甘蓝型油菜依赖焦磷酸的磷酸果糖激酶(PFK)基因片段克隆及hpRNAi载体构建  

Cloning and hpRNAi Vector Construction of Brassica napus Pyrophosphate-dependent Phosphofructokinase (PFK)

作　　者：汪承刚[1] 张菊红[1] 谢妤[1] 刘幼琪[1] 黄邦全[1] 

机构地区：[1]湖北大学生命科学学院植物遗传育种实验室,武汉430062

出　　处：《分子植物育种》2006年第2期199-204,共6页Molecular Plant Breeding

基　　金：湖北省科技厅国际合作重点项目（2005CA020）;湖北省教育厅国际合作项目（G200510001）资助.

摘　　要：依赖焦磷酸的磷酸果糖激酶(PFK)是糖酵解途径的关键酶。本研究首先构建带有种子特异性napin启动子和Nos终止子的植物表达载体2300-nap及带有组成型35S启动子和Nos终止子的植物表达载体2300-35S;然后用PCR法从甘蓝型油菜(BrassicanapusL.)中油119总基因组中扩增出依赖焦磷酸的磷酸果糖激酶(PFK)基因片段,再以扩增出的PFK基因片段作模板设计引物扩增出一个相应的小片段。将两个PFK基因片段反向连接,插入到植物表达载体2300-nap的napin启动子和nos终止子之间,植物表达载体2300-35S的35S启动子和nos终止子之间,分别构建成可转录表达出发夹RNA(hairpinRNA,hpRNA)结构的种子特异型和组成型油菜RNA干扰载体,为今后油菜利用RNA干扰(RNAi)提高含油量的基因工程研究奠定了基础。Pyrophosphate-dependent phosphofructokinase （PFK） plays an important role in the EMP pathway in rapeseed （Brassica napus L.）. Firstly we constructed plant expression vector 2300-nap harboring seed-specific napin promoter and nos terminator, and plant expression vector 2300-35S harboring constitutive 35S promoter and nos terminator. PFK gene fragment was PCR-amplified from B. napus genomic DNA and was used as template to amplify a smaller fragment. These two PFK fragments were ligated in opposite directions into plant expression vector 2300-nap and 2300-35S respectively to produce hpRNAi vector hp-PFK-nap and hp-PFK-35S. This is the first step to increase oil content in rapeseed using RNA interference.

关 键 词：甘蓝型油菜 依赖焦磷酸的磷酸果糖激酶(PFK) 发夹RNA(hpRNA) 载体构建 

分 类 号：S565.4[农业科学—作物学] Q782[生物学—分子生物学]