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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.
2010 Project Description
Genes involved in TAG biosynthesis in soybeans and high oil accumulating plants and a model system have been further characterized. The entire soybean genome has been "BLAST" searched with three classes of genes that can synthesize oil and we find three DGAT1, five DGAT2 and six PDAT1 homologues in the soybean genome.
We have shown that two of the DGAT1s are expressed at a time of maximum TAG biosynthesis in soybean seeds and have TAG synthesis activity. These were again grown out in the field in Lexington, 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. Crosses of these lines with other high oil soybeans and soybeans with large reductions in phytate and oligosaccharides have been grown out and F2 seeds collected.
Preliminary yield data was collected in 2010 and no significant reduction in yield is seen with some of the high oil lines. As much as 20% more oil per acre may be possible with some of our soybean lines with protein yield per acre as much as conventional lines.
Genes involved in TAG biosynthesis in soybeans and high oil accumulating plants and a model system have been further characterized. The entire soybean genome has been "BLAST" searched with three classes of genes that can synthesize oil, DGAT1, DGAT2 and PDAT1. We find three DGAT1, five DGAT2 and six PDAT1 homologues in the soybean genome. We have shown that two of the DGAT1s are expressed at a time of maximum TAG biosynthesis in soybean seeds and have TAG synthesis activity. These have been deposited in GenBank and assigned accession #s AB257589 and AF257590.
New soybean lines with much higher oil levels and total oil + protein levels have been further characterized and found to hold true for another generation and in field production. Preliminary yield trials were conducted in 2010 and no significant reduction in yield is seen with some of the high oil lines. As much as 20% more oil per acre may be possible with some of our soybean lines with protein yield per acre as conventional lines.
This increased oil production could make more than $2 billion of renewable oil produced by US soybean growers per year and increase this renewable resource for edible, fuel and renewable chemical applications without requiring more land for the production.
Li, R., K. Yu, T. Hatanaka and D.F. Hildebrand. 2010. Vernonia DGATs increase accumulation of epoxy fatty acids in oil. Plant Biotechnology Journal 8: 184-195.
Li, R., K. Yu and D.F. Hildebrand. 2010. DGAT1, DGAT2 and PDAT Expression in Seeds and Other Tissues of Epoxy and Hydroxy Fatty Acid Accumulating Plants. Lipids 45:145-157.
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 9: 1-17.
Wilson, R.F. and D. Hildebrand. 2010. Engineering Status, Challenges and Advantages of Oil crops. Chapter 8 In: Plant Biotechnology for Sustainable Production of Energy and Coproducts, P.N. Mascia, J. Scheffran and J.M. Widholm (Eds.), Springer, New York, pp. 209-259.
Hildebrand, D.F., R. Li and T. Hatanaka. Diacylglycerol Acyltransferase Sequences and Related Methods. Patent Appl No. 12/622,045 filed Nov. 19, 2009.
Hildebrand, D., J.R. Thoguru, R. Li, T. Hatanaka and S. Rao. 2010. Production and Accumulation of Unusual Fatty Acids in Plant Tissues. Chapter 3 In Biocatalysis and Molecular Engineering, Hou, C.T. and J.F. Shaw eds., John Wiley & Sons pp. 43-56.
Hildebrand, D. 2010. Production of unusual fatty acids in plants. 2010. Online Plant Lipid Biochemistry Book, http://lipidlibrary.aocs.org/plantbio/unusualfa/index.htm.
Hilker, B.L., H. Fukushige, C. Hou and D. Hildebrand. 2009. Some properties of a self-sufficient cytochrome P-450 from Bacillus megaterium strain ALA2. Chapter In "Biocatalysis and Bioenergy", Hou, C.T. and J.F. Shaw eds., John Wiley & Sons, NY, pp. 291-308.
Hildebrand, D.F., R. Li and T. Hatanaka. Method for Increasing Renewable Oil Production. Provisional patent filed July, 2010.