Cloning And Characterization Of Glycerol-3-phosphate Dehydrogenase Gene (RcGPDH) From Castor Bean

The castor plant (Ricinus communis), a species of Euphorbiaceae family, is a perennial shrub. Its seeds accumulate 60% oil in the form of triacylglycerol (TAG). Castor oil is unique in that 90% of its fatty acid content is ricinoleate (12-hydroxy-9-cis-octadecenoic acid), which make castor oil a vital industrial raw material for numerous products. Now, the castor plant is an important industrial oil crop of the world. Rencnetly, many researches focused on the acyltransferase in the Kennedy pathway of triacylgycerol biosynthesis were reported. A few studies were performed to analysis the function of the glycerol-3-phosphate dehydrogenase which catalyses dihydroxyacetone phosphate to produce the glycerol-3-phosphate, a glycerol backbone in TAG biosynthesis. The function of glycerol-3-phosphate dehydrogenase in plant oil biosynthesis is unclear. In present study, a glycerol-3-phosphate dehydrogenase gene (RcGPDH) was cloned from castor bean and characterized.According to the Express Sequence Tags (EST) of castor bean, primers were designed and the full cDNA of RcGPDH gene was cloned by RACE (rapid-amplification of cDNA ends). High levels of expression were detected by real-time PCR in the castor seeds during later stages of developments. To elucidate the function of RcGPDH gene, a yeast expression vector PYES2.1/V5-His-TOPO-RcGPDH was constructed and transformed to the wild yeast strain BY4742. The growth curve of transgenic yeasts was figured using spectrophotometry, and the oil contents and fatty acid compositions were measured using vanillin method and GC (gas chromatography) respectively. Meanwhile, a plant expression vector, which contained a seed specific promoter, was constructed and transformed to Arabidopsis thaliana. Results showed that the transgenic yeasts grew stably after cultured 18 hours, but the strain with RcGPDH gene grew slower than the control strain. The oil content and the fatty acids composition weren't various between the two yeast strains, which suggested that RcGPDH gene didn't play a positive role on the yeast oil accumulation. Total lipid content decreased and seeds weight increased in RcGPDH-overexpressing plants, and apparent changes in total fatty acid composition were also detected in the transgenic seeds, saturated fatty acids reduced and polyunsaturated fatty acids increased. So we speculated that RcGPDH gene has no positive contribution to oil accumulation, however maybe has positive role to adapt to low temperature stress. To further verify the function of RcGPDH gene in the oil accumulation, in vitro propagation also was performed to establish a stable genetic transformation system of castor.