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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.
2011 Project Description
Diacylglycerol acyltransferases (DGAT1 and 2) and phospholipid:diacylglycerol acyltransferases (PDATs)are rate-limiting steps in plant storage lipid accumulation and play an essential role in controlling flux of fatty acids into storage TAGs. DGAT1, DGAT2 and PDAT expression in developing seeds of Cat's claw and macadamia indicate that three genes are important for 16:1∆9 accumulation.
A full-length cDNA of DGAT1 was isolated from developing seeds of cat's claw (Dolichandra unguis-cati). Previously, we developed several transgenic soybean lines expressing a yeast CoA-∆9 desaturase. One line with much higher total oil and protein levels was selected for yield trials on a UK farm in Lexington in 2011.
We also developed a number of VgDGAT-transgenic soybean lines with high oil + protein. Three good lines of VgDGAT1 transgenic soybean were planted for large-scale yield trials. A number of other good lines of VgDGAT1 transgenic soybean were also planted on a UK farm in 2011 for increasing seed stocks for further yield trials and other analyses in the future. All the transgenic soybeans for field study have been harvested. Currently, the genotyping and phenotyping are in process including analysis of the yield, protein and oil data of the target transgenic soybean lines.
For increasing oil levels further, numerous soybean crosses were made previously, including MIPs mutant line x VgDGAT1-transgenic line (high oil and normal protein) and NC 381 (high oil) x VgDGAT1. Hundreds of the crossed seeds were genotyped. A number of true crossed seeds were identified and then planted in the greenhouse and field for increasing these seed for further analyses and soybean improvement work. All soybean plants derived from those true crossed seeds have been harvested. We are currently genotyping and phenotyping these seeds. Soybean lines with good expression of cat's claw ∆9-ACP desaturase are being selected.
We already obtained target soybean lines expressing a ∆9-CoA desaturases. Both cat's claw ∆9-ACP and yeast or mushroom ∆9-CoA desaturases can be combined in the engineered soybean line by crossing these single-transgenic lines. By doing this, soybeans with high accumulation of 16:1Δ9 in seed oil should be obtained. This has worked in Arabidopsis.
Fatty acyl-ACP thioesterase B (FatB) is responsible for 16:0 to release from ACP and transport to the cytosol for incorporation into seed oil triacylglycerol. Four candidate soy FatBs were identified,Glyma05g08060, Glyma17g12940, Glyma06g23560 and Glyma04g21910. Based on the conserved regions for these soybean FatBs, three pairs of special primers were designed for development of an RNAi construct to down regulate expression of the FatB gene family in soybean. We selected three different small RNA sequences from the conserved region of soybean FatBs. Such expression constructs can express enough small RNAs to effectively down regulate the soybean FatBs.
Genes involved in TAG biosynthesis in soybeans and high oil accumulating plants and a model system have been further characterized. 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.
Full-scale yield trials were conducted in 2011 of some of our new high oil + protein lines for which sufficient seed was available 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.
We have identified four candidate genes for improving soybean oil quality and reducing the saturated fatty acid levels in soybean oil. Two RNAi primers were designed for down-regulating expression of genes providing saturated fatty acids for soybean oil biosynthesis. These are being used in soybean metabolic engineering for improved soybean oil quality as well as a better understanding of soybean oil biosynthesis.
Li, R., K. Yu, Y. Wu, T. Hatanaka and D. Hildebrand. 2012. Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds. Metabolic Engineering (in press)
Armstrong, P.R., J.G. Tallada, C. Hurburgh and D.F. Hildebrand. 2012. Development of single-seed near-infrared spectroscopic predictions of corn and soybean constituents using bulk reference values and mean spectra. Transactions of the ASABE (in press)
Jamboonsri, W., T.D. Phillips, R.L. Geneve, J.P. Cahill and D. Hildebrand. 2012. Extending the range of an ancient crop; a new ω3 source. Gen. Res. Crop Evol. (in press).
Hildebrand, D. 2011. Lipid Biosynthesis, Chapter 2 in Plant Metabolism and Biotechnology. Hiroshi Ashihara, Alan Crozier and Atsushi Komamine, eds., John Wiley & Sons, Ltd., West Sussex, UK, pp 25-62.
Hildebrand, D. 2011. PRODUCTION OF UNUSUAL FATTY ACIDS IN PLANTS, chapter in Plant Lipid Biochemistry in the online book, The AOCS Lipid Library, http://lipidlibrary.aocs.org/plantbio/unusualfa/index.htm.
Hildebrand, D.F., R. Li and T. Hatanaka. Method for Increasing Renewable Oil Production. US patent application # 13/206,361 filed August 9, 2011.
Hildebrand, D.F., S. Rao and J.R. Thoguru. FUNGAL DESATURASES AND RELATED METHODS. U.S. Patent # 8,053,633 Patent issued Nov. 8, 2011.