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Triacylglycerol Biosythesis in Soybeans
Department of Plant and Soil Sciences
This proposal addresses CREES goals 1, 2, 4 and 5 by providing fundamental information that can lead to increased economic opportunities for soybean producers, ways to improve the nutritional value of plant oils and enhanced production of environmentally friendly renewable resources. Plant oils are mainly composed of triacylglycerol (TAG) which represent an important edible and industrial resource. Oil from crops such as soybeans is the main substrate for biodiesel and can be an important source of renewable chemicals for the future.
The basis of selective accumulation of specific fatty acids and the final synthesis of TAG in seed oil are not fully understood. Current evidence indicates that Acyl-CoA: diacylglycerol acyltransferase (DGAT) catalyzes the synthesis of most oilseed TAG. There are two classes of DGATs with no homology, DGAT1 and DGAT2. The goal of this research is a much improved understanding of seed oil biosynthesis in soybeans that should be applicable to other oilseeds. The objectives are
1.) A detailed analysis of the expression of DGATs during soybean and Arabidopsis seed development and in other tissues in relation to seed oil synthesis.
2.) The effects of inhibiting expression of DGATs in developing seeds on oil accumulation.
3.) Subcellular localization of DGATs in relation to TAG biosynthesis in developing seed cotyledons.
To accomplish these, soybean DGAT2 gene(s) will be cloned and expression of DGAT transcripts will be determined by northern blotting and Real-time PCR will be performed. Gene inhibition will be achieved by RNAi suppression and TILLING of specific DGAT genes. Subcellular localization will utilize immuno-gold and transmission electron microscopy.
2009 Project Description
Two full-length acyl-CoA diacylglycerol acyltransferase (DGAT1s) have been cloned from developing soybean cDNA designated GmDGAT1a (GenBank # AB257589) and GmDGAT1b (GenBank # AB257590). Soybean DGAT1a looks to be the same as GenBank entry # AY496439 Submitted (08-DEC-2003) from the Institute of Genetics and Developmental Biology, Beijing, China (Wang et al., 2006). They have 99% identity with only two amino acid differences with our clone having a gly instead of asp and a his instead of gln both toward the amino terminus and underlined below. This may be due to allelic differences in the genotypes used (GmDGAT1a cDNA is from the Group II cultivar `Jack' and Wang et al. used cv. `8904'). GmDGAT1b does not match anything previously reported.
Interestingly GmDGAT1a and GmDGAT1b show somewhat different expression patterns although both show maximum transcript levels at stages of high triacylglycerol (TAG) biosynthesis. DGAT2 and phosphatidylcholine diacylglycerol acyltransferase (PDAT) do not show expression patterns consistent with a role in oil accumulation in soybean seeds. The activity of these soybean DGAT1 cDNAs was analyzed in a yeast expression system. Little increase in DGAT activity with GmDGAT1a compared to the vector control is seen and much greater activity is evident with expression of GmDGAT1b. Interestingly we see much higher TAG biosynthetic activity with a DGAT from a much higher oil accumulating plant, Vernonia galamensis, designated VgDGAT1a, than DGAT1s from soybeans.
We have provided initial characterization of the accumulation of TAG in macadamia seeds and cloned three genes encoding TAG biosynthetic enzymes from stages of seed development of very high oil accumulation. The genomic sequence of soybean DGAT1a was recently reported by (Wang et al., 2006) and we have sequenced the full genomic sequence of DGAT1b. DGAT1a is 7575 bp and DGAT1b is 8164 bp. Both have 14 introns and 15 exons. The 2nd, 6th and 13th introns have the same length with small to large length differences seen with the other introns with most being longer in DGAT1b. Exons 1, 2, 3, 5 and 10 also show length differences between DGAT1a and DGAT1b.
A plant transformation vector with VgDGAT1a driven by a seed-specific promoter was constructed and introduced into soybean somatic embryo cultures. Good expressing lines were selected and some resulting matured somatic embryos were found to accumulate increased oil or TAG levels. Plants were regenerated and grown out in a greenhouse and good expressing lines were found to have 4 - 5% higher oil levels than controls many without reductions in protein levels. These were grown out in the field in Lexington and harvested and the protein and oil contents determined. These lines produced in the field were again found to have 4 - 5% higher oil levels than controls without reductions in protein levels. These have been crossed with other high oil soybeans and soybeans with large reductions in phytate and oligosaccharides.
The synthesis of oil (TAG) in soybeans and high oil accumulating plants and a model system has been characterized. The genes involved in TAG biosynthesis in soybeans and high oil accumulating plants and a model system have also been characterized. New soybean lines with much higher oil levels and total oil + protein levels have been produced and characterized. Soybean lines with higher protein levels in meal have been produced. Breeding of soybean lines with further seed compositional improvements has been advanced.
Li, R., K. Yu, T. Hatanaka, and D.F. Hildebrand. 2009. Vernonia DGATs increase accumulation of epoxy fatty acids in oil. Plant Biotechnology Journal (in press).
Li, R., K. Yu, and D.F. Hildebrand. 2009. DGAT1, DGAT2 and PDAT Expression in Seeds and Other Tissues of Epoxy and Hydroxy Fatty Acid Accumulating Plants. Lipids (in press).
Rao, S., J.R. Thoguru and D. Hildebrand. 2009. Cloning and characterization of a Pleurotus ostreatus (oyster mushroom) delta-9 desaturase active with palmitic acid, Bioscience, Biotechnology, and Biochemistry (in press).
4. Rao, S., L. Mamadou, M. McConnell, R. Polisetty, P. Kwanyuen and D. Hildebrand. 2009. Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent, BMC Biotechnology (in press).
Rao, S. and D. Hildebrand. 2009. Changes in Oil Content of Transgenic Soybeans Expressing the Yeast SLC1 Gene. Lipids: 44: 945-951.
Hildebrand, D., J.R. Thoguru, R. Li, T. Hatanaka and S. Rao. 2010. Production and Accumulation of Unusual Fatty Acids in Plant Tissues. In Biocatalysis and Molecular Engineering, Hou, C.T. and J.F. Shaw eds., John Wiley & Sons (In press).
Hildebrand, D., R. Li and T. Hatanaka. 2010. Genomics of soybean oil traits. In Soybean Genomics, G. Stacey, ed. Elsevier (In Press).
Hildebrand, D., R. Li, K. Yu. & T. Hatanaka. 2009. Accumulation of Epoxy Fatty Acids in Plant Oils. Chapt. 3 In: Biocatalysis and Agricultural Biotechnology, C.T. Hou and J.F. Shaw, eds., CRC Press, Boca Raton.
Hildebrand, D.F., S. Rao and J.R. Thoguru. FUNGAL DESATURASES AND RELATED METHODS. U.S. Patent Appl No 12/346,234 Patent filed in Jan. 2009.
Hildebrand, D.F. and S. Rao. SOYBEAN SELECTION SYSTEM BASED ON AEC RESISTANCE. Patent Issued April 28, 2009 as U.S. Patent Number 7,525,013.