棉花GbCBF2基因的克隆、互补表达载体的构建及遗传转化

Cloning,Complementary Expression Vector Construction and Transformation of GbCBF2 Gene from Cotton

为研究棉花CBF2基因在植物抗逆中的作用,构建了p CAMBIA1300-At CBF2P-Gb CBF2-At CBF2T植物互补表达载体,并对拟南芥cbf2突变体进行遗传转化。以新海16 c DNA为材料,采用PCR的方法克隆4个棉花CBF2基因,同时克隆拟南芥CBF2基因启动子At CBF2P和终止子At CBF2T,并将这些基因与p Bluescript SK载体连接,构建p Bluescript SK-At CBF2P-Gb CBF2-At CBF2T中间过渡载体,用SacⅠ和Bam HⅠ双酶切中间载体,获得At CBF2P-Gb CBF2-At CBF2T核心片段,将此片段插入到p CAMBIA1300表达载体中,构建p CAMBIA1300-At CBF2P-Gb CBF2-At CBF2T植物互补表达载体。以拟南芥cbf2突变体为材料,采用农杆菌介导的方法进行遗传转化,获得转棉花CBF2基因群体。获得转基因株系后,将为筛选出棉花的CBF2抗冻负调控基因及棉花抗逆分子育种奠定基础。

In order to study the function of cotton CBF2 gene in plant resistance,the complementary expression vector p CAMBIA1300-At CBF2P-Gb CBF2-At CBF2 T was constructed,and transformed to Arabidopsis cbf2 mutant by genetic engineering. The four CBF2 genes were cloned from c DNA of the cotton Xinhai 16 and Arabidopsis CBF2 gene promoter At CBF2 P and termination At CBF2 T were also cloned by PCR method. Firstly,these genes were connected with p Bluescript SK,and obtained the intermediate transition vector p Bluescript SK-At CBF2P-Gb CBF2-At CBF2 T,then the At CBF2P-Gb CBF2-At CBF2 T fragment was obtained by cutting the intermediate transition vector with SacⅠ and Bam HⅠ,after the fragment inserted into p CAMBIA1300,the complementary expression vector p CAMBIA1300-At CBF2P-Gb CBF2-At CBF2 T was constructed. The cotton CBF2 gene was transformated into Arabidopsis cbf2 mutant by Agrobacterium mediated method and regenerative seedlings were obtained. These results will lay the foundation for screening cotton negative regulator CBF2 gene and stress-resistance molecular breeding.