Studies On Yeast Glycerol-3-phosphate Dehydrogenase Gene Transformation In Rapeseed (Brassica Napus L.)

Oilseed rape is the most important oilseed crop planted widely in China, as well as a major source of edible vegetable oil for human. Meanwhile, the vegetable oil of rapeseed is a kind of sustainable use of biological resources. Regulating gene expression of fatty acids synthesis in Brassica napus L. by genetic engineering can hopefully increase the content of oil, improve its quality and produce new germplasm resources. In this paper, Saccharomyces cerevisiae were used as materials from which the fatty acids synthesis related genes GPD were cloned. The sequences were bioinformatically analyzed, and the new expression vectors with seed-specific promoter napin gene were constructed. Then, the high frequency regeneration system and genetic transformation of Xiangyou15were studied. The main results were as followings:1. Cloning and sequence analysis of GPD gene.A pair of primers was designed according to the DNA sequence reported in GenBank. The GPD1and GPD2gene were isolated from the genomic DNA of Saccharomyces cerevisiae by PCR amplification. The PCR products were cloned into pGEN-T vector and sequenced. The results indicated that the cloned fragments contained1175and1324nucleotides respectively, and shared a homology of99.4%and99.5%with the reported yg3and NM001183314.2. Construction of the plant expression vector earring GPD gene.The products that had been cloned into pGEM-T vector digested by BamHI/SacI. The fragments were inserted into the same enzyme site of pB1121and pB1121.N.Then,the expression vectors G1、G2、NG1、NG2were constructed. Transformation of Agrobacterium strains LBA4404with them were done and vertified by plaque-PCR.3. The genetic transformation and obtaining the transgenic Brassica napus L.The cotyledon petiols and hypocotyls of oilseed rape(Brassica napus L)were used as recipient of transformation with G1、G2、NG1and NG2.By infecting and co-cultured with Agrobacterium tumefaciens, transgenic plant had been obtained by selected from Kanamycin-resistant. The PCR detection proved that the GPD gene was introduced into Xiangyou15genome.