拟南芥MDH2基因GFP载体构建及转基因植株的筛选鉴定  

Construction of Arabidopsis MDH2-GFP Vector and Identification of Transgenic Lines

作　　者：王媛媛 韩洋洋 耿庆鎏 朱香豫 曹树青[1] 樊婷婷[1] WANG Yuan-yuan;HAN Yang-yang;GENG Qing-liu(School of Food and Biological Engineering,Hefei University of Technology,Hefei,Anhui 230009)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230009

出　　处：《安徽农业科学》2019年第2期96-98,共3页Journal of Anhui Agricultural Sciences

基　　金：中央高校基本科研业务费专项资金资助(JZ2018HGTB0248)

摘　　要：[目的]以野生型拟南芥为材料构建MDH2基因GFP载体及GFP转基因植株,研究MDH2基因在植物响应镉胁迫机制中的功能。[方法]通过提取野生型拟南芥的RNA,反转录成为cDNA,并以cDNA为模板,利用PCR扩增MDH2基因,将扩增所获得MDH2基因和pXB94-GFP质粒双酶切后连接,并转化进大肠杆菌感受态细胞中,鉴定正确后再转入农杆菌感受态细胞中,通过菌落PCR鉴定出阳性单菌落。再通过浸花法转入野生型拟南芥中,最后利用抗性筛选和PCR鉴定获得MDH2转基因阳性植株。[结果]成功克隆MDH2基因,并构建了MDH2-GFP重组质粒,抗性筛选获得了MDH2-GFP转基因植株。[结论]成功获得MDH2-GFP转基因植株,为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] In order to further study the function of MDH2 gene in response to cadmium stress in plant,the vector of Arabidopsis MDH2-GFP was built up and transgenic lines were constructed.[Method]The RNA of wild-type Arabidopsis was extracted and reverse transcribed into cDNA,the full-length MDH2 coding sequence was amplified by PCR.The amplified MDH2 gene and pXB94-GFP plasmid were digested and connected by double enzymes.The connected product was transformed into E.coli competent cells,then the true recombinant plasmid was transformed into Agrobacterium tumefaciens competent cells and the positive single colony was identified by colony PCR,then it was transferred into wild-type Arabidopsis by the floral-dip method.Finally,the positive transgenic plants were obtained by resistance screening and PCR identification.[Result]MDH2 gene was successfully cloned and the positive transgenic lines were obtained by selective medium with resistance.[Conclusion]The successful acquisition of MDH2-GFP transgenic lines laid the foundation for further research on the function of MDH2 gene.

关 键 词：拟南芥 MDH2 载体 转基因植株 

分 类 号：Q939.9[生物学—微生物学]