI/PstI fragment of pKR1266 (SEQ ID NO. 123), which contains the GM FAD2-TE2-TE2loop-TE2-FAD2, was cloned into the BsiWI/SbfI fragment of pKR278 (SEQ ID NO. 104), to produce pKR1267 (SEQ ID NO: 124).

CONSTRUCTION OF PKR1269 COMPRISING GM FAD2-TE2 DOWN TUNE UP ASSEMBLY:

[0454] The NotI fragment from pKR1261 (SEQ ID NO: 121), which contains GM FAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR457 (SEQ ID NO: 125), which was previously described in WO 2005/047479 (the contents of which are incorporated herein by reference), to produce pKR1264 (SEQ ID NO: 126).

[0455] The PstI fragment of pKR1264 (SEQ ID NO. 126), which contains the GM FAD2-TE2-TE2loop-TE2-FAD2 was cloned into the SbfI subfragment of pKR277 (SEQ ID NO. 127), which was described previously in the document WO 2004/071467 to produce pKR1269 (SEQ ID NO: 128).

[0456] Coexpression of GM downregulation constructs FAD2-TE2-TE2loop-TE2-FAD2 alone or with YL DGAT2:

[0457] Soybean embryogenic suspension culture (cv. Jack) was transformed with pKR 1267 (SEQ ID NO. 124) alone and which has experiment number MSE2213 or with the BsiWI fragment of pKR1269 (SEQ ID NO. 128 ) and pKR1274 (SEQ ID NO. 105) and which has experiment number MSE2210. Events were selected and somatic embryos matured in SHaM as described in Example 5.

[0458] Oil concentrations and fatty acid profiles were determined as described in Example 4 of MSE2213 and MSE2210 and the results of each experiment are displayed in Table 35 and Table 36, respectively. TABLE 35 OIL CONCENTRATIONS AND FATTY ACID PROFILES FOR MSE2213 EVENTS TABLE 36 OIL CONCENTRATIONS AND FATTY ACID PROFILES FOR MSE2210 EVENTS

[0459] Comparison of the results in Tables 35 and 36 demonstrates that the combination of YL DGAT1 expression with the downregulation of GM FAD2-1 and GM TE2 alters the fatty acid profile to an extent that exceeds the change observed when only FAD2-1 and TE2 genes are suppressed.

Claims (34)

1. METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A SOYBEAN SEED, characterized by the fact that it comprises: (a) transforming at least one soybean cell with a recombinant construct that contains at least one DGAT sequence as defined in SEQ ID NO: 9, 66, 82, 87 or 92; and (b) selecting the transformed soybean cell(s) from step (a) containing an increased total fatty acid content of at least 10% when compared to the total fatty acid content of a seed of non-transgenic zero segregating soy. 2. METHOD, according to claim 1, characterized by the fact that the percentage of fatty acids in said soybean seed, which is represented by linolenic acid, is decreased. 3. The method of any one of claims 1 to 2, further comprising the steps of: (a) regenerating a transgenic plant from said transformed soybean cell(s); and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct, and wherein said transgenic soybean seed has an increased total fatty acid content of at least 10% when compared to the total fatty acid content of a non-transgenic null segregant soybean seed. 4. METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF A CORN SEED, characterized by the fact that it comprises: (a) transforming at least one corn seed with a recombinant construct that has at least one DGAT sequence as defined in SEQ ID NO: 9, 66, 82, 87 or 92; and (b) selecting the processed corn seed(s) from step (a) that contains an increased total fatty acid content of at least 10% when compared to the total fatty acid content of a non-transgenic null segregating corn seed. 5. METHOD, according to claim 4, characterized by the fact that the percentage of fatty acids in said corn seed, which are represented by linolenic acid, is decreased. 6. METHOD, according to any one of claims 4 to 5, characterized by the fact that it additionally comprises the steps of: (a) regenerating a transgenic plant from said corn seed(s) transformed(s); and (b) obtaining a transgenic corn seed from said transgenic plant, wherein said transgenic corn seed comprises said recombinant construct, and wherein said transgenic corn seed has a total fatty acid content increased by at least 10% when compared to the total fatty acid content of a non-GMO null segregating corn seed. 7. METHOD, according to any one of claims 1 to 6, characterized by the fact that step (b) comprises the selection of the transformed soy cell(s) from step (a) containing( êm) a total fatty acid content increased by at least 10% and an oleic acid content increased by at least 25% when compared to the total fatty acid content and the oleic acid content of a non-segregating soybean seed. transgenic. 8. The method of claim 7, further comprising the steps of: (a) regenerating a transgenic plant from said transformed soybean cell(s); and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct, and wherein said transgenic soybean seed has an increased total fatty acid content of at least 10% and an increased oleic acid content of at least 25% when compared to the total fatty acid content and oleic acid content of a non-transgenic null segregant soybean seed. 9. METHOD, according to any one of claims 1 to 2, characterized by the fact that step (b) comprises the selection of the transformed soy cell(s) from step (a) containing( êm) a total fatty acid content increased by at least 10% and a linolenic acid content decreased by at least 25% when compared with the total fatty acid content and the linolenic acid content of a non-segregating soybean seed. transgenic. 10. METHOD, according to claim 9, characterized by the fact that it additionally comprises the steps of: (a) regenerating a transgenic plant from said transformed soy cell(s) ; and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct, and wherein said transgenic soybean seed has a total fatty acid content increased by at least 10% and a decreased linolenic acid content of at least 25% when compared to the total fatty acid content and linolenic acid content of a non-GMO null segregating soybean seed. 11. METHOD, according to any one of claims 1 to 2, characterized by the fact that step (b) comprises the selection of the transformed soy cell(s) from step (a) that contains ( êm) a total fatty acid content increased by at least 10% and a linolenic acid content decreased by at least 4% when compared with the total fatty acid content and the linolenic acid content of a non-segregating soybean seed. transgenic. 12. METHOD, according to claim 11, characterized by the fact that it further comprises the steps of: (a) regenerating a transgenic plant from said transformed soy cell(s) ; and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct, and wherein said transgenic soybean seed has a total fatty acid content increased by at least 10% and a decreased linolenic acid content of at least 4% when compared to the total fatty acid content and linolenic acid content of a non-GMO null segregating soybean seed. 13. METHOD, according to any one of claims 1 to 2, characterized by the fact that step (b) comprises the selection of the transformed soy cell(s) from step (a) that contains ( êm) a total fatty acid content increased by at least 10% and a palmitic acid content decreased by at least 8% when compared with the total fatty acid content and the palmitic acid content of a non-segregating soybean seed. transgenic. 14. METHOD, according to claim 13, characterized by the fact that it additionally comprises the steps of: (a) regenerating a transgenic plant from said transformed soy cell(s) ; and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct and wherein said transgenic soybean seed has a total fatty acid content increased by at least less 10% and a decreased palmitic acid content of at least 8% when compared to the total fatty acid content and the palmitic acid content of a non-transgenic zero segregating soybean seed. 15. METHOD, according to any one of claims 1 to 2, characterized by the fact that step (b) comprises the selection of the transformed soy cell(s) from step (a) that contains ( êm) a total fatty acid content increased by at least 10% and a stearic acid content increased by at least 14% when compared to the total fatty acid content and the stearic acid content of a non-segregating soybean seed. transgenic. 16. METHOD, according to claim 15, characterized by the fact that it additionally comprises the steps of: (a) regenerating a transgenic plant from said transformed soy cell(s) ; and (b) obtaining a transgenic soybean seed from said transgenic plant, wherein said transgenic soybean seed comprises said recombinant construct, and wherein said transgenic soybean seed has a total fatty acid content increased by at least 10% and an increased stearic acid content of at least 14% when compared to the total fatty acid content and stearic acid content of a non-GMO null segregating soybean seed. 17. RECOMBINANT POLYNUCLEOTIDE, characterized by the fact that it comprises: (a) a nucleotide sequence as defined in SEQ ID NO: 82, 87 or 92; or (b) a complement of the nucleotide sequence of (a), in which the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. 18. CONSTRUCTION OF RECOMBINANT DNA, characterized by the fact that it comprises the recombinant polynucleotide, as defined in claim 17, operably linked to at least one regulatory sequence. 19. METHOD FOR INCREASING THE TOTAL FATTY ACID CONTENT OF AN OILSEED PLANT SEED, characterized in that it comprises: (a) transforming at least one oilseed plant cell with the recombinant polynucleotide, as defined in the claim 17; and (b) selecting the transformed oilseed plant cell(s) from step (a) that contain an increased total fatty acid content as compared to the total fatty acid content total of one non-transgenic null segregating oilseed. 20. METHOD, according to claim 19, characterized by the fact that it additionally comprises the step of obtaining a transformed oilseed plant cell, in which said transformed oilseed plant cell comprises in its genome the recombinant polynucleotide, as defined in claim 17. 21. METHOD, according to claim 19, characterized by the fact that it additionally comprises the steps of: (a) regenerating a transgenic oilseed plant from said oilseed plant cell(s) processed oilseed(s); and (b) obtaining a transgenic oilseed plant seed from said transgenic oilseed plant, wherein said transgenic oilseed plant seed comprises said recombinant polynucleotide as defined in claim 17. 22. METHOD, according to any one of claims 19 to 21, characterized by the fact that said oilseed plant seed is selected from the group consisting of soybeans, corn, canola, sunflower, flax, cotton and saffron . 23. METHOD FOR PRODUCING A SOYBEAN OR CORN SEED, having an increase in the total fatty acid content, characterized by the fact that the method comprises: (a) supplying a transgenic soybean seed or a transgenic corn seed, wherein said transgenic soybean seed or said transgenic corn seed comprises a recombinant construct having at least one DGAT sequence as defined in SEQ ID NO: 9, 66, 82, 87 or 92; (b) cultivating said transgenic soybean seed or said transgenic corn seed to regenerate a transgenic soybean plant or a transgenic corn plant; (c) obtaining a soybean seed or a corn seed from said regenerated transgenic soybean plant or said regenerated transgenic corn plant, wherein said soybean seed or said corn seed has a content of total fatty acid increased by at least 10% when compared to the total fatty acid content of a non-GMO null-segregating soybean seed or a non-GMO null-segregating corn seed. 24. METHOD, according to claim 23, characterized by the fact that the percentage of fatty acids in said soybean seed or in said corn seed, which is represented by linolenic acid, is decreased. 25. METHOD, according to any one of claims 23 to 24, characterized by the fact that said soybean seed or said corn seed has an increased total fatty acid content of at least 10% and an oleic acid content increased by at least 25% when compared to the total fatty acid content and the oleic acid content of a soybean seed or a non-GMO zero segregating corn seed. 26. METHOD, according to any one of claims 23 to 24, characterized by the fact that said soybean seed or said corn seed has an increased total fatty acid content of at least 10% and a linolenic acid content decreased by at least 25% when compared to the total fatty acid content and linolenic acid content of a soybean seed or a non-GMO zero segregating corn seed. 27. The method of any one of claims 23 to 24, wherein said soybean seed or said corn seed has an increased total fatty acid content of at least 10% and a decreased linolenic acid content of at least 4% when compared to the total fatty acid content and linolenic acid content of a non-transgenic null segregant soybean seed or corn seed. 28. METHOD, according to any one of claims 23 to 24, characterized by the fact that said soybean seed or said corn seed has an increased total fatty acid content of at least 10% and a palmitic acid content decreased by at least 8% when compared to the total fatty acid content and the palmitic acid content of a soybean seed or a non-GMO zero segregating corn seed. 29. METHOD, according to any one of claims 23 to 24, characterized by the fact that said soybean seed or said corn seed has an increased total fatty acid content of at least 10% and a stearic acid content increased by at least 14% when compared to the total fatty acid content and stearic acid content of a soybean seed or a non-GMO null segregating corn seed. 30. METHOD, according to any one of claims 23 to 29, characterized by the fact that it additionally comprises the step of using said soybean seed or said corn seed to produce a product and/or a by-product. 31. METHOD, according to claim 30, characterized by the fact that said product is a food or feed. 32. METHOD, according to any one of claims 23 to 31, characterized by the fact that said seed is a soybean seed. 33. METHOD FOR PRODUCING A PRODUCT, from a soybean seed or a corn seed, as defined in any one of claims 23 to 29, characterized by the fact that the method comprises: (a) processing said soybean seed soybeans or said corn seed to produce a processed product; and (b) obtaining the processed product from step (a). 34. METHOD, according to claim 33, characterized by the fact that it additionally comprises the step of producing food or feed using said processed product.