Isolation And Functional Analysis Of The Glycerol-3-Phosphate Acyltransferase Genes In Jatropha Curcas L.

Jatropha curcas L.(Euphorbiaceae), is native of tropical America, but isnow found abundantly in many tropical and sub-tropical regions throughout Africa andAsia. Jatropha has a potential as a biofuel plant replaceing fossil fuels in future, because ofits easy propagation, drought endurance, rapid growth, adoption to wide agro-climaticcondition, high oil content. Worldwide, introduction of jatropha for the purpose biodieselproduction met with limited success due to unrelaiable seed and oil yields. Investigatingthe molecular basis of storage lipid accumulation during the seed development is animmediate need to improve the oil content in jatropha. Glycerol-3-phosphateacyltransferase (GPAT; EC2.3.1.15) catalyzes the first step of glycerolipid biosynthesis inchloroplasts by transferring the acyl group of acyl-(acyl-carrier protein)(ACP) to the sn-1position of glycerol-3-phosphate. GPAT, the rate-limiting enzyme in the pathway ofglycerolipid biosynthesis, plays a critical role in biosynthesis of lipids in plants. Therefore,genetic engineering of the GPAT may allow us to modify the fatty acid composition oftriglycerides and to enhance oil content in plant.In this study, we isolated two GPAT genes from the developing seeds of Jatrophacurcas, designated JcGPAT1and JcGPAT2respectively. Homology analysis showed thatthe deduced amino acid sequence of the two genes exhibited high identity to the GPATgene in other plant. The expression patterns were analyzed in different tissues and differentstages in the developing seeds. Those results showed the expression level of GPAT in levelof transtription and translation. The function of the two genes was identified byheterologous expression in Saccharomyces cerevisiae wild strain BY4742and mutantstrain Y15983, using plasmid of vector pYES2.1/V5-His/lacZ as control. Seed specificplant expression vectors were constructed successfully in order to study the functions ofJcGPAT1and JcGPAT2. The main results are as follows:Based on the plastidial GPAT sequence available from NCBI (ACR61638), specificprimers were designed and the full sequences of a GPAT gene were amplified against thefirst-strand cDNA, named JcGPAT1. The full length cDNA is1389bp, encoding462amino acids which have a high identity (ranging from63%to79%) with GPAT genes in other plants reported such as castor (Ricinus communis) and safflower (Carthamustinctorius). The results of real-time quantitative PCR show that JcGPAT1increased itsexpression levels in leaf compared to the expression in developing seeds.Based on the conserved regions of GPAT genes available from GenBank database, wedesigned degenerate primers and obtained the cDNA sequences of GPAT gene by RACEtechnology from Jatropha curcas, named JcGPAT2(HQ395225). The full length cDNA is1672bp, encoding375amino acids which have a high identity (ranging from78%to95%)with GPAT genes in other plants reported such as castor (Ricinus communis) and tung tree(Vernicia fordii). RT-PCR analysis showed that JcGPAT2was expressed in differenttissues including the developing seeds, leaf, root tip and callus.Heterologous expression in yeast strain (Saccharomyces cerevisiae) was performedfor JcGPAT1and JcGPAT2. We obtained the growth curves of transformed yeast strainswith spectrophotometer at600nm, and determined their oil content using chemical vanillinmethod. Based on the growth curves and oil content, there is no a difference between theyeast strain transformed with JcGPAT2and the control, suggesting that JcGPAT2gene didnot play a positive role on the yeast lipid accumulation. On the contrary, the straintransformed with JcGPAT1grew slower than the control. In the meanwhile, the transgenicstrain accumulated more lipids, strongly indicating that JcGPAT1may be involved inglycerolipid synthesis in organisms.This study provided not only the initial information of the two GPAT genes inJatropha, but also a first glimpse of the gene expression and regulation patterns, which arecritical to understand the molecular basis of lipid biosyntheses, and to identify therate-limiting role of the gene during seed developmentand.