拟南芥ProWRKY12-GUS转基因植株的构建及其染色分析  

Construction and staining analysis of Arabidopsis thaliana ProWRKY12-GUS transgenic plants

作　　者：陶曼芝 吴席 朱香豫 王婷婷 贾亚峰 曹树青 TAO Manzhi;WU Xi;ZHU Xiangyu;WANG Tingting;JIA Yafeng;CAO Shuqing(School of Food and Biological Engineering,Hefei University of Technology,Hefei 230601,China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230601

出　　处：《合肥工业大学学报（自然科学版）》2022年第9期1248-1251,共4页Journal of Hefei University of Technology：Natural Science

基　　金：国家自然科学基金资助项目(31872803);省级大学生创新训练资助项目(S202010359218);校级大学生创新训练资助项目(X202110359437)。

摘　　要：为了解析植物缺铁的分子机制,文章对拟南芥突变体库缺铁胁迫进行筛选,并获得了缺铁胁迫响应突变体wrky12。为了进一步探究WRKY12基因的表达模式及其对缺铁胁迫的响应模式,构建WRKY12-GUS重组质粒,再以野生型拟南芥为模板构建重组植株。从野生型拟南芥植株中克隆WRKY12启动子片段,将双酶切过后的WRKY12启动子片段连接到同样双酶切后的pART27-GUS质粒上,再将连接好的重组质粒转化到大肠杆菌中,通过聚合酶链式反应(polymerase chain reaction,PCR)鉴定出阳性单克隆,测序确定后即得到ProWRKY12-GUS重组质粒,再将重组质粒转入农杆菌中,同样通过菌落PCR得到阳性单克隆。最后采用浸花法侵染野生型拟南芥植株,通过抗性筛选和PCR鉴定的方法得到纯合的ProWRKY12-GUS转基因植株。为进一步研究WRKY12基因功能奠定基础。In order to study the molecular mechanism of iron deficiency in plants,this paper screened for iron deficiency stress in the Arabidopsis thaliana mutant library and obtained the mutant wrky12 in response to iron deficiency stress.To explore the expression pattern of WRKY12 gene and its response to iron deficiency stress,the paper constructed a WRKY12-GUS recombinant plasmid,and further constructed the recombinant plant using wild-type Arabidopsis thaliana as the template.The WRKY12 promoter fragment was cloned from wild-type Arabidopsis thaliana plants,and the WRKY12 promoter fragment after the double enzyme digestion was connected to the pART27-GUS plasmid,and then the recombinant plasmid was transformed into Escherichia coli,identifying positive monoclones with polymerase chain reaction(PCR).After sequencing the ProWRKY12-GUS recombinant plasmid was gotten,and then the recombinant plasmid was transformed into agrobacterium,obtaining positive monoclones with colony PCR.Finally,the wild-type Arabidopsis thaliana was infected by floral dip method and ProWRKY12-GUS transgenic plants were obtained by means of resistance screening and PCR identification.It lays the foundation for the future research on the function of WRKY12 gene.

关 键 词：拟南芥 WRKY12 载体 转基因植株 GUS染色分析 

分 类 号：Q939.9[生物学—微生物学]