Production And Molecular Detection Of TiERF1Transgenic Rapeseeds

Rapeseed is an important oilseed crop in China. The disease rape sclerotinia rot, mainly caused by Sclerotinia sclerotiorum, has become one of the most serious diseases of rapeseed in China and seriously challenging the quantity and quality of rapeseed production in recent years. The most safety, economical and effective way is to breed and utilize disease-resistant rapeseed cultivars. However, no resistant cultivar has been identified. It is difficult to breed rape sclerotinia-rot-resistant varieties using traditional methods. Hence, genetic engineering provides an alternative approach to improve the level of resistance to this disease.In the study, the expression vector pBI121-TiERF1was constructed basing on the vector pA20-TiERF1. The high effective regeneration system and genetic transformation system were established. The transformation vector pBI121-TiERF1was introduced into Xiangyou15and Zhongshuang9by agrobacterium tumefacience-mediated cotyledons node transformation system. The transgenic rapeseed lines were obtained.The main results were as follows:1. Construction of transformation vector pBI121-TiERF1.Schema of the transformation vector pBI121-TiERF1was prepared by sub-cloning the full-length coding sequence of TiERFl into the BamH Ⅰ and Sac Ⅰ sites of the original vector pEASY-T1, after the sequencing analyzed, both the recombinant pEASY-T1-TiERF1and original vector pBI121were digested by BamH Ⅰ and Sac Ⅰ. It was then sub-cloned in-frame to the BamH Ⅰ and Sac Ⅰ sites of the ORF of TiERFl in the pBI121vector, which resulted in the expressing vector pBI121-TiERF1. The pBI121-TiERF1vector was transformed into Agrobacterium strains LBA4404by freeze-thaw method. The presence of the introduced TiERF1gene in Agrobacterium strains was detected by colony PCR using primers specific designed.2. Establishment of regeneration system Zhongshuang9The best pre-medium of Zhongshuang9was MS medium supplemented with 1.0mg/L6-BA and1.0mg/L2.4-D, and the best differentiation medium was MS medium supplemented with3.0mg/16-BA,0.02mg/1NAA and4.5mg/1AgNO3. The regeneration rate was92.5%.3. Establishment of the optimized systems of genetic transformation for Zhongshuang9and Xiangyou15The experiment was conducted to investigate the effects of the parameters on agrobacterium tumefacience-mediated cotyledons node transformation system. The results indicated that Xiangyou15seedling age were5days, pre-culture3days, the OD600of LBA44040.6, infection5minutes, coculture2days and adding AS200μmol/L incoculture medium. For Zhongshuang9, seedling age were5days, pre-culture3days, the OD600of LBA44040.4, infection5minutes, coculture2days and also adding AS200μmol/L in coculture medium.4. Obtaining regeneration plants and PCR detection8and9regenerated seedlings with Kanamycin resistant were obtained from Xiangyou15and Zhongshuang, respectively. PCR analysis showed that the TiERF1gene was introduced into genome of4Xiangyou15plants and5Zhongshuang.9TiERF1transgenic rapeseeds were obtained.