2种启动子驱动下的VaCBF3基因植物表达载体构建

为了探索山葡萄CBF3基因(VaCBF3)对杨树的遗传转化的影响,及组成型启动子和诱导型启动子对转基因植株生长的影响,分别构建由2种类型启动子调控的植物表达载体.根据GenBank上公布的rd29A基因序列设计特异性引物,利用PCR方法从拟南芥基因组DNA中克隆rd29A基因启动子片段,利用限制性内切酶通过酶切、连接构建由组成型启动子35S和诱导型启动子rd29A基因启动子驱动的VaCBF3基因植物表达载体,利用冻融法完成重组质粒对根癌农杆菌LBA4404的转化.试验结果表明,所克隆的rd29A基因启动子片段序列分析表明该启动子片段长度为945bp,与GenBank中基因序列同源性达99％,具有DRE、ABRE、TATAbox和CAAT box4个完整的顺式作用元件,具有启动子功能.经过酶切、连接成功地将目的基因与载体质粒连接,并将重组质粒导入至根癌农杆菌LBA4404中,为下一步利用农杆菌介导法对杨树的遗传转化及研究2种启动子的启动效率奠定了一定的基础.

Construction of Plant Expression Vector of VaCBF3 Gene Regulated Separately by Two Promoters

VaCBF3 gene was isolated and identified from Vitis amurensis.To study its effect on Poplar genetic transformation and the effect of constitutive promoter and inducible promoter on the growth of transgenic plants,plant expression vectors regulated by two different promoters respectively were constructed.According to the sequence of rd29A published on GenBank,the promoter of rd29A gene was cloned from Arabidopsis thaliana by PCR and the VaCBF3 gene plant expression vectors including constitutive promoter 35S and inducible rd29A gene promoter were constructed respectively by restriction enzyme,and the recombinant plasmids was transferred into Agrobacterium tumefaciens LBA4404 by Freeze-thaw method.Sequence analysis showed that the length of the cloned rd29A gene promoter was 947bp,and the homology of the promoter compared with the sequences on GenBank was 99%,including four integrated cis-acting elements DRE,ABRE,TATA box and CAAT box.The target gene was connected with the vector plasmid successfully by restriction enzyme,and the recombinant plasmids were transferred into Agrobacterium tumefaciens LBA4404,and these findings would be used for the genetic transformation of poplar using Agrobacterium-mediated technique and researched for the efficiency of the two promoters.