拟南芥 ProNAS4-GUS 转基因植株的构建及表达模式

为了进一步研究 NAS4 基因的表达模式及其在缺铁胁迫下的响应模式,文章利用从野生型拟南芥中提取的基因组 DNA 作为模板,克隆获得 NAS4 启动子片段,将片段与 pART27-GUS 质粒进行酶切处理,然后将片段与质粒进行连接,将连接产物通过热激转化法转入大肠杆菌中,通过聚合酶链式反应(polymerase chain reaction, PCR)鉴定获得阳性菌株。质粒测序正确后将重组质粒通过电击转化法转入农杆菌中,经过 PCR 鉴定得到阳性菌株后,用浸花法侵染野生型拟南芥;通过抗性筛选和 PCR 鉴定得到 ProNAS4-GUS 转基因植株,并对获得的转基因植株进行 GUS 染色分析,发现 NAS4 基因在幼苗的根部和叶脉中都有所表达。实验结果为研究 NAS4 基因在植物缺铁胁迫下的响应模式奠定了基础。

In order to further study the expression pattern of NAS4 gene and its response pattern under iron deficiency stress, DNA extracted from wild-type Arabidopsis thaliana was used as template to clone the NAS4 promoter fragment. Then, the fragment and pART27-GUS were double-digested, the fragment was ligated with the pART27-GUS and transformed into Escherichia coli by heat shock method, and the positive strain was obtained by polymerase chain reaction (PCR) identification. After correct sequencing, the recombinant plasmid was transformed into Agrobacterium using electroporation transformation method. Identified by PCR positive monoclonal, the positive strain was obtained. Finally, the wild-type Arabidopsis thaliana was infected by floral dip method and the ProNAS4-GUS transgenic positive plants were obtained through resistance screening, and the obtained transgenic plants were subjected to GUS staining analysis, showing that NAS4 gene was expressed in the roots and veins of seedlings. It lays a foundation for further exploring the response pattern of NAS4 gene under iron deficiency stress in plants.