Cloning And Functional Research Of Glycerol-3-Phosphate Dehydrogenase Gene From Brassica Napus L

Oilseed (Brassica napus L.) is a major oil-producing crop, which provided people with vegetable oil and industrial production of biodiesel with ideal of raw material. Along with the growth of the population and the development of industry, rapeseed oil consumption increase year by year, it is an urgent need for improving rapeseed oil yield which ecided by two factors:rapeseed yield and oil content.Average yield in rape has improved greatly these years, thus increasing oil content of the rape become the key factor to improve the production benefit of oilseed rape.The process of formation storage lipids in oilseed is really complex, it involves fatty acid and acyl-CoA synthesis, glycerol-3-phosphate generation and incorporation of the acyl-CoA into glycerol backbone leading to triglyceride acyl groups three stages. Previous attempts to manipulate oil synthesis in plants and have mainly concentrated on the process involved in the biosynthesis and use of fatty acids, neglecting the possible role of glycerol-3-phosphate metabolism on the rate of triacylglycerol synthesis. On the basis of gene chip research, we screened a gene, which catalyze glycerol-3-phosphate dehydrogenase, called G3PDH, subsequent, this study cloned, analysised the expression and preliminary functional research of the gene.The main results were summarized as follows:1. On the basis of gene chip research screened the gene glycerol-3-phosphate dehydrogenase that related the seed oil accumulation, and got two copies of the complete coding sequence areas in brassica napus L.by homologous cloning method; the complete coding sequence of the two copies have1338base pairs, code445amino acids; through the genome walking method got the5’non-coding regions upstream the coding regions of the two copies,and through rapid amplification of cDNA3’ends method got the3’ non-coding regions downstream of the coding regions of the two copies; used full length coding region primers amplification and got the genomic sequence of the two copies, genes structure analysis showed that, both of the two copies have five exons and four introns; bioinformatics analysis showed that the genes are highly conserved with higher plant in the evolution, and two copies of the gene encoding amino acid sequences have typical structure domain of glycerol-3-phosphate dehydrogenase, the cell positioning analysis showed that the two proteins were cytoplasm protein.2. Using Realtime Fluores-cence Quantitative PCR method analyzed BnG3PDH expression. The results showed that BnG3PDH mainly express in pistil, but has a low expression in roots and leaves, we detected it did’t expressed in the stem, petals, sepals and stamens; according to the expression analysis of BnG3PDH at different development period of the pods, we found the gene had a high expression at peak of oil accumulation in seeds, but not in the skin of the pod; we analyzed BnG3PDH expression in high and low oil content materials, the results showed that the gene expression in high oil material significantly higher than low oil content material at peak of oil accumulation in seeds. In summary, these results suggest BnG3PDH may relate to the rate of TAG synthesis in seeds.3.In order to further study the function of the cloned gene, this study constructed the hairpin interference vector and over-expression vector drived by the napin promoter, the construction of the vectors lay the foundation for functional research G3PDH through transgenic approach.4. These constructs were transformed into Agrobacterium tumefaciens, then used for the transformation of Arabidopsis thaliana and Brassica napus L.by Agrobacterium-Mediated Floral-Dip method, we screened genetically modified Arabidopsis thaliana plants by antibiotics, these genetically modified plants were used to study the function of BnG3PDH, and the results of the study provided a new way for genetic improvement of seed oil content.