转录因子DREB1A基因的克隆与植物表达载体的构建

Cloning of transcription factor DREB1A gene and the construction of plant express vector

根据GenBank中已发表的转录因子DREB1A基因的cDNA序列设计并合成了一对引物 ,通过RT PCR的方法从低温处理的拟南芥总RNA中扩增出DREB1A基因的全长cDNA片段。将其克隆到 pMD1 8T vector中。经测序证明该片段与GenBank上报道的序列具有 99.8%的同源性。 2个碱基的置换导致了一处氨基酸的差异 ,但这一氨基酸并不在基因的功能结构域上 ,推测其不会影响基因功能。以植物表达载体pBch为基础 ,构建了由组成型启动子 35S调控的DREB1A基因的植物表达载体pBDR35S 。

According to the cDNA sequence of transcription factor DREB1A gene in GenBank a pair of primer was designed and synthesized. Using the total RNA of low temperature treated Arabdopsis thaliana seedling as templet, the full length of cDNA of DREB1A gene was amplified through method of RT-PCR. It was cloned into pMD18 T vector. DNA sequencing indicated that the cloned fragment showed 99.8% identity to the sequence of the gene in GenBank. The Two bases change leads to only one amino acid changed. But the changed amino acid is not in the function domain so the cDNA will have the normal physiology function. The plant express vector pBch was digested and ligated with the cloned cDNA fragment and got the DREB1A gene express vector pBDR35s regulated by 35S promoter.