DREB2A基因的克隆、序列分析及其植物表达载体的构建

Cloning and sequencing of DREB2A gene and construction of its plant expression vector

研究提取盐处理的拟南芥植株叶片总RNA,用DREB2A基因特异引物通过RT-PCR扩增出1050bp的片段,并将该片段克隆至pUC18上。序列分析结果表明,该克隆序列与GenBank上的DREB2A基因序列同源性达100%。进一步将DREB2A基因分别克隆至植物表达载体卡盒pCCE12和pCC29A上,构建了分别由E12启动子、rd29A启动子调控的DREB2A基因植物表达载体pCDRE12和pCDR29A。通过冻融法将重组质粒导入根癌农杆菌LBA4404中,为农杆菌介导的DREB2A基因对植物的遗传转化奠定基础。

A 1 050 bp DNA fragment which was amplified by RT-PCR using DREB2A gene special primer from Arabidopsis thaliana leaf treated by 200 mmol·L-1 NaCl was cloned into pUC18. The result of sequencing showed that the sequence of this fragment was 100% homologous with that of DREB2A gene in GenBank. After that DREB2A gene was cloned into plant expression vector cassette pCCE12 and pCC29. Thus two plant expression vectors, pCDRE12 and pCDR29A were constructed, in which DREB2A gene was controlled by E12 promoter and rd29A promoter respectively. Then the recombination plasmids were introduced into Agrobacterium LBA4404 by freezing -melting transformation method. This work provides a foundation for the transformation of DREB2A gene to plant mediated by Agrobacterium.