拟南芥CTSP3基因GUS载体构建及转基因植株的筛选鉴定

Construction of Arabidopsis CTSP3-GUS Vector and Identification of Transgenic Lines

[目的]深入研究植物基因CTSP3的基因表达模式及其对重金属镉胁迫的响应机制,以野生型拟南芥为材料构建CTSP3-GUS重组质粒及GUS转基因植株。[方法]通过提取野生型拟南芥的DNA,克隆其启动区基因片段,将基因片段和pART27-GUS质粒双酶切后连接,转化到大肠杆菌感受态细胞中,菌落PCR和测序获得阳性单克隆。然后将CTSP3-GUS重组质粒转入农杆菌感受态GV3101,获得阳性单菌落。接着采用浸花法侵染野生型拟南芥,最后通过抗性筛选和PCR鉴定获取CTSP3-GUS转基因植株。[结果]成功克隆CTSP3启动区基因片段,构建出重组质粒,获得了CTSP3-GUS转基因植株。[结论]获得CTSP3-GUS转基因植株,为接下来进一步研究该基因在植物响应镉胁迫机制中的功能奠定了基础。

[Objective]To study the expression pattern of CTSP3 gene and its response mechanism to the heavy metal cadmium stress,we build the CTSP3-GUS recombinant vector and GUS transgenic plants.[Method]The Arabidopsis(wild-type)DNA was extracted,and the promoter region gene fragment was cloned.The gene and pART27-GUS plasmid were digested and ligated into E.coli competent cells,positive clones were obtained by colony PCR and sequencing.The CTSP3-GUS recombinant plasmid was then transferred into Agrobacterium competent GV3101 to obtain a positive single colony.Then,wild-type Arabidopsis thaliana was digested by dip flower method,and finally CTSP3-GUS transgenic plants were obtained by resistance screening and PCR identification.[Result]The gene fragment of CTSP3 promoter region was successfully cloned,and the recombinant plasmid was constructed to obtain CTSP3-GUS transgenic plants.[Conclusion]The CTSP3-GUS transgenic plants were obtained,which laid a foundation for further studying the function of this gene in response to cadmium stress in plants.