# Patent

## Title
Diacylglycerol acyltransferase gene from plants

## Abstract

The present invention relates to the isolation, purification, characterization and use of the plant diacylglycerol acyltransferase (DGAT) gene and genetic products. The invention includes isolated and purified DGAT DNA and relates to methods of regulating seed oil content, the ratio of diacylglycerol/triacylglycerol proportions in the seed oil, fatty acid synthesis, seed oil acryl composition, seed size/weight and carbon flux into other seed components, using the gene, and to tissues and plants transformed with the gene. The invention also relates to transgenic plants, plant tissues and plant seeds having a genome containing an introduced DNA sequence of the invention, and a method of producing such plants and plant seeds.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 09/623,514 filed Mar. 29, 2001 now U.S. Pat. No. 7,015,373, issued on Mar. 21, 2006, which is a national phase entry under 35 U.S.C. § 371 of PCT International Patent Application No. PCT/CA99/01202, filed on Dec. 16, 1999, designating the United States of America, and published, in English, as PCT International Publication No. WO 00/36114 on Jun. 22, 2000, which claims priority to U.S. Provisional Patent Application 60/112,812, filed Dec. 17, 1998, each of the contents of the entirety of which is incorporated by this reference.


TECHNICAL FIELD
This invention relates to plant genes useful for the genetic manipulation of plant characteristics. More specifically, the invention relates to the identification, isolation and introduction of acyl-CoA:diacylglycerol acyltransferase (DGAT) genes useful, for example, for altering the seed oil content, the ratio of diacylglycerol/triacylglycerol proportions in the seed oil, fatty acid synthesis, seed oil acyl composition, seed size/weight and carbon flux into other seed components, in commercial or crop plants.
BACKGROUND OF THE INVENTION
Plant seed oils are major sources of essential polyunsaturated fatty acids for human diets and renewable feedstocks for chemical industries. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids that are channeled into the cytosolic acyl-CoA pool to sustain triacylglycerol accumulation. Triacylglycerol (TAG) biosynthesis is located in the endoplasmic reticulum with glycerol-3-phosphate and fatty acyl-CoAs as the primary substrates. There are three acyltransferases involved in the plant storage lipid bioassembly, namely the glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), the lyso-phosphatidic acid acyltransferase (LPAT, EC 2.3.1.51) and the diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). These three acyltransferases catalyze the stepwise acylation of the glycerol backbone with the final step being the acylation of sn-1,2-diacylglycerol (DAG) by DGAT into the formation of TAGs, a biochemical process generally known as the Kennedy pathway (Stymne and Stobart, 1987).
Among the three ER-based fatty acyl-CoA acyltransferases, only LPAT gene(s) have been cloned from plants (Knutzon et al., 1995, Lassner et al., 1995). Like several other enzymes involved in storage lipid biosynthesis, acyltransferases are intrinsic ER proteins and are extremely difficult to purify. The research on plant DGAT has been largely limited to studies of activity profiles by using the particulate fractions generated by differential centrifugation of seed or microspore-derived embryo homogenates (Weselake et al., 1993). Although partial purification of DGAT from cotyledons of germinating soybean seeds was reported (Kwanyuan and Wilson, 1986), detailed molecular characterization of this enzyme is lacking.
Accordingly, while the Kennedy pathway is known and shows the steps in the biosynthesis of TAGs in plants, there has not been any identification and use of a genetic element that can be used reliably in plants to modify the DGAT step in TAG synthesis and composition in a way that may be exploited commercially.
SUMMARY OF THE INVENTION
An object of the invention is to identify, isolate and clone a genetic element that may be used to modify the natural formation of triacylglycerols in plants in order to increase the yield of commercial plant oils, or to modify their composition to achieve specific commercial improvements of plants and plant products.
Another object of the invention is to identify, isolate and characterize diacylglycerol acyltransferase (DGAT) gene and cDNA sequences from Arabidopsis and to utilize these sequences in the genetic manipulation of plants.
Another object of the invention is to provide a vector containing the full-length DGAT cDNA sequence from Arabidopsis in a sense orientation under the control of a seed-specific promoter (e.g. napin; See Josefsson et al., 1987; Radke et al., 1988; Voelker et al., 1992), for re-introducing into Arabidopsis or for introducing into other plants.
Another object of the invention is to provide a vector containing a genomic fragment from Arabidopsis consisting of the full-length DGAT gene under the control of its own 5′ upstream regulatory sequences, for re-introducing into Arabidopsis or for introducing into other plants.
Another object of the invention is to provide a method to construct a vector containing the full-length DGAT sequence or a significant portion of the DGAT sequence from Arabidopsis, in an antisense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants.
Another object of the invention is to provide a method of modifying Arabidopsis and other plants to change their seed oil content.
Another object of the invention is to provide a method of modifying Arabidopsis and other plants to change the acyl composition of their seed oil.
Another object of the invention is to provide a method of modifying Arabidopsis and other plants to change their average seed weight or size.
According to one aspect of the present invention, there is provided a vector containing isolated and purified deoxyribonucleic acid (cDNA) of SEQ ID NO:1 (pDGATcDNA; ATCC No PTA-989), for introduction of the cDNA in a sense orientation into a plant cell.
As another aspect of the present invention, there is provided a vector containing isolated and purified genomic deoxyribonucleic acid (genomic DNA) of SEQ ID NO:3 (PDGAT gene; ATCC No PTA-988), for introduction of the gene in a sense orientation into a plant cell.
According to yet another object of the invention, there is provided a method for preparing a vector containing SEQ ID NO: 1 or a part thereof, for introduction of the gene or partial gene in an antisense orientation, into a plant cell.
According to yet another object of the invention, there is provided seed of Arabidopsis thaliana ecotype Columbia mutant AS11 (ATCC No. PTA-1013) and characterization of its lipid phenotype (Katavic et al., 1995; Zou et al. 1999). The AS II mutant seed line has an insertion mutation at the TAG1 locus on chromosome II, and produces plants exhibiting reduced DGAT activity (FIG. 4) and a reduced TAG/DAG ratio during seed development (Table 1), resulting in an altered seed fatty acyl composition (FIG. 2), reduced oil content (Table 1), and increased seed oil diacylglycerol content during development (FIG. 3) and at maturity (lower TAG/DAG ratio cf Table 1). The cDNA sequence of the AS11 DGAT is shown in SEQ ID NO:23, the genomic DNA sequence is shown in SEQ ID NO:24 and the translated protein sequence of the AS11 DGAT is shown in SEQ ID NO:25.
The invention also relates to transgenic plants and plant seeds having a genome containing an introduced DNA sequence of SEQ ID NO:1 or SEQ ID NO:3, and a method of producing such plants and plant seeds.
The invention also relates to SEQ ID NO:1 or SEQ ID NO:3, or a part of SEQ ID NO:1 or SEQ ID NO:3, or SEQ ID NO:1 containing an 81 bp insertion (SEQ ID NO: 23) or SEQ ID NO:3 containing a 147 bp insertion (SEQ ID NO:24) such that the deduced amino acid sequence of the encoded protein contains the repeated sequence SHAGLFNLCVVVLIAVNSRLIIENLMK (SEQ ID NO:25) where the spacing and identity of the underlined amino acids are identical or are replaced by conserved substitutions, characterized in that said sequence has been introduced in sense or antisense orientation, and a method of producing such plants and plant seeds.
As will be appreciated by persons skilled in the art, the invention also relates to substantially homologous DNA sequences from plants encoding proteins with deduced amino acid sequences of 25% or greater identity, and 40% or greater similarity, isolated and/or characterized and/or designed by known methods using the sequence information of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:1 containing an 81 bp insertion (SEQ ID NO:23) such that the deduced amino acid sequence of the encoded protein contains the repeated sequence SHAGLFNLCVVVLIAVNSRLIIENLMK (SEQ ID NO:25) where the spacing and identity of the underlined amino acids are identical or are replaced by conserved substitutions, and to parts of reduced length that are still able to function as inhibitors of gene expression by use in an anti-sense, co-suppression (Transwitch; Jorgensen and Napoli 1994) or other gene silencing technologies (i.e., RNAi). It will be appreciated by persons skilled in the art that small changes in the identities of nucleotides in a specific gene sequence may result in reduced or enhanced effectiveness of the genes and that, in some applications (i.e., anti-sense, co-suppression or RNAi), partial sequences often work as effectively as full length versions. The ways in which the gene sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. All such variations of the genes are therefore claimed as part of the present invention.
Other preferred degrees of identity to the indicated sequences are at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%; and other preferred degrees of similarity are at least 50%, 60%, 70%, 80%, 90% and 95%. The inventors have used a computer program known as MegAlign®, DNASTAR® of DNASTAR Inc., 1228 South Park Street, Madison, Wis. 53715, USA, for assessing homology. This program is based on the Clustal V algorithm (Higgins and Sharp (1998): A package for performing multiple sequence alignment on a microcomputer; GENE 73:237-244). For each gap introduced in the alignment, the program deducts a penalty from the score. A higher gap penalty suppresses gapping; a lower value promotes it. The program also assesses penalties based on the length of the gap. The more residues the gap spans, the greater the penalty. The program deducts these penalties from the overall score of the alignment.
Stated more generally, the present invention relates to the isolation, purification and characterization of a diacylglycerol acyltransferase (DGAT) gene from the Brassicaceae (specifically Arabidopsis thaliana) and demonstrates its utility in regulating fatty acid synthesis, seed oil content, diacylglycerol/triacylglycerol ratios and seed size/weight. Until now, no concrete data is available on the gene structure of plant DGATs, or their utility in altering oil content or composition through genetic manipulation.
When considering altered oil contents or compositions, results from averages of statistically-significant numbers of plants or seeds according to the invention are best compared with results from averages of statistically-significant numbers of untransformed (control) plants or seeds of the same genotype grown under identical conditions at the same time. This allows for the variability of individual plants of the same genotype, particularly when such plants are grown under different conditions. The actual number of plants or seeds used to form the required average may vary, but should be enough to provide a generally constant average whenever such number is selected. Generally, the number should be at least 10, and is more preferably at least 20, 30, 50 or 100.
The DGAT gene was cloned, characterized and authenticated from Arabidopsis by: (1) selection and characterization of plant ESTs sharing some homology to mammalian acyl-CoA: cholesterol acyltransferases; (2) the functional expression of a full-length cDNA in yeast; (3) the characterization and isolation of the DGAT (TAG 1) gene from Arabidopsis mutant AS11 containing an insertion mutation in the DGAT gene and a seed oil phenotype which consists of an altered DAG/TAG ratio, and an altered oil content and acyl composition; (4) complementation of the AS11 mutant by insertion of the DGAT cDNA sequence to restore a wild-type fatty acid composition; (5) the over-expression of the DGAT cDNA in wild-type A. thaliana transgenic plants which produce seeds with an increased oil content, increased average seed weight and altered seed oil acyl composition.
The A. thaliana DGAT structure is significantly homologous (over 40% identical over a region of more than 400 amino acids) to its mammalian counterparts, and is highly homologous to subsequently reported putative B. napus DGATs at both the nucleotide (92%) and the deduced amino acid (90%) levels (Nikyiforuk et al, 1999; GenBank/EMBL Accession No AF155224; AF164434).
The DGAT of the current invention is useful in manipulating DGAT activity, and triacylglycerol bioassembly in plants. For example, by transforming plants with a construct containing the DGAT gene in a sense orientation, under the control of a tissue-specific promoter (e.g. seed-specific promoter napin), the expression of DGAT and accumulation of seed oil can be enhanced or the acyl composition of the seed oil altered. Yet another example would be to express the DGAT cDNA under the control of a constitutive promoter (e.g. 35S (Datla et al., 1993)) to increase the TAG content of vegetative tissues (leaves, roots, stems). This may have particular advantages for altering the starch/oil ratio in root crops.
Alternatively, DGAT expression can be silenced to some degree by anti-sense or co-suppression (Transwitch) phenomena (De Lange et al., 1995; Mol et al., 1990; Jorgensen and Napoli, 1994; Kinney, 1995; Vaucheret et al, 1998; Taylor, 1998). For example, silencing DGAT in a seed specific manner may result in a reduction in TAG accumulation. This could have applications in reducing the oil content in seed barley to enhance stability during storage. As a second example, seed-specific silencing may lead to a relatively high accumulation of DAG or an increase in the DAG/TAG ratio in the developing or mature seed. As yet another example, the expression of the mutated DGAT gene which results in a 27 amino acid repeat insertion in the mutant DGAT protein (See FIG. 5a) can be used to alter the DAG/TAG ratio in developing and mature seed. Such manipulations can lead to edible seed oils produced naturally in the plant, containing enhanced relative levels of DAG/reduced levels of TAG (See FIG. 3; Table 1) to act as all-natural emulsifiers in the food and confections industries, or to enhance the nutritional/health profile of vegetable oils as functional foods (e.g. as cooking oils, stir fry oils, in salad dressings, margarines etc.) by inhibiting neutral fat deposition in humans. Processed oils produced from canola and soybean which contain increased proportions of diacylglycerol have been cited by the Kao Corporation of Japan (e.g. Econa Cooking Oil; Kao Corporation K1, 1-14-10 Nihonbashi-Kayabacho, Chuoku, Tokyo 103 Japan; e-mail: 210064@kastanet.kao.cojp) as a product making it difficult for blood neutral fat to increase after a meal, and for fat to cling to the body, thereby assisting individuals who are overweight or who suffer from high neutral fat levels. As a third example, silencing or reducing the activity of DGAT in a seed specific manner (as observed in mutant AS11; e.g. by over-expressing SEQ ID NO:23 or silencing expression of SEQ ID NO:1 or SEQ ID NO:3), and combining this trait with the capacity to produce polyhydroxyalkanoates (PHAs; e.g. polyhydroxybutyrate) in seeds (Poirier et al., 1992; 1995) will allow an increased flow of unesterified fatty acids towards β-oxidation (Poirier et al., 1999). By recycling or diverting the unesterified fatty acids into β oxidation, the resulting acetyl-CoA moieties will lead to a significant increase in polyhydroxyalkanoates (PHAs) or a change in PHA composition (Poirier et al., 1999). Transgenic plants producing PHAs in seeds have potential for utility as biodegradable thermoplastics. However, up to now, the levels of PHAs produced have been relatively small (Poirier et al, 1992; 1995). The utility of transgenically reducing the DGAT activity to significantly enhance PHA production (e.g. 10-fold increase) in PHA-producing seeds is now possible, due to the current DGAT invention.
Some of the manipulations and deliverables which are possible using the DGAT gene or a part thereof, include, but are not limited to, the following: seeds with increased or decreased oil content; seeds containing oils with an enhanced diacylglycerol content, seed oils with an altered acyl composition; plants producing larger or heavier seeds; plants exhibiting an enhanced or altered capacity to accumulate storage compounds in other storage organs (e.g. tubers, roots).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the Kennedy pathway for the bioassembly of triacylglycerols in plants, and shows the critical role played by DGAT as the final step of the Kennedy pathway.
FIG. 2 is a graph showing the comparison of the fatty acid composition of seed oil from wild-type (WT) and DGAT mutant AS 11 lines of Arabidopsis thaliana. Proportions of fatty acids are reported as Mol % of the total fatty acid composition of the seed oil from each line. Error bars are ±SE (n=10 plants of each line sampled, with 50 seeds per sample per analysis).
FIG. 3 is a graph showing a comparison of the DAG content in developing seed of wild-type (WT) and DGAT mutant AS11 lines of Arabidopsis thaliana. More specifically, it is a comparison of the fatty acid content of DAG pool in wild-type green developing seeds compared to that of the AS11 mutant.
FIG. 4 is a graph showing a comparison of the DGAT activity (pmol/min/mg protein) in developing seeds at the milky, early green, mid-green and brown-green stages of embryo development in wild-type (WT) and DGAT mutant AS 11 lines of Arabidopsis thaliana. Developing seeds at each stage were selected and DGAT enzyme analyses conducted as described previously by Katavic et al., 1995).
FIG. 5(a) shows the amino acid sequence alignment of the Arabidopsis DGAT (AtTAG1) (SEQ ID NO:2) with mammalian (mouse and putative human) DGATs. MDGAT, mouse DGAT (SEQ ID NO:4); GenBank/EMBL Accession No. AF078752 (Cases et al., 1998); HARGP1, human ARGP1 protein (SEQ ID NO:5); GenBank/EMBL Accession No. AF059202; Oelkers et al., 1998). Dots indicate gaps. Identical amino acid residues are highlighted in black. Conserved residues are shaded. The 27-amino acid repeat found in Arabidopsis thaliana mutant AS11 and generated by the insertion mutation (81 bp) found in SEQ ID NO:23 (SHAGLFNLCVVVLIAVNSRLIIENLMK) is overlined thus:




The putative diacylglycerol binding site is overlined thus:





The SnRK1 targeting site is overlined thus:





with an asterisk (*) over the serine (S) residue as the targeted phosphorylation site.

FIG. 5(b) shows the Kyte-Doolittle hydropathy plot of the DGAT protein.
FIG. 6(a) shows the results of a Northern analysis of TAG1 gene expression in Arabidopsis thaliana. Total RNA was extracted from roots (RT), leaves (LF), flowers (FL), young seedlings (YS), developing siliques (SL), and germinating seeds (GS).
FIG. 6(b) shows the results of Southern blot analysis of the TAG1 gene in Arabidopsis thaliana. Genomic DNA was digested with restriction enzymes BglII (Lane 1), EcoRI (Lane 2), and HindIII (Lane 3). The TAG1 DNA probe was 32P labeled by random priming.
FIG. 7(a) is a diagrammatic representation of the TAG1 gene structure. The boxes indicate the 16 exons (solid boxes for coding regions, open box for untranslated regions), and the lines represent the 15 introns. A, B and C denote the positions of the primers used for PCR amplifications of the segments from wild type (WT) and AS11. The specific primers A, B and C are described in Experimental Procedures: Primer Strategy (found later in this specification).
FIG. 7(b) shows gel separation of the PCR products amplified from wild type (WT) and AS11. Lane 1, PCR product with primers A and B using WT genomic DNA as template. Lane 2, PCR product with primers A and B using AS11 genomic DNA as template. Lane 3, PCR product with primers C and B using WT genomic DNA as template. Lane 4, PCR product with primers C and B using AS11 genomic DNA as template. Lane 5, RT-PCR with primers A and B using RNA prepared from WT seedling RNA. Lane 6, RT-PCR with primers A and B using RNA prepared from AS11 seedling RNA.
FIG. 8 is a graph showing microsomal DGAT activity in Yeast Strain YMN5 Transformed with empty plasmid (PYES Con) and with the A. thaliana DGAT cDNA (pYES:DGAT). This illustrates the expression of the TAG1 cDNA in yeast. Host cultures of strain YMN5 were transformed with pYES2 plasmid only (pYES2; without TAG1 insert) or with pYES2 containing the TAG1 cDNA insert (pYES2:TAG1). Following induction in the presence of galactose, transformants were lysed and assayed for DGAT activity as described in the Experimental Procedures. The results of two separate DGAT activity experiments are shown.
FIG. 9 is a map of plasmid pSE129A which may be used as a vector. The vector contains the following salient features for plant transformation in the current invention: the seed-specific napin promoter and NOS terminator between which is a multiple cloning site.
FIG. 10 is a graph showing the complementation of the AS11 DGAT mutation with the wild-type cDNA. Transformation of Arabidopsis thaliana mutant line AS11 with the DGAT cDNA [SEQ ID NO:1] under the control of a napin promoter, leads to a restoration of the wild-type (WT) fatty acid composition in the seed oil of the transformant lines 3-4, 9-1, 14-2 and 9-4. Fatty acid composition (wt %) was determined on the seed oil extracted from 100-seed samples from A. thaliana non-transformed controls (WT), mutant line AS 11, and T2 seeds of napin:DGAT transgenic lines.
FIG. 11 is a graph showing the seed oil content of non-transformed WT control, and pRD400 control (empty plasmid) and napin:DGAT T2 transgenic Arabidopsis thaliana seed lines. More particularly, the graph shows the transformation of wild type (WT) Arabidopsis thaliana with the DGAT cDNA (SEQ ID NO:1) under the control of a napin promoter, leads to a higher seed oil content in the DGAT transgenic lines. Oil content is expressed as μg total fatty acids (FAs) per 100 seeds from A. thaliana non-transformed controls (WT Con), and T2 seeds of pRD400 control (empty plasmid) transgenic, and napin:DGAT transgenic lines 1′, 2′, 9, 10 and 11. Standard error bars for the control lines are indicated; n=10.
FIG. 12 is a graph showing the average 100-seed weight of non-transformed WT control, and pRD400 control (empty plasmid) and napin:DGAT T2 transgenic Arabidopsis thaliana seed lines. More specifically, the graph shows the over-expression of the DGAT cDNA under the control of a napin promoter, in wild-type (WT) Arabidopsis thaliana leads to a higher average seed weight. The average weight of 100-seed samples from A. thaliana non-transformed controls (WT Con), and T2 seeds of pRD400 control (empty plasmid) transgenic, and napin:DGAT transgenic lines 1′, 2′, 9, 10 and 11 are reported as mg dry weight (DW).
FIG. 13 is a graph showing the positive correlation between oil content (expressed as μg Total fatty acids (FAs) per 100 seeds) and average seed weight (expressed as average mg DW per 100-seed samples) from A. thaliana non-transformed controls (WT Con ♦), and T2 seeds of pRD400 control (empty plasmid) transgenic (0 and napin:DGAT transgenic lines 1′, 2′, 9, 10 and 11(▪) are reported as mg dry weight (DW).



BEST MODE OF THE INVENTION
FIG. 1 is a diagram illustrating the Kennedy pathway for the biosynthesis of TAGs in plants. Of the various illustrated enzymes, DGAT is the only enzyme in the Kennedy pathway that is exclusively committed to TAG biosynthesis, and its key role is apparent from the scheme of FIG. 1. sn-1,2-DAG, generated as a result of either the catalytic action of PA phosphatase (EC 3.1.3.4) or CPTase (EC 2.7.8.2), can be used in the biosynthesis of TAG.
For this reason, the inventors of the present invention decided to investigate DGAT to see if the corresponding gene in plants could be sequenced and cloned and used to modify the seed oil content and fatty acid composition of plants in a way that could be commercially useful.
The acyl-CoA dependent acylation of sn-1,2-DAG is catalyzed by DGAT (Stymne and Stobart, 1987). In developing and germinating seeds of oilseed plants, TAG accumulation and DGAT activity have been shown to associate with the endoplasmic reticulum (ER; high speed microsomal fraction) (Stobart et al., 1986; Cao and Huang, 1986; Stymne and Stobart, 1987; Frentzen, 1993; Settlage et al, 1995; Lacey and Hills, 1996). The biochemical properties of microsomal DGAT have been examined in a number of plant systems (Frentzen, 1993) including developing seeds (Bernerth and Frentzen, 1990; Vogel and Browse, 1996; Cao and Huang, 1987) and embryo cultures (Taylor et al., 1991; Weselake et al., 1991; Taylor et al., 1992; Little et al., 1994) of B. napus L. In general, studies with developing seeds indicate that DGAT activity increased rapidly during the active phase of oil accumulation and then decreases markedly as seed lipid content reaches a plateau (Tzen et al., 1993; Weselake et al., 1993).
A number of studies with both mammalian (Mayorek et al., 1989; Tijburg et al., 1989) and plant (Ichihara et al., 1988; Perry and Harwood, 1993 a and 1993 b; Settlage et al. 1995) systems have suggested that DGAT may catalyze a rate-limiting reaction in TAG bioassembly. However, this hypothesis has not been rigorously tested, and has not been reduced to practice by transgenic expression of any DGAT gene in plant or animal systems, until now. Developing seeds of B. napus L., cv Shiralee, have been shown to produce significant levels of DAG during the active phase of oil accumulation suggesting that DGAT catalyzed reaction may regulate the flow of carbon into TAG (Perry and Harwood, 1993 a and 1993 b). In addition, an ethyl methanesulfonate-induced (EMS-induced) mutant of A. thaliana, designated AS11, has been shown to have a reduced DGAT activity that correlated with both an increased DAG pool and decreased accumulation of TAG (Katavic et al. 1995). Given its possible rate-limiting role in TAG bioassembly, the inventors of the present invention have identified DGAT as a potential target in the genetic modification of plant lipid biosynthesis.
Previously, the partial characterization of an EMS-induced Arabidopsis thaliana mutant, AS11, with altered fatty acid composition was reported (Katavic et al., 1995). In comparison to wild type plant seeds, AS11 seeds have reduced levels of the very long chain fatty acid eicosanoic acid (20:1) and reduced oleic acid (18:1) and accumulate α-linolenic acid (18:3) as the major fatty acid in triacylglycerols (FIG. 2). The AS11 mutant has a consistently lower ratio of TAG/DAG in developing seeds, and it accumulates an elevated amount of seed DAG (FIG. 3), the substrate of the diacylglycerol acyltransferase. Through a series of biochemical analyses, it was shown that AS11 had reduced diacylglycerol acyltransferase activities throughout seed development (FIG. 4). AS11 also had a reduced (by 25-30%) oil content phenotype, providing some evidence that DGAT may be controlling flux into TAG biosynthesis, as shown in Table 1 below. The AS11 did not have a wrinkled-seed phenotype as described in other low-seed-oil mutants (Focks and Benning, 1998).







TABLE 1







Comparison of AS11 [Katavic et al., (1995) and wild-type


A. thaliana seeds with respect to lipid profiles at


mid-development, and the relative TAG, DAG and sterol


ester contents in AS11 and WT seeds at maturity.












TAG/DAG

Relative TAG
Sterol Esters at



ratio
TAG/DAG
content at
maturity (% of


Seed
at mid-
ratio at
maturityb
Totol Lipid


Type
developmenta
maturitya
c(nmol/mg DW)
Extract)d














WT
17
90
1.00b
0.8





c(255)


AS11
5
20
 0.6b
1.15





c(174)





aEmbryos staged and lipids measured as described in Katavic et al., (1995);


bRelative TAG content of 200-seed samples of AS11 and WT were measured by MASS-1H-NMR according to the method of Rutar (1989). The integration response for resonances attributable to liquid-like oil were summed and the value for AS11 seed is reported relative to the response for the WT control seed sample (the latter set at a value of 1.00);


cTAG content (nmoles/mg DW) measured by transmethylation of a TLC-purified TAG fraction, followed by GC analysis of fatty acid methyl esters;


dA total lipid extract was prepared as described by Taylor et al., (1991; 1992), and sterol esters isolated and characterized as described in the Experimental Procedures.






Genetic analysis indicated that the fatty acid phenotype is caused by a semidominant mutation in a nuclear gene, designated TAG 1. The mutation was mapped to chromosome II, and was estimated to lie in the region approximately 17.5±3 cM from the sti locus and 8±2 cM from the cp2 locus.
Because a DGAT gene has not heretofore been cloned from any plant, until now, it has not been possible to address the possibility of genetic modifications to alter carbon flux, increase fatty acid synthesis, oil content, oil acyl composition, or seed size, by modulating plant DGAT activity.
However, there are many examples of successful modifications to plant metabolism that have been achieved by genetic engineering to transfer new genes or to alter the expression of existing genes, in plants. It is now routinely possible to introduce genes into many plant species of agronomic significance to improve crop performance (e.g. seed oil or tuber starch content/composition; meal improvement; herbicide, disease or insect resistance; heavy metal tolerance etc.) (MacKenzie and Jain, 1997; Budziszewski et al., 1996; Somerville, 1993; Kishore and Somerville, 1993).
For example, increases in the proportions of some strategic fatty acids and in the quantities of seed oil have been achieved by the introduction of various fatty acid biosynthesis and acyltransferase genes in oilseed crops. These include the following demonstrations: Expression of an anti-sense construct to the stearoyl-ACP Δ9 desaturase in Brassicaceae led to an increase in the stearic acid content (Knutzon et al., 1992). Expression of a medium chain fatty acyl-ACP thioesterase from California Bay, in Brassicaceae was demonstrated to increase the lauric acid (12:0) content (Voelker et al., 1992; 1996). Expression of a Jojoba β keto-acyl-CoA synthase in low erucic acid Brassicaceae led to an increase the level of erucic acid (22:1); the effect following expression in high erucic acid cultivars was negligible (Lassner et al., 1996). Increased proportions of oleic acid in Brassica napus and in soybean have been achieved by silencing the microsomal FAD2 (Δ12) desaturase (Hitz et al., 1995; Kinney, 1995; 1997). Transformation of Arabidopsis thaliana and rapeseed (B. napus) with a yeast sn-2 acyltransferase resulted in seed oils with increased proportions of 22:1 and other very long-chain fatty acids and significant increases in seed oil content (Zou et al., 1997).
Starch deposition has also been altered by genetic engineering. By expression of a mutant E. coli glgC16 gene encoding an ADP glucose pyrophosphorylase in potato tubers, an increase in starch accumulation was achieved (Stark et al., 1992).
The inventors therefore considered the DGAT gene to hold great promise for the desired modification of TAGs in plants.
The best modes for carrying out the invention will be apparent from the following description of the results of tests and experiments that have been carried out by the inventors.
The inventors chose to use the well-accepted model plant system Arabidopsis thaliana for the cloning of DGAT, as a host system for genetic engineering to alter DGAT expression, and to study the effects of altering DGAT expression on seed triacylglycerol bioassembly. This is because, over the past several years, Arabidopsis thaliana, a typical flowering plant, has gained increasing popularity as a model system for the study of plant biology. As a result of the ease with which this plant lends itself to work in both classical and molecular genetics, Arabidopsis has come to be widely used as a model organism in plant molecular genetics, development, physiology and biochemistry (Meyerowitz and Chang, 1985; Meyerowitz, 1987; Goodman et al., 1995). This model dicotyledonous plant is also closely related to Brassica crop species and it is increasingly apparent that information concerning the genetic control of basic biological processes in Arabidopsis will be transferable to other species (Lagercrantz et al., 1996).
Indeed, there are numerous examples wherein studies of the molecular biology and biochemistry of a particular metabolic pathway or developmental process and the possibility of genetically engineering a plant to bring about changes to said metabolic pathway or process, has first been tested in the model plant Arabidopsis, and then shown to yield similar phenotypes in other plants, particularly crop plants.
For example, the extra-plastidial membrane associated oleate (18:1) Δ12 (ω-6) desaturase gene, FAD2, was originally studied and eventually cloned from Arabidopsis thaliana, by identifying the lesion found in an A. thaliana mutant defective in desaturating oleate to produce linoleate (18:2) on the phosphatidylcholine backbone. This resulted in a high oleic acid phenotype in the A. thaliana seed oil (Okuley et al., 1994). Genetic engineering of both soybean (Glycine max.) and canola B. napus to silence the indigenous FAD2 gene(s) in a seed-specific manner by anti-sense or co-suppression approaches, resulted in similar high oleic acid seed oil phenotypes (Kinney, 1995; 1997).
Transgenic expression of a yeast sn-2 acyltransferase (SLC1-1) gene to achieve enhanced seed oil and very long-chain fatty acid content was first performed in Arabidopsis and later shown to yield similar phenotypes in transgenic rapeseed (B. napus) experiments (Zou et al., 1997). Arabidopsis thaliana has repeatedly shown itself to be a useful model system for metabolic engineering of metabolic pathways (e.g. lipid biosynthesis, photosynthesis) or processes (organogenesis, reproductive development etc.) common to all higher plants.
In the area of secondary metabolism/signal transduction, an anthocyanin pathway-specific transcriptional activator from the monocot maize designated as R (the myc transcription factor involved in activation of biosynthetic genes for anthocyanin production in the aleurone cells of maize kernels), was expressed in the dicot Arabidopsis, causing augmented anthocyanin pigmentation in the inflorescences. Subsequent expression in another dicot, tobacco (Nicotiana tabacum), resulted in similar floral pigmentation changes (Lloyd et al., 1992). These experiments demonstrate that whole pathways common to all flowering plants can be coordinately controlled through the introduction of transcriptional regulators, and that the mechanisms are common to diverse plant species.
In the context of the current invention, all plant seeds accumulate some triacylglycerol (oil) and this ubiquitous process is affected, at least in part, by the activity of a microsomal DGAT, as explained previously. Thus, many of the effects observed following genetic engineering to modulate DGAT expression in Arabidopsis can be expected and predicted to result in similar phenotypes when carried out in all other plants. For example, after the present invention was made, information has become available that supports the findings of the present inventors by showing that B. napus has a highly homologous DGAT gene (Nikiforuk et al., 1999), and thus B. napus is a clear target for similar genetic modifications as those shown for A. thaliana. 
There are a number of ways by which genes and gene constructs can be introduced into plants, and a combination of plant transformation and tissue culture techniques have been successfully integrated into effective strategies for creating transgenic crop plants. These methods, which can be used in the present invention, have been extensively reviewed elsewhere (Potrykus, 1991; Vasil, 1994; Walden and Wingender, 1995; Songstad et al., 1995), and are well known to persons skilled in the art. For example, one skilled in the art will certainly be aware that, in addition to Agrobacterium-mediated transformation of Arabidopsis by vacuum infiltration (Bechtold et al., 1993) or wound inoculation (Katavic et al., 1994), it is equally possible to transform other plant and crop species, using Agrobacterium Ti-plasmid-mediated transformation (e.g. hypocotyl (DeBlock et al., 1989) or cotyledonary petiole (Moloney et al, 1989) wound infection), particle bombardment/biolistic methods (Sanford et al., 1987; Nehra et al., 1994; Becker et al., 1994) or polyethylene glycol-assisted protoplast transformation (Rhodes et al., 1988; Shimamoto et al., 1989) methods.
As will also be apparent to persons skilled in the art, and as extensively reviewed elsewhere (Meyer, 1995; Datla et al., 1997), it is possible to utilize plant promoters to direct any intended up- or down-regulation of transgene expression using constitutive promoters (e.g. those based on CaMV35S), or by using promoters which can target gene expression to particular cells, tissues (e.g. napin promoter for expression of transgenes in developing seed cotyledons), organs (e.g. roots), to a particular developmental stage, or in response to a particular external stimulus (e.g. heat shock).
Particularly preferred plants for modification according to the present invention include Arabidopsis thaliana, borage (Borago spp.), Canola, castor (Ricinus communis), cocoa bean (Theobroma cacao), corn (Zea mays), cotton (Gossypium spp), Crambe spp., Cuphea spp., flax (Linum spp.), Lesquerella and Limnanthes spp., Linola, nasturtium (Tropaeolum spp.), Oenothera spp., olive (Olea spp.), palm (Elaeis spp.), peanut (Arachis spp.), rapeseed, safflower (Carthamus spp.), soybean (Glycine and Soja spp.), sunflower (Helianthus spp.), tobacco (Nicotiana spp.), Vernonia spp., wheat (Triticum spp.), barley (Hordeum spp.), rice (Oryza spp.), oat (Avena spp.) sorghum (Sorghum spp.), rye (Secale spp.) or other members of the Gramineae.
The present invention is particularly useful when used to modify the yield or composition of oilseed produced from oilseed crops. Oilseed crops are plant species that are capable of generating edible or industrially useful oils in commercially significant yields, and include many of the plant species listed above. Such oilseed crops are well known to persons skilled in the art.
EXAMPLES
Example 1
Isolation of the TAG 1 (DGAT) cDNA from Arabidopsis thaliana 
Since one of the most likely defects in AS11 mutant is at the DGAT itself (Table 1; FIG. 4), the inventors attempted cloning strategies based on sequence information of enzymes that share common substrates with DGAT. One of the candidate enzymes that would serve this purpose is the acyl-CoA: cholesterol acyltransferase (ACAT, EC 2.3.1.26) (Chang et al., 1997). Like DGAT, ACAT is an ER protein functioning as an O-acyltransferase by using acyl-CoA as the fatty acyl donor for the esterification of free cholesterol to generate sterol esters. Through a BLAST database search, the inventors identified an Arabidopsis thaliana expressed sequence tag (EST) (accession no. AA042298; SEQ ID NO:6) with a deduced amino acid sequence showing 41% identity to that of the yeast acyl-CoA: cholesterol acyltransferase (Yang et al., 1996, Yu et al., 1996), within the short sequence (104 amino acids) that was available for the EST.
The corresponding cDNA (E6B2T7) clone was obtained from the Arabidopsis Biological Resource Center, Columbus, Ohio. Upon complete sequencing, the 878 bp E6B2T7 clone was found to be a partial cDNA. However, the ORF prediction from this partial cDNA confirmed the initial EST search results in that the encoded product is structurally similar to ACAT, especially in the regions at the C-terminus. The inventors were confident that the cDNA contained the 3′ untranslated region through an ORF search, although the polyA tail was missing.
The inventors further used the partial cDNA sequence to search against Arabidopsis thaliana genomic sequence information. An Arabidopsis ‘IGF’ BAC clone ‘F27F23’ (accession no. AC003058) was identified to include a region that matched the cDNA, and therefore it was concluded that this was the region encompassing the corresponding gene. Moreover, this BAC clone ‘F27F23’, is contained in the YAC clone, CICO6EO8, which, according to the published map position (http://weeds.mgh.harvard.edu/goodman/c2_b.html), represents a region between centimorgan 35.9 and centimorgan 38.7 on chromosome II; this position is similar to the estimated location for TAG1, and the lesion identified by the mutation in AS11 (Katavic et al., 1995). In view of our previous results on the characterization of the AS 11 mutant, the map position of this gene strongly suggested that it may encode a DGAT.
To clone a full-length cDNA, a series of oligonucleotide primers were designed, based on the genomic sequences located at different positions 5′ upstream of the region covering the partial cDNA. We used these primers in combination with a primer located at the 3′ UTR of the partial cDNA (E6B2T7) to perform PCR reactions with cDNA phagemid prepared from an Arabidopsis thaliana (ecotype Columbia) silique-specific cDNA library (Giraudat et al., 1992) as a template. The longest cDNA amplified was 1904 bp, which we subsequently designated as TAG1, and deposited into the Genbank under accession AJ238008 (SEQ ID NO:1). We believe this cDNA represents a full-length clone because its size is in agreement with that of the transcript detected in the northern blot (see FIG. 6a). The longest open reading frame is flanked by a 134-nucleotide 5′ untranslated region and a 203-nucleotide 3′ untranslated region. There is an in-frame stop codon (TGA at position nt43) which is followed by an in-frame ATG at position nt-139. It is thus inferred that the ATG at position nt-139 is the initiation codon.
Example 2
The primary structure of TAG1 predicts a DGAT-related enzyme
The predicted open reading frame of the TAG1 cDNA encodes for a polypeptide of 520 amino acids with a calculated molecular weight of 58993 Daltons. With the BLAST search program (Altschul et al. 1990), it was found that the recently reported mouse diacylglycerol acyltransferase (accession no. AF078752) (Cases et al., 1998) is a protein which showed the highest sequence similarity to the deduced amino acid sequence of TAG1 (FIG. 5a). TAG1 was also similar to a human acyl CoA: cholesterol acyltransferase-related enzyme (accession no. AF059202). The human acyl CoA: cholesterol acyltransferase-related enzyme, also known as ARGP1, is most likely to be a DGAT with no significant ACAT activity, although the true nature of the enzyme awaits further confirmation (Oelkers et al., 1998). The similarity between TAG1 and the mammalian DGAT extends over a region of more than 400 amino acids with a sequence identity of about 41%. A putative diacylglycerol/phorbol ester-binding motif, H-K-W-X-X-R-H-X-Y-X-P (SEQ ID NO:26), a signature sequence observed to be unique to DGAT while absent in the ACATs (Oelkers et al., 1998), is located at amino acids 414-424 (SEQ ID NO:7); FIG. 5a). This diacylglycerol binding motif is also found in the subsequently published B. napus DGAT sequences (Nikyiforuk et al., 1999; GenBank/EMBL Accession Nos. AF155224, SEQ ID NO:8; AF164434, SEQ ID NO:9). Among other cloned acyltransferases (e.g. GPATs, LPATs, dihydroxyacetone phosphate acyltransferases) it has been reported that there is an invariant proline in a highly hydrophobic block IV that may participate in acyl-CoA binding (Lewin et al., 1999). In the TAG1 sequence, the hydrophobic block from residues 221-229 containing an invariant proline at residue 224, might constitute such a motif.
TAG1 showed some sequence similarity to other acyl CoA: cholesterol acyltransferases from a number of species (Chang et al., 1997). However, the similarity is largely confined to the C-terminus and is lower (around 30%) than is the similarity of TAG1 to the mammalian DGAT.
The TAG1 protein has multiple hydrophobic domains (FIG. 5b) and an analysis by the PC Gene program predicted that the protein has 5 possible transmembrane segments (amino acids 178-195, 233-253, 363-388, 433-476, 486-507). In the mammalian DGAT, a putative tyrosine phosphorylation motif was observed (Cases et al., 1998), but no apparent tyrosine phosphorylation site could be found in TAG 1. However, a visual examination revealed a consensus sequence (X-L200-X-K202-X-X-S205-X-X-X-V209, SEQ ID NO:10) identified as a targeting motif typical of members of the SNRK1 protein kinase family, with serine residue 205 being the residue for phosphorylation. The SNRK1 (SNF1-related protein kinase-1) proteins are a class of Ser/Thr protein kinases that have been increasingly implicated in the global regulation of carbon metabolism in plants (Halford and Hardie, 1998). This consensus SnRK1 targeting motif is also found in the subsequently published B. napus DGAT sequences (Nikyiforuk et al, 1999; GenBank/EMBL Accession Nos. AF155224; AF164434). Interestingly, similar SnRK1 targeting motifs could also be identified in the lyso-phosphatidic acid acyltransferases (LPATs) from coconut (Knutzon et al., 1995) and meadowfoam (Lassner et al., 1995), respectively.
Example 3
The TAG1 gene is Ubiquitously Expressed in Arabidopsis 
Northern blot analyses were performed to investigate the expression profile of the TAG1 gene. Total RNA was extracted from different tissues, including roots, leaves, flowers, developing siliques, young seedlings and germinating seeds. The highest steady-state level accumulation of TAG1 transcript was in RNA isolated from germinating seeds and young seedlings (FIG. 6a). TAG1 transcripts were also detected in root, leaf and flower tissues, albeit at lower levels. Surprisingly, the TAG1 gene is expressed in developing siliques at a level that is comparable to that of other vegetative tissues, but lower than that of germinating seeds and young seedlings. This expression profile in general is not inconsistent with the notion that DGAT is present in all plant tissues capable of TAG biosynthesis (Kwanyuan and Wilson, 1986). It has been shown in a number of plant species, including soybean and safflower, that germinating seeds actively synthesize TAGs (Ichihara and Noda, 1981; Kwanyuan and Wilson, 1986; Wilson and Kwanyuan, 1986). The relatively high level of expression in roots is also consistent with the fact that root plastids are capable of synthesizing large amounts of triacylglycerol (Sparace et al., 1992).
Southern blot hybridization (Southern, 1975) was performed with genomic DNA digested with several restriction enzymes including BglII, EcoRI and HindIII. The TAG1 gene has no internal BglII and HindIII site, while one internal EcoRI site exists. Our Southern analysis suggested that TAG1 most likely represents a single copy gene in the Arabidopsis genome (FIG. 6b).
Example 4
An Insertion Mutation is Found in the TAG1 Gene in mutantASli
Alignment of the genomic sequence (accession no. AC003058; SEQ ID NO:3) with that of the TAG1 cDNA (SEQ ID NO:1) revealed that the TAG1 gene contains 16 exons and 15 introns, spanning a region of about 3.4 kb (FIG. 7a). DNA containing the TAG1 allele from AS11 was PCR-amplified and completely sequenced. The AS11 TAG1 allele has a 147-bp insertion located at the central region of intron 2. The insertion is a duplication of a segment that is composed of 12 bp from the 3′ end of intron 1, the entire sequence of exon 2 (81 bp) and 54 bp from the 5′ end of intron 2 (FIG. 7a).
In order to rule out the possibility of PCR artifacts, two sets of primers were used to perform further PCR amplifications. Primers A and B (see Experimental Procedures, Primer Strategy) located in exons 1 and 3, respectively, amplified a DNA fragment that is about 150 bp longer from AS11 (FIG. 7b, lane 2) than that from the wild type (FIG. 7b, lane 1). The second pair of primers, C and B (Experimental Procedures), with one to be found in both exon 2 and the insertion segment, and the other located in exon 3, generated two amplified fragments from AS11 (FIG. 7b, lane 4), while only one from the wild type (FIG. 7b, lane 3). Hence these results confirmed that the insertion mutation the inventors identified through sequencing, reflected the true nature of the mutation in the TAG1 gene in the AS11 genome.
Example 5
The AS11 TAG1 Transcript has an 81-bp Insertion in Its Open Reading Frame
Northern blot analyses indicated that there was no difference in the expression profiles of the TAG1 gene, between the AS 11 mutant and wild type A. thaliana. In order to investigate the effect of the mutation at the transcript level, reverse-transcription PCR (RT-PCR) was performed to amplify the TAG1 transcript from RNA extracted from germinating seedlings of mutant AS11. Sequencing analysis revealed that there is an 81-bp insertion composed entirely of exon 2 in the transcript from AS11. The exon 2 in the repeat is properly spliced. The alteration of the transcript thus does not disturb the reading frame. However, this additional exon 2 sequence in the AS11 transcript would result in an altered DGAT protein with the 27 amino acid insertion 131SHAGLFNLCVVVLIAVNSRLIIENLMK157 (SEQ ID NO:11). The inventors' data shows that this insertion results in a 40-70% reduction in DGAT activity throughout seed development (Katavic et al., 1995). The 81 bp insert responsible for reduced DGAT activity in AS11 is visible in the comparison of RT-PCR products (FIG. 7b: Compare lane 5 (WT) and lane 6 (AS11).) The DNA aberration observed in the AS11 mutant was unexpected, since ethyl methanesulfonate (EMS) generally causes point mutations. Although we cannot rule out the possibility that this AS11 mutant was the result of a spontaneous mutation event, EMS-induced deletions and insertions have been reported in other systems (Mogami et al., 1986, Okagaki et al., 1991).
Example 6
The TAG1 Gene Insertion in Arabidopsis Mutant AS11 Affects Seed Triacylglycerol Accumulation, but not Sterol Ester Accumulation in Seeds
Because TAG1 also showed some sequence homology to acyl CoA: cholesterol acyltransferases (ACATs) from a number of species (Chang et al., 1997), the inventors compared both triacylglycerol and sterol ester accumulation in seeds of the wild-type A. thaliana and AS11 mutant. While the triacylglycerol content and TAG/DAG ratios were reduced in AS11 (i.e. increased proportion of seed oil DAGs,) in contrast, the proportions of sterol esters in WT and AS11 seeds were similar, at 0.8 and 1% of the total lipid extract, respectively (Table 1). If the TAG1 lesion affected ACAT-like activity, one might expect a reduction in seed sterol esters, but this was not observed. These results indicated that TAG1 is not involved in sterol-ester homeogenesis, and thus not an acyl CoA: sterol acyltransferase.
Example 7
TAG1 Expression in Yeast
The TAG1 cDNA overexpressed in yeast resulted in a 3.5 to 4-fold increase in microsomal DGAT activity compared to plasmid only (pYES2) control transformants (FIG. 8), confirming that the TAG1 gene product functions as a DGAT. When 14C18:1-CoA was added to the yeast lysates, sterol esters were also labeled in vitro (data not shown), but there was no significant difference in the 14C-labeled sterol esters produced by lysates from the pYES2 GAL-induced control and the pYES2:TAG1 Gal-induced transformant. This confirms that the TAG1 product does not encode an acyl-CoA: sterol acyltransferase (like ACAT).
Example 8
Complementation of the A. thaliana AS11 Mutant Line by Transformation with the DGAT cDNA
The cloned full-length DGAT cDNA was used as a template for PCR amplification with the primers DGATXbaI (CTAGTCTAGAATGGCGATTTTGGA; SEQ ID NO:12) and DGATXhoI (GCGCTCGAGTTTCATGACATCGA; SEQ ID NO:13) to provide new restriction sites on each end of the sequence as described in Experimental Procedures. A 1.6 kb fragment was excised by a XbaI/KpnI digestion and ligated into the corresponding sites of the pSE129 vector (provided by Dr. P. Covello, PBI/NRC). pSE129A is a vector derived from the plant transformation vector pRD400 (Datla et al. 1992). The vector pSE129A contains the seed-specific napin promoter and the nos terminator cloned into the EcoRI and HindIII sites of the pRD400 plasmid (FIG. 9). Hence in the DGAT-pSE129A construct, the Arabidopsis DGAT cDNA is under the control of the napin promoter. The construct integrity was confirmed by sequencing.
The pSE129A containing the napin:DGATcDNA was introduced into A. tumefaciens, used to transform A. thaliana mutant AS11, and progeny analyzed as described in Experimental Procedures. A number of T2 transgenic lines were isolated which complemented the fatty acid mutant phenotype found in AS11 (reduced 20:1 and elevated polyunsaturated C18s), restoring the wild-type seed fatty acid profile (FIG. 10). This finding confirms the nature of the lesion in AS11 and directly ties the AS11 lipid phenotype to this mutation.
Example 9
Over-Expression of the DGAT cDNA in Wild-Type A. thaliana 
The cloned fill-length DGAT cDNA was used as a template for PCR amplification with the primers DGATXbaI (CTAGTCTAGAATGGCGATTTTGGA; SEQ ID NO:12) and DGATXhoI (GCGCTCGAGTTTCATGACATCGA: SEQ ID NO:13) to provide new restriction sites on each end of the sequence as described in Experimental Procedures. A 1.6 kb fragment was excised by a XbaI/KpnI digestion and ligated into the corresponding sites of the pSE129 vector (provided by Dr. P. Covello, PBI/(NRC). pSE129A is a vector derived from the plant transformation vector pRD400 (Datla et al. 1992). The vector pSE129A contains the seed-specific napin promoter and the nos terminator cloned into the EcoRI and HindIII sites of the pRD400 plasmid (FIG. 9). Hence in the DGAT-pSE129A construct, the Arabidopsis DGAT cDNA is under the control of the napin promoter. The construct integrity was confirmed by sequencing.
The pSE129A containing the napin:DGATcDNA was introduced into A. tumefaciens, used to transform wild-type A. thaliana, and progeny analyzed as described in Experimental Procedures. A number of T2 transgenic lines were isolated which exhibited an increased oil content (FIG. 11) an increased average 100-seed weight (FIG. 12) and a strong linear correlation between the two traits (FIG. 13).
In terms of fatty acyl composition, wild type lines containing over-expressed DGAT cDNA showed a decrease in the total saturates, and increases in the monounsaturates and in the 18:1/[18:2+18:3] index, as shown in Table 2 below. Such changes are all towards a “healthier” oil profile, and can be applied directly to canola, other oilseeds in the Brassicaceae and other edible oil crops to produce similar oil composition improvements.







TABLE 2







Fatty acid composition of seed oil from A. thaliana non-transformed


wild-type controls (WT Con) and three T2 transgenic lines (2′,


9 and 11) of wild type transformed with the DGAT cDNA under the


control of a napin promoter (napin: DGAT).











Total





Saturatesa
Monounsaturatesb
18:1/[18:2 + 18:3]


Line
Wt %
Wt %
indexc





WTControl
15.1 ± 0.1
36.7 ± 0.2
29.9 ± 0.6


2′ napin:DGAT
13.4
38.6
34.1


9 napin:DGAT
13.1
39.3
35.6


11 napin:DGAT
13.1
38.3
33.0





aIncludes 16:0, 18:0, 20:0, 24:0


bincludes 18:1, 20:1, 22:1, 24:1


c([Wt % 18:1] + [Wt % 18:2 + Wt % 18:3]) × 100






EXPERIMENTAL PROCEDURES
Example 10
Plant Material
Arabidopsis thaliana ecotype Columbia and mutant AS11 were grown under conditions described previously (Katavic et al., 1995). The A. thaliana mutant line AS11 was generated and characterized relative to wild type (WT) A. thaliana ecotype Columbia, as described by Katavic et al., (1995); (ATCC NO: PTA-1013).
Example 11
DNA Manipulation
Standard methods and procedures were used for DNA preparation, plasmid propagation and isolation (Sambrook et al., 1989). Sequencing was conducted on an Applied Biosystems Model 373A DNA Sequencing System using the Taq DyeDeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.). The nucleotide and the deduced amino acid sequences were compared with sequences available in databanks using the BLAST program (Altschul et al., 1990).
Example 12
Southern and Northern Analysis
Total RNA was extracted from different tissues at various developmental stages, using the method of Lindstrom and Vodkin (1991). RNA samples were denatured with formaldehyde and separated on 1.2% formaldehyde-agarose gels. About 5 μg of total RNA was loaded, and the amount of RNA per lane was calibrated by the ethidium bromide-staining intensity of the rRNA bands. Genomic DNA was isolated, digested with restriction enzymes and a Southern blot analysis was performed according to Sambrook et al. (1989). The TAG1 DNA probe was 32P labeled by random-priming according to protocols of the manufacture (BRL).
Example 13
PCR Strategy
Primers used for the amplification of the TAG1 gene were as follows:

    DGAT1 (AGACACGAATCCCATTCCCACCGA; SEQ ID NO:14),
    DGAT2 (AGTGGTGACAACGCAGGGATGATG; SEQ ID NO:15),
    DGAT3 (ATGGTCGCTCCCACATTGTGT; SEQ ID NO:16),
    DGAT4 (CATACAATCCCCATGACATTTATCA; SEQ ID NO:17).

DGAT1 and DGAT2 amplify the 5′ half of the TAG1 gene and DGAT3 and DGAT4 amplify the 3′ end of the TAG1 gene. Genomic DNA from AS11 was used as template for PCR amplification of the mutant TAG1 allele using the thermal profile: 94° C. 3 min; 40 cycles of 94° C. 30 seconds, 62° C. 45 seconds, 72° C. 1 min; and 72° C. 15 min. To further confirm the mutation, primers A (CGACCGTCGGTTCCAGCTCATCGG; (SEQ ID NO:18)) and B (GCGGCCAATCTCGCAGCGATCTTG; (SEQ ID NO:19)), as well as primers C (TAAACAGTAGACTCATCATCG; (SEQ ID NO:20)) and B, were used in pairs, respectively, to amplify the internal fragment containing the mutation. The primers DGAT1 and DGAT4 were used for PCR amplification of the cDNA with an A. thaliana silique cDNA library as template. Primers A and B were also used in RT-PCR amplification of the cDNA fragment encompassing the insertion segment.



Example 14
Construction of TAG1 Multicopy Vector and Transformation and Characterization of DGAT Expression in Yeast
The TAG1 cDNA was cloned into pBluescript SK as described (Hadjeb and Berkowitz, 1996). The cDNA was cut out from the vector with KpnI/XbaI, and subsequently cloned into the respective sites of the yeast expression vector pYes2 (Invitrogen). The construct was confirmed by sequencing. Constructs with TAG1 transcription under the control of the GAL1 promoter released a fragment of approximately 1.9 kb. Because the TAG1 fragment has its own initiating ATG codon, the product expressed is not a fusion protein. As a host for yeast expression, an SLC deletion strain (YMN5 [slc1Δ2::LEU2 ura3]) (kindly provided by M. M. Nagiec and R. C. Dickson, University of Kentucky, Lexington, Ky.; Nagiec et al., 1993) was used; we reasoned that in this mutant, the endogenous DAG pool may be lower than in WT yeast, and that this would allow us to maximize the activity from over-expressed TAG1 in the presence of exogenously supplied 14C-DAG during in vitro DGAT assays of transformant lysates. Yeast transformation was performed according to Elble (1992). YMN5 transformants containing vector only (pYES2) were used as controls. Single colonies were cultured overnight in 20 mL of SD medium (Synthetic Dextrose medium with glucose and without uracil, as described by Ausubel et al., 1995, Vol. 2, p. 13.1.3) on a rotary shaker (270 rpm) at 28° C. Cells were pelleted from the overnight culture and resuspended in 50 mL of medium for induction of expression (SD medium containing galactose and without uracil). Cells were reincubated at 28° C., with shaking at 270 rpm, and harvested after 4-6 hr. GAL-induced yeast transformants were harvested by centrifugation at 5000 rpm for 5 min and resuspended in 100 mM Hepes-NaOH, pH 7.4, containing 1 mM EDTA and 1 mM DTT. Cell lysates were prepared using acid-washed glass beads as described by Ausubel et al. (1995). Protein in yeast lysates was measured using the Bradford (1976) assay, protein levels in each lysate were normalized and aliquots (250 μg protein) were assayed for DGAT activity as described below.
Example 15
Lipid Substrates and DGAT Analyses
14C-labeled diolein [1-14C oleic] (Sp. activity 55 mCi/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, Mo.). The 14C-labeled sn-1,2-diolein isomer was purified by TLC on borate-impregnated plates and emulsified in Hepes buffer the presence of 0.2% Tween-20 as described by Taylor et al., (1991). 20:1-CoA, CoASH, ATP, and all other biochemicals were purchased from Sigma.
DGAT assays were conducted at pH 7.4, with shaking at 100 rpm in a water bath at 30° C. for 30-60 min. Assay mixtures (0.5 mL final volume) contained lysate protein (250 μg), 90 mM Hepes-NaOH, 0.5 mM ATP, 0.5 mM CoASH, 1 mM MgCl2, 200 μM sn-1,2 diolein (sp. activity 2 nC/nmol) in 0.02% Tween 20, and 18 μM 20:1-CoA as the acyl donor. The 14C-labeled TAGs were isolated by TLC and quantified as described by Taylor et al (1991).
Example 16
Further Lipid and Sterol Ester Analyses in AS11 and WT
Total lipid extracts (TLEs), and lipid class analyses in WT and the AS11 mutant were performed as described by Taylor et al., (1991; 1992) and by Katavic et al., (1995). Relative seed oil content was also measured by magic angle sample spinning 1H-NMR, according to the method of Rutar (1989). Analyses were conducted with 200-seed samples of intact wild-type and AS11 seeds using a Bruker AM wide-bore spectrometer (Bruker Analytische Masstechnik GHBH, Silberstreifen D-76287, Rheinstetten4/Karlstuhe, Germany) operating at 360 MHz. To reduce anisotropic line broadening, the seed sample was rotated at 1 kHz in a zirconium rotor oriented 54.7° to the magnetic field. The integration response for resonances attributable to liquid-like oil were summed and the value for AS11 seed was recorded relative to the response for the WT control seed sample, the latter set at a value of 1.00.
Sterol esters were purified from the TLEs by thin layer chromatography (TLC) on Silica H plates developed in hexane:diethyl ether:formic acid (80:20:2, v/v/v). After elution from the silica H with chloroform:methanol (2:1, v/v), the sterol esters were quantified by saponification followed by methylation of the resulting fatty acids with 3N methanolic-HCl. The fatty acid methyl esters (FAMEs) were analyzed by GC as described previously (Taylor et al., 1991). The free sterols released by saponification were also analyzed by GC on a 30 m DB-5 column; GC temperature program: initial temp: 180° C., increasing at 10° C./min to 300° C. and held at this temperature for 15 min. The sterol ester content was reported as a % of the TLE; i.e. FAMEs released from sterol esters calculated as proportion of the FAMEs released by transmethylation of the total lipid extract (TLE).
Example 17
Construction of Plant Transformation Vector Containing the Wild-Type DGAT Gene for Over-Expression in WT A. thaliana and Complementation of the A. thaliana AS11 Mutant










Two primers:
(SEQ ID NO:21)









Gen 1 (GAGAGGATCCACGCTCACGACCCATTCTTCCCG;),



 



and



 







(SEQ ID NO:22)









Gen 2 (AAGAAGGATCCATCCCCAAAACGGGACCACCAA)






were synthesized according to sequences upstream and downstream of the TAG1 gene. These primers were used to PCR amplify a genomic fragment of 5.1 kb from wild-type A. thaliana. The PCR fragment was purified and digested with BamHI and inserted into the corresponding site in plasmid pRD400 (Datla et al. 1992) to generate the plant transformation vector DGATg-pRD400.

Example 18
Construction of DGAT cDNA Plant Transformation Vector for Seed-Specific Expression
The cloned full-length DGAT cDNA was used as a template for PCR amplification with the primers DGATXbaI (CTAGTCTAGAATGGCGATTTTGGA; SEQ ID NO:12) and DGATXhoI (GCGCTCGAGTTTCATGACATCGA; SEQ ID NO:13) to provide new restriction sites on each end of the sequence. The PCR profile was as follows: 94° C. 1 min; 30 cycles of 94° C. 30 seconds, 55° C. 30 seconds, 72° C. 1 min; and 72° C. 5 min. The PCR product was then ligated into the PCR-2.1 vector (InVitrogen). A 1.6 kb fragment was excised by a XbaI/KpnI digestion and ligated into the corresponding sites of the pSE129 vector (provided by Dr. P. Covello, PBI/NRC). pSE129A is a vector derived from the plant transformation vector pRD400 (Datla et al. 1992). The vector pSE129A contains the seed-specific napin promoter and the nos terminator cloned into the EcoRI and HindIII sites of the pRD400 plasmid (See FIG. 9). Hence in the DGAT-pSE129A construct, the Arabidopsis DGAT cDNA is under the control of the napin promoter. The construct integrity was confirmed by sequencing.
Example 19
Transformation of Agrobacterium with Plant DGAT Vector Construct
Electrocompetent Agrobacterium cells, GV3101 (pMP90) strain, were prepared as follows: An Agrobacterium culture was grown 24 to 48 hrs in 2YT, and when the absorbance at 600 nm reached 0.5 to 0.7, the cells were chilled on ice and pelleted by centrifugation (5,000×g, 10 min in a GSA rotor at 4° C.). The pellet was washed in 1, 0.5 and 0.02 volumes of cold 10% sterile glycerol and resuspended in 0.01 volume of cold 10% glycerol. The electrocompetent cells were then frozen in liquid N2 and stored at −70° C. The Agrobacterium cells were transformed by electroporation with 20-50 ng of transforming DNA (either DGATg-pRD400 or DGAT-pSE129A) according to the manufacturer's instructions, plated on a selective medium (LB with 50 μg/mL kanamycin) and incubated overnight at 28° C. Single transformed cells were grown overnight (28° C., 225 r.p.m.) in 5 mL LB with 50 μg/mL Kanamycin and 25 μg/mL Gentamycin. DNA extraction and purification were performed with a Qiaprep Spin Miniprep kit (Qiagen). The fidelity of the construct was re-checked by DNA sequencing before plant transformation.
Example 20
Transformation of Arabidopsis thaliana 
The transformation protocol was adapted from that described by Clough and Bent (1998). Seeds of Arabidopsis thaliana ecotype Columbia and mutant AS 11 (Katavic et al., 1995) were grown at 22° C. under fluorescent illumination (120 μE·m−2.S−1) in a 16 h light/8 hour dark regime. Typically, four to six plants were raised in a 10 cm2 pot in moistened Terra-lite Redi-earth (W. R. Grace & Co. Canada Ltd. Ajax, ON, Canada). To prevent the soil mix in the pot from falling into the inoculation media, soil was mounded as a platform with seeds sown on top, and the whole pot covered by a nylon window screen and secured by a rubber band. Plants were vacuum infiltrated in an Agrobacterium suspension when the first flowers started opening.
To grow Agrobacterium, a 5 mL suspension in LB medium containing 50 μg/mL kanamycin and 25 μg/mL gentamycin was cultured overnight at 28° C. The day before infiltration, this “seed culture” was divided into four flasks containing 250 mL of LB medium supplemented with 50 μg/mL kanamycin and 25 μg/mL gentamycin. These cultures were grown overnight at 28° C. The next morning after the absorbance at 600 nm was checked (approximately
=1.0), the cells were harvested by centrifugation (5,000×g, 10 min in a GSA rotor at room temperature) and resuspended in the infiltration medium (sucrose 5%; Silwet-77 0.005% in water) to obtain an optical density at 600 nm of 0.8.
The Agrobacterium suspension was poured into a beaker and the potted plants inverted into the beaker so that the flowers and bolts were submerged. The beaker was placed into a large Bell jar and a vacuum drawn using a vacuum pump, until bubbles formed on the stem surfaces and the solution started to bubble slightly, and then the vacuum was released rapidly. The necessary time and pressure will vary from one lab setup to the next, but good infiltration is visibly apparent as uniformly darkened, water-soaked tissue. Pots were removed from the beaker, laid on their side in a plastic tray and covered with a plastic dome, to maintain humidity. The following day, the plants were uncovered, set upright and allowed to grow for approximately four weeks in a growth chamber under continuous light conditions as described by Katavic et al., (1995). When the siliques were mature and dry, seeds were harvested and selected for positive transformants.
Example 21
Selection of Putative Transformants (Transgenic plants) and Analysis of Transgenic Plants
For each construct, seeds were harvested in bulk. Seeds were surface-sterilized by submerging them in a solution containing 20% bleach and 0.01% Triton X-100 for 20 min, followed by three rinses with sterile water. Sterilized seeds were plated by resuspending them in sterile 0.1% phytagar at room temperature (about 1 mL phytagar for every 500-1000 seeds), and applying a volume containing 2,000-4,000 seeds onto 150×15 mm kanamycin selection plate. Plates were incubated for 2 days in the cold without light, and grown for 7-10 days in a controlled environment (22° C. under fluorescent illumination (120 μE·m−2·s−1) in a 16 h light/8 hour dark regime). The selection media contained 1⁄2 MSG medium, 0.8% phytagar, 3% sucrose, 501g/mL kanamycin and 50 μg/mL Timentin. Petri dishes and lids were sealed with a MicroporeTM surgical tape (3M Canada, London, ON, Canada). After 7-10 days, drug-resistant plants that had green leaves and well established roots within the medium were identified as transformants and at the 3-5 leaf stage, selected transformants were transplanted into flats filled with heavily moistened soil mix. Transformants were grown to maturity and mature seeds (T2 generation as defined in Katavic et al., (1994)) were harvested from individual plants, and further analyzed.
Genomic DNA was isolated from individual T1 plants following the protocol of Dellaporta et al., (1983). A PCR amplification using the paired primers described previously for the DGAT cDNA or for the DGAT gene, was performed to confirm the presence of the cDNA or the gene, respectively, in the T1 transformants. Southern analyses (Southern, 1975) were performed to select the transformants containing a single copy of the inserted fragment. DNA samples were digested with restriction enzymes (BglII for the DGAT cDNA and EcoRI for the DGAT gene), resolved by electrophoresis on a 1% agarose gel, and Southern blotting performed using a nylon filter (Hybond-N+, Amersham) according to Sambrook et al. (1989). The DGAT cDNA fragment, labeled with α-[32P] dCTP (NEN/DuPont) using the Random Primer DNA labeling kit (Gibco BRL), was used as a probe. Hybridization was performed at 60° C. according to Church and Gilbert (1984). The filter was then exposed to Kodak X-OMAT-AR film.
DEPOSIT INFORMATION
The following biological material has been deposited at the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va., 20110-2209, U.S.A. All of these deposits were made on behalf of the Applicant/Assignee (National Research Council of Canada) under the terms of the Budapest Treaty on the dates indicated, and have been given the accession numbers shown below.















Deposited Material
Date of Deposit
Accession No.









Arabidopsis DGAT gene
Nov. 29, 1999
PTA-988



Arabidopsis DGAT cDNA
Nov. 29, 1999
PTA-989



Arabidopsis AS11 seeds
Dec. 3, 1999
PTA-1013










SEQUENCE LISTING FREE TEXT
The Sequence Listing provided below contains free text entries in respect of SEQ ID NOs:12 to 22. The free text used in the Sequence Listing is repeated as follows:
















SEQ ID NO: 12
Primer of DGATXbal



SEQ ID NO: 13
Primer of DGATXhol



SEQ ID NO: 14
Primer DGAT1



SEQ ID NO: 15
Primer DGAT2



SEQ ID NO: 16
Primer DGAT3



SEQ ID NO: 17
Primer DGAT 4



SEQ ID NO: 18
Primer A



SEQ ID NO: 19
Primer B



SEQ ID NO: 20
Primer C



SEQ ID NO: 21
Primer Gen 1



SEQ ID NO: 22
Primer Gen 2.










A summary of all of the listed sequences is provided below for ease of review:

SEQ ID NO:1—(pDGAT; vector containing isolated and purified deoxyribonucleic acid cDNA; ATCC No PTA-989), Genbanl/EMBL Accession No. AJ238008.
SEQ ID NO:2—The deduced amino acid sequence of the Arabidopsis DGAT (AtTAG1) protein.
SEQ ID NO:3—(pgenomic DGAT; vector containing isolated and purified genomic deoxyribonucleic acid (genomic DNA) ATCC No PTA-988).
SEQ ID NO:4—MDGAT, mouse DGAT [GenBank/EMBL Accession No. AF078752 (Cases et al., 1998)].
SEQ ID NO:5—HARGP1, human ARGP1 protein [GenBank/EMBL Accession No. AF059202; Oelkers et al., 1998].
SEQ NO:6—Arabidopsis thaliana expressed sequence tag (EST) [accession no. AA042298).
SEQ ID NO:7—A diacylglycerol/phorbol ester-binding motif found in SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:9 (414HKWMVRHIYFP424).
SEQ ID NO:8—B. napus DGAT amino acid sequence GenBank EMBL Accession No AF155224.
SEQ ID NO:9—B. napus DGAT amino acid sequence GenBank/EMBL Accession No. AF164434.
SEQ ID NO:10—Targeting motif typical of members of the SnRK1 protein kinase family found in SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:9 X-L200-X-K202-X-X-S205-X-X-X-V209 
SEQ ID NO:11—A 27 amino acid insertion repeat in SEQ ID NO:2 found in the Arabidopsis thaliana AS11 mutant.
131SHAGLFNLCVVVLIAVNSRLIIENLMK157 
SEQ ID NO:12—CTAGTCTAGAATGGCGATTTGGA (nucleotide sequence of Primer DGATXbaI).
SEQ ID NO:13—GCGCTCGAGTTTCATGACATCGA (nucleotide sequence of Primer DGATXhoI).
SEQ ID NO:14—AGACACGAATCCCATTCCCACCGA (nucleotide sequence of Primer DGAT1).
SEQ ID NO:15—AGTGGTGACAACGCAGGGATGATG (nucleotide sequence of Primer DGAT2).
SEQ ID NO:16—ATGGTCGCTCCCACATTGTGT (nucleotide sequence of Primer DGAT3).
SEQ ID NO:17—CATACAATCCCCATGACATTTATCA (nucleotide sequence of Primer DGAT4).
SEQ ID NO:18—CGACCGTCGGTRCCAGCTCATCGG (nucleotide sequence of Primer A).
SEQ ID NO:19—GCGGCCAATCTCGCAGCGATCTTG (nucleotide sequence of Primer B).
SEQ ID NO:20—TAAACAGTAGACTCATCATCG (nucleotide sequence of Primer C).
SEQ ID NO:21—GAGAGGATCCACGCTCACGACCCATTCTTCCCG (nucleotide sequence of primer Gen 1).
SEQ ID NO:22—AAGAAGGATCCATCCCCAAAACGGGACCACCAA (nucleotide sequence of primer Gen 2).
SEQ ID NO:23—AS11 mutant DGAT cDNA nucleotide sequence.
SEQ ID NO:24—AS11 mutant DGAT genomic DNA nucleotide sequence.
SEQ ID NO:25—the deduced amino acid sequence of SEQ ID NO:23.
SEQ ID NO:26—A putative diacylglycerol/phorbol ester-binding motif found in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9
REFERENCES


    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Stuhl, K., eds (1995). Current Protocols in Molecular Biology, Vols 1, 2, and 3. Wiley, New York.
    Bechtold, N., Ellis, J., and Pelletier, G. (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Sciences de la vie/Life sciences 316: 1194-1199.
    Becker, D., Brettschneider, R. and Lörz, H. (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J. 5: 299-307.
    Bernerth R, Frentzen M (1990) Utilization of erucoyl-CoA by acyltransferases from developing seeds of Brassica napus (L.) involved in triacylglycerol biosynthesis. Plant Sci 67: 21-28.
    Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
    Budziszewski, G. J., Croft, K. P. C. and Hildebrand, D. F. (1996) Uses of biotechnology in modifying plant lipids. Lipids 31: 557-569.
    Cao, Y-Z and Huang, AHC (1986) Diacylglycerol acyltransferase in maturing oil seeds of maize and other species. Plant Physiol. 82: 813-820.
    Cao Y-Z, Huang A H C (1987) Acyle coenzyme A preference of diacylglycerol acyltransferase from maturing seeds of Cuphea, maize, rapeseed and canola. Plant Physiol. 84: 762-765
    Cases, S., Smith, J. S., Zheng, Y-W., Myers, H. M., Lear, S. R., Sande, E., Novak, S., Collins, C.,
    Welch, C. B., Lusis, A. J., Erickson, S. K. and Farese, R. V., JR. (1998) Identification of a gene encoding a acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc. Nat'l. Acad. Sci. USA, 95, 13018-13023.
    Chang, T. Y., Chang, C. C. Y. and Cheng, D. (1997) Acyl-Coenzyme A: Cholesterol Acyltransferase. Annu. Rev. Biochem. 66, 613-38.
    Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA. 81, 1991-95.
    Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735-43
    Datla, R. S. S., Hammerlindl, J. K., Panchuk, B., Pelcher, L. E. and Keller, W. A. (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211: 383-384.
    Datla, R. S. S., Bekkaoui, F., Hammerlindl, J., Pilate, G., Dunstan, D. I. and Crosby, W. L. (1993) Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci. 94: 139-149.
    Datla, R., Anderson, J. W. and Selvaraj, G. (1997) Plant promoters for transgene expression. Biotechnology Annual Review 3: 269-296.
    DeBlock, M., DeBrouwer, D. and Tenning P. (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91: 694-701.
    De Lange, P., Van Blokland, R., Kooter, J. M. and Mol, J. M. N. (1995) Suppression of flavenoid flower pigmentation genes in Petunia hybrida by the introduction of antisense and sense genes. In: Gene Silencing in Higher Plants and Related Phenomena in Other Eukaryotes. P. Meyer (Ed.), Springer-Verlag, Berlin, pp. 55-75.
    Dellaporta, S. L., Wood, J. and Hicks, J. B. (1983) A plant DNA minipreparation:Version II. Plant Mol. Biol. Rep. 1, 19-21
    Elble, R. (1992). A simple and efficient procedure for transformation of yeasts. Biotechniques 13, 18-20.
    Focks, N. and Benning, C. (1998) wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism. Plant Physiol. 118, 91-101.
    Frentzen M (1993) Acyltransferases and triacylglycerols. In: Moore, Jr. TS, editor, Lipid Metabolism in Plants, pp. 195-230. CRC Press, Ann Arbor,.
    Giraudat, J., Hauge, B. M., Valon, C., Smalle, J., Parcy, F., Goodman, H. M. (1992) Isolation of the Arabidopsis AB13 gene by positional cloning. Plant Cell 4, 1251-1261.
    Goodman, H. M., Ecker, J. R. and Dean, C. (1995) The genome of Arabidopsis thaliana. Proc. Nat'l. Acad. Sci. USA 92: 10831-10835.
    Hadjeb N and Berkowitz G A (1996) Preparation of T-over-hang vectors with high PCR product cloning efficiency. Biotechniques 20: 21-22.
    Halford, N. G. and Hardie, D. G. (1998) SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37, 735-748.
    Hitz, W. D., Mauvis, C. J., Ripp, K. G., Reiter, R. J., DeBonte, L. and Chen, Z. (1995) The use of cloned rapeseed genes for cytoplastic fatty acid desaturases and the plastid acyl-ACP thioesterases to alter relative levels of polyunsaturated and saturated fatty acids in rapeseed oil. Proc. 9th International Cambridge Rapeseed Congress UK, pp. 470-472.
    Ichihara, K. and Noda, M. (1981) Lipid synthesis in germinating safflower seeds and protoplasts. Phytochemistry 20, 1245-1249.
    Ichihara, K., Takahashi, T. and Fujii, S. (1988) Diacylglycerol acyltransferase in maturing safflower seeds: its influences on the fatty acid composition of the triacyiglycerol and on the rate of triacylglycerol synthesis. Biochim. Biophys. Acta 958, 125-129.
    Jorgensen, R. A. and Napoli, C. A. (1994) Genetic engineering of novel plant phenotypes. U.S. Pat. No. 5,283,184.
    Josefsson, L-G, Lenman M, Ericson M L and Rask L (1987) Structure of a gene encoding the 1.7S storage protein, napin, from Brassica napus. J biol Chem 262: 12196-12201.
    Katavic, V., Haughn, G. W., Reed, D., Martin, M. and Kunst, L. (1994) In planta transformation of Arabidopsis thaliana. Mol. Gen. Genet. 245: 363-370.
    Katavic, V., Reed, D. W., Taylor, D. C., Giblin, E. M., Barton, D. L., Zou, J-T., MacKenzie, S. L., Covello, P. S. and Kunst, L. (1995) Alteration of fatty acid composition by an EMS-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity. Plant Physiol. 108, 399-409.
    Kinney, A. J. (1995) Improving soybean seed quality. In: Induced Mutations and Molecular Techniques for Crop Improvement. International Atomic Energy Agency, Vienna, Austria., pp. 101-113.
    Kinney, A. J. (1997) Genetic engineering of oilseeds for desired traits. In: Genetic Engineering, Vol. 19 (J. K. Setlow, ed.), Plenum Press, NY., pp. 149-166.
    Kishore G. M. and Somerville, C. R. (1993) Genetic engineering of commercially useful biosynthetic pathways in transgenic plants. Current Opinion in Biotechnology. 4: 152-158.
    Knutzon, D. S., Thompson, G. A., Radke, S. E., Johnson, W. B., Knauf, V. C., and Kridl, J. C. (1992) Modification of Brassica seed oil by anti-sense expression of a stearoylacyl carrier protein desaturase gene. Proc. Nat'l Acad. Sci. USA, 89: 2624-2628.
    Knutzon, D. S., Lardizabal, K. D., Nelson, J. S., Bleibaum, J. L., Davis, H. M and Metz, J. (1995) Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium chain length substrates. Plant Physiol. 109, 999-1006.
    Kwanyuen, P. and Wilson, R. F. (1986) Isolation and purification of diacylglycerol acyltransferase from germinating soybean cotyledons. Biochim. Biophys. Acta 877, 238-245.
    Lacey D J, Hills M J (1996) Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L. Planta 199: 545-551.
    Lagercrantz, U., Putterill, J., Coupland, G. and Lydiate, D. (1996) Comparative mapping in Arabidopsis and Brassica, fine scale genome collinearity and congruence of genes controlling flowering. Plant J. 9:13-20.
    Lassner, M. W., Levering, C. K., Davis, H. M. and Knutzon, D. S. (1995) Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol. 109, 1389-1394.
    Lassner, M. W., Lardizabal, K, and Metz, J. G. (1996) A jojoba β-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants. The Plant Cell, 8: 281-292.
    Lewin T M, Wang P and Coleman R A (1999) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38: 5764-5771.
    Lindstrom J. T. and Vodkin L. O. (1991) A soybean cell wall protein is affected by seed color genotype. Plant Cell 3:561-571
    Little D, Weselake R J, Pomeroy M K, Furukawa-Stoffer T and Bagu J (1994) Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape. Biochem J 304: 951-958.
    Lloyd, A. M., Walbot, V. and Davis, R. W. (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators Rand C1. Science 258: 1773-1775.
    MacKenzie, S. L. and Jain, R. K. (1997) Improvement of oils crops via biotechnology. Recent Res. Dev. In Oil Chem. 1:149-158.
    Mayorek, N, Grinstein I, and Bar-Tana J (1989) Triacylglycerol synthesis in cultured rat hepatocytes. The rate-limiting role of diacylglycerol acyltransferase. Eur J Biochem 182: 395-400.
    Meyer, P. (1995) Understanding and controlling transgene expression. Trends in Biotechnology, 13: 332-337.
    Meyerowitz, E. M. (1987) Arabidopsis thaliana. Ann. Rev. Genet. 21: 93-111.
    Meyerowitz, E. M. and Chang, C. (1985) Molecular biology of plant growth and development: Arabidopsis thaliana as an experimental system. In: Developmental Biology, Vol. 5, Plenum Press, NY., pp. 353-366.
    Mogami, K., O'Donnell, P. T., Bernstein, S. I., Wright, T. R. F and Emerson, C. P., JR. (1986) Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function. Proc. Nat'l. Acad. Sci. USA. 83, 1393-1397.
    Mol, J. M. N., Van der Krol, A. R., Van Tunen, A. J., Van Blokland, R., De Lange, P. and Stuitje, A. R. (1990) Regulation of plant gene expression by antisense RNA. FEBS Lett. 268: 427-430.
    Moloney, M. M., Walker, J. M. and Sharma, K. K. (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep. 8: 238-242.
    Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993). A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J. Biol. Chem. 268, 22156-22163.
    Nehra, N. S., Chibbar, R. N., Leung, N., Caswell, K., Mallard, C., Steinhauer, L. Baga, M. and Kartha K. K. (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant J. 5: 285-297.
    Nykiforuk C, Laroche A and Weselake RJ (1999) Isolation and sequence analysis of a novel cDNA encoding a putative diacylglycerol acyltransferase from a microspore-derived cell suspension culture of Brassica napus L. cv Jet Neuf (Accession No. AF155224).), Plant Physiology 120: PGR99-123.
    Oelkers, P., Behar, A., Cromley, D., Billheimer, J. T. and Sturley, S. T. (1998) Characterization of two human genes encoding acyl Coenzyme A: cholesterol acyltransferase-related enzymes. J. Biol. Chem. 273, 26765-26771.
    Okagaki, R. J., Neuffer, M. G. and Wessler, S. R. (1991) A deletion common to two independently derived Waxy mutations in maize. Genetics 128, 425-431.
    Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E. and Browse, J. (1994) Arabidopsis fad2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. The Plant Cell 6:147-158.
    Perry, H. Y. and Harwood, J. L. (1993a) Changes in the lipid content of developing seeds of Brassica napus. Phytochemistry 32: 1411-1415.
    Perry, H. Y. and Harwood, J. L. (1993b) Use of [2-3H] glycerol precursor in radiolabeling studies of acyl lipids in developing seeds of Brassica napus. Phytochemistry 34: 69-73.
    Poirier, Y., Dennis D E, Klomparens K and Somerville C (1992) Polyhydroxybutyrate, a biodegradable thermoplastic produced in transgenic plants. Science 256: 520-523.
    Poirier, Y., Nawrath C and Somerville C (1995) Production of polyhydroxyalkanoates, a family of biodegradeable plastics and elastomers in bacteria and plants butyrate, a biodegradable thermoplastic produced in transgenic plants. Bio-Technology, 13: 142-150.
    Poirier, Y., Ventre, G and Caldelari, D(1999) Increased flow of fatty acids toward β oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids. Plant Physiology 121: 1359-1366.
    Potrykus, I. (1991) Gene transfer to plants: Assessment of published approaches and results. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225.
    Radke S E, Andrews B M, Moloney M M, Crouch M L, Kridl J C, and KnaufVC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor. Appl. Genet. 75: 685-694.
    Rhodes, C. A., Pierce, D. A., Mettler, I. J., Mascarenhas, D. and Detmer, J. J. (1988) Genetically transformed maize plants from protoplasts. Science 240: 204-207.
    Rutar, V. (1989) Magic angle sample spinning NMR spectroscopy of liquids as a nondestructive method for studies of plant seeds. J. Agric. Food. Chem. 37, 67-70.
    Sambrook J., Fritsch E. F. and Maniatis T. (1989) In Molecular Cloning, A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press.
    Sanford, J. C., Klein, T. M., Wolf, E. D. and Allen, N. (1987) Delivery of substances into cells and tissues using a particle bombardment process. J. Part. Sci. Technol. 5: 27-37.
    Settlage, S H, Wilson R F and Kwanyuen, P. (1995) Localization of diacylglycerol acyltransferase to oil body associated endoplasmic reticulum. Plant Physiol. Biochem. 33: 399-407.
    Shimamoto, K., Terada, R., Izawa, T. and Fujimoto, H. (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274-276. Somerville, C. R. (1993) Future prospects for genetic modification of the composition of edible oils from higher plants. Am. J. Clin. Nutr. 58 (2 Suppl.): 270S-275S.
    Songstad, D. D., Somers, D. A. and Griesbach, R. J. (1995) Advances in alternative DNA delivery techniques. Plant Cell, Tissue and Organ Culture 40:1-15.
    Southern E. M. (1975) Detection of specific sequences among DNA fragments separated by gele electrophoresis. J. Mol. Biol. 98: 503-517.
    Sparace S. A., Kleppinger-Sparace K. F., Stahl R. J., Xue L. and Qi, Q. (1992) Lipid biosynthesis in pea root plastids and some effects of glycolytic intermediates. In SL MacKenzie and DC Taylor, eds. Seed Oils for the Future, AOCS Press, Champaign, Ill. pp 52-60.
    Stark, D. M., Timmerman, K. P., Barry, G. F., Preiss, J. and Kishore, G. M. (1992) Regulation of the amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science 258: 287-292.
    Stobart A K, Stymne S, H{umlaut over (g)}lund S. (1986) Safflower microsomes catalyse oil accumulation in vitro: A model system. Planta 169: 33-37.
    Stymne, S. and Stobart, A. K. (1987) Triacylglycerol Biosynthesis. In Stumpf, P. K. ed, The Biochemistry of Plants, Academic Press, New York. 9, 175-214.
    Taylor. CB. (1998) Comprehending cosuppression. The Plant Cell 9:1245-1249.
    Taylor, D. C., Weber, N., Barton, D. L., Underhill, E. W., Hogge, L. R., Weselake, R. J. and Pomeroy, M. K. (1991) Triacylglycerol bioassembly in microspore-derived embryos of Brassica napus L. cv. Reston. Plant Physiol. 97, 65-79.
    Taylor, D. C., Barton, D. L., Rioux, K. P., MacKenzie, S. L., Reed, D. W., Underhill, E. W., Pomeroy, M. K. and Weber, N. (1992) Biosynthesis of acyl lipids containing very-long chain fatty acids in microspore-derived embryos of Brassica napus L. cv. Reston. Plant Physiol. 99, 1609-1618.
    Tijburg, L B, Geelen, M J and van Golde L M (1989) Regulation of the biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanloamine in the liver. Biochim BiophysActa 1004:1-19.
    Tzen T C, Cao Y, Laurent P, Ratnayake C and Huang H C (1993) Lipids, proteins and structures of seed oil bodies from diverse species. Plant Physiol. 101: 267-276.
    Vasil, I. K. (1994) Molecular improvement of cereals. Plant Mol. Biol. 25: 925-937.
    Vaucheret, H., Beclin C, Elmayan T, Feuerbach, F., Godon C, Morel J-B, Mourrain, P., Palauqui, J-C and Vernhettes S (1998) Transgene-induced gene silencing in plants. The Plant Journal 16: 651-659.
    Voelker, T. A., Worrell, A. C., Anderson, L., Bleibaum, J., Fan, C., Hawkins, D. J., Radke, S. E., and Davies, H. M. (1992) Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Science 257: 72-74.
    Voelker, T. A., Hayes, T. R., Cramner, A. M., Turner, J. C., and Davies, H. M. (1996) Genetic engineering of a quantitative trait: metabolic and genetic parameters influencing the accumulation of laurate in rapeseed. The Plant Journal 9: 229-241.
    Vogel, G and Browse, J (1996) Cholinephosphotransferase and diacylglycerol acyltransferase: Substrate specificities at a key branch point in seed lipid metabolism. Plant Physiol. 110: 923-931.
    Walden, R. and Wingender, R. (1995) Gene-transfer and plant regeneration techniques. Trends in Biotechnology 13: 324-331.
    Weselake, R. J., Taylor, D C, Pomeroy, M. K., Lawson S L, and Underhill E W (1991) properties of diacylglycerol acyltransferase from microspore-derived embryos of Brassica napus L. Photochemistry: 30: 3533-3538.
    Weselake, R. J., Pomeroy, M. K., Furukawa, T. L., Golden, J. L., Little, D. B. and Laroche, A. (1993) Developmental profile of diacylglycerol acyltransferase in maturing seeds of oilseed rape and safflower and micro-spore-derived cultures of oilseed rape. Plant Physiol. 102, 565-571.
    Wilson, R. F. and Kwanyuan P. (1986) Triacylglycerol synthesis and metabolism in germinating soybean cotyledons. Biochim. Biophys. Acta 877, 231-237.
    Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T. M., Rothstein, R. and Sturley, S. L. (1997) Sterol esterification in yeast: a two-gene process. Science 272, 1353-1356.
    Yu, C., Kennedy, N. J., Chang, C. C. Y. and Rothblatt, J. A. (1996) Molecular cloning and characterization of two isoforms of Saccharomyces cerevisiae Acyl-CoA: Sterol Acyltransferase. J. Biol. Chem. 271, 24157-24163.
    Zou, J-T., Katavic, V., Giblin, E. M., Barton, D. L., MacKenzie, S. L., Keller, W. A., Hu, X. and Taylor, D. C. (1997) Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. The Plant Cell 9: 909-923.


The teachings of the above references are hereby incorporated by reference.

## Claims

1. A process for producing a plant, plant seed or progeny thereof, the process comprising:
transforming a plant cell with a nucleic acid sequence encoding a diacylglycerol acyltransferase protein, wherein the nucleic acid sequence comprises the sequence according to SEQ ID NO: 1; growing a plant from the plant cell until the plant produces seed; and harvesting the seed from the plant, wherein the seed comprise the nucleic acid sequence.

2. The process of claim 1, wherein the nucleic acid sequence encoding the diacylglycerol acyltransferase protein is integrated into the genome of the plant cell.

3. The process of claim 1, further comprising selecting the seed having the nucleic acid sequence encoding the diacylglycerol acyltransferase protein integrated into the genome of the plant cell.

4. The process of claim 1, wherein the diacylglycerol acyltransferase protein comprises SEQ ID NO:2.

5. The process of claim 1, further comprising:
planting the seed in soil;
growing a second plant from the seed; and
harvesting the seed from the second plant, wherein the seed comprise the nucleic acid sequence.

6. The process of claim 1, wherein transforming the plant cell with the nucleic acid sequence encoding the diacylglycerol acyltransferase protein comprises:
transforming an Agrobacterium cell with the nucleic acid sequence encoding the diacylglycerol acyltransferase protein; and
transforming the plant cell with the Agrobacterium cell.

7. The process of claim 3, wherein selecting the seed having the nucleic acid sequence encoding the diacylglycerol acyltransferase protein integrated into the genome of the plant cell comprises:
isolating genomic nucleic acid from the seed; and
amplifying the nucleic acid sequence encoding the diacylglycerol acyltransferase protein from the isolated genomic nucleic acid from the seed.

8. The process of claim 1, wherein the plant is selected from the group consisting of Arabidopsis thaliana, Borago spp., Canola, Ricinus spp., Theobroma spp., Zea spp., Gossypium spp, Crambe spp., Cuphea spp., Linum spp., Lesquerella spp., Limnanthes spp., Linola, Tropaeolum spp., Oenothera spp., Olea spp., Elaeis spp., Arachis spp., rapeseed, Carthamus spp., Glycine spp., Soja spp., Helianthus spp., Nicotiana spp., Vernonia spp., Triticum spp., Hordeum spp., Oryza spp., Avena spp., Sorghum spp., Secale spp., Brassicaceae, and other members of the plant family Gramineae.

9. The process according to claim 1, further comprising extracting oil from the harvested seed.