甘蓝型油菜S-GT基因克隆、hpRNAi载体构建及遗传转化  

Cloning and hpRNAi vector construction for gene of thiohydroximate S-glucosyltransferase and genetic transformation in Brassica napus

作　　者：李长春[1] 乔富兴[1] 严晓锋[1] 丁全如[1] 黄邦全[1] 

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2008年第3期290-293,共4页Journal of Hubei University：Natural Science

基　　金：湖北省科技厅;湖北省教育厅国际合作重点项目(2005CA020G200510001);Swedish Research Links Programme(348-2006-6684)资助

摘　　要：根据发表的甘蓝型油菜S-GT基因cDNA序列设计引物,以甘蓝型油菜总DNA为模板进行PCR扩增,获得S-GT基因全长.根据获得的基因序列设计引物扩增出S-GT基因序列相同但是带有不同酶切位点的两个片段,将两个片段反向插入到带有本实验所构建的种子特异表达载体内含子的两端,成功构建了油菜S-GT基因的种子特异性hpRNAi载体.通过根瘤农杆菌EHA105介导,转化甘蓝型油菜‘hubu 11’的子叶柄,诱导愈伤组织分化出绿芽,进而再生出完整的植株.通过PCR初步鉴定获得了58株转基因阳性植株.PCR primers were designed according to the cDNA sequence of S-GT gene in Brassica napus and full length of the S-GT gene in B. napus was obtained by using the genomic DNA as PCR template. Two small fragments with identical sequences but different restriction sites were amplified from the cloned S-GT sequence and ligated in opposite orientations into the seed-specific vector 2300- nap-intron to produce hpRNAi vector to silence the internal S-GT activities in seeds of B. napus to lower the glucosinolate level in the seeds and not in the vegetative organs. Cotyledons from Brassica napus cv. hubu 11 were infected with Agrobacterium tumefaciens EHA105 containing plasmid 2300- nap-hpBnSGT-Intron. Plantlets were regenerated on selective media containing kanamycin and 58 transgenic plantlets were confirmed to be positive by PCR amplification.

关 键 词：甘蓝型油菜 硫甙 thiohydroximate SIglucosyltransferase hpRNAi载体 转基因 

分 类 号：Q785[生物学—分子生物学]