甘蓝型油菜WRI1基因cDNA克隆与种子特异性表达载体的构建  

Cloning of WRI1cDNA fromBassica napus and Construction of Its Seed-Specific Expression Vector

作　　者：施春霖[1] 刘聪[1] 肖旦望 邬克彬[1] 陈社员[1] 熊兴华[1] 

机构地区：[1]作物基因工程湖南省重点实验室,湖南农业大学,长沙410128

出　　处：《西北农业学报》2014年第1期120-125,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家863课题(2012AA101107-3);湖南省科技厅课题(2007RS4014)

摘　　要：WRI1基因所编码的转录因子参与糖酵解和脂肪酸合成,在种子油的积累过程中起着关键作用。在种子中超表达WRI1基因,对提高油菜种子含油量具有重要意义。本研究利用RT-PCR技术,从甘蓝型油菜湘油15号cDNA中分离克隆了5个WRI1基因全长cDNA序列,选取2种存在明显差异(碱基缺失)的cDNA序列构建植物种子特异性表达载体。为后续转基因研究这种差异是否会造成WRI1基因在提高含油量作用上的差异作准备。通过双酶切和连接反应,分别将WRI1基因和napin启动子基因插入到pBI121载体的GUSA基因和CaMV35启动子基因位置。酶切及PCR检测结果显示,napin启动子和WRI1已插入相应位置,完成2种载体种子特异性表达WRI1基因重组载体pBI121+napin+WRI1的构建,命名为pBI121NW2、pBI121NW4。WRI1 is involved in the portant role in oil accumulation. 5 pus） by RT-PCR and ligated into struct seed-specific expression vect regulation of glycolysis and fatty acid biosynthesis. It plays an im- WRI1 cDNAs was amplified from Xiangyou No. 15 （Brassica ha- pMD18-T or. The recom P b e vector. Two different clones were used to con pMD18-T Simple vector which contain WRH cDNA sequence and recombinant pGEM-T Easy vector which contain Napin promoter sequence was digested by BamH I/Sac I （the new restriction sites were added in the PCR amplification primers ） and Hind Ⅲ/BamH I respectively. The fragments were inserted into the corresponding sites in the pBI121. Recombinant seed-specific expression vectors were identified by restriction enzyme digestion and PCR analysis.

关 键 词：甘蓝型油菜 懈朋 克隆 种子特异性表达载体 

分 类 号：S565.4[农业科学—作物学]