拟南芥bZIP23基因过量表达载体的构建及过表达植株的筛选

目的]以拟南芥为材料克隆bZIP23基因,构建bZIP23基因的过量表达载体和筛选过表达植株,为验证其功能奠定基础.方法]提取拟南芥总RNA和RT-PCR克隆bZIP23基因,用限制性内切酶切割和T4 DNA连接酶连接,使bZIP23基因连接到35S强启动子的pART27载体上;将连接产物转化到Trans1-T1感受态细胞中,筛选阳性单克隆进行菌落PCR鉴定并测序验证,获得重组质粒.将该重组质粒电激转化至根瘤农杆菌GV3101菌株,浸花法转化拟南芥野生型植株.结果]通过单菌落PCR鉴定和DNA测序结果显示,bZIP23基因与35S过量表达载体已连接,获得了重组载体;抗性筛选与遗传鉴定获得相应的转基因过量表达阳性植株.结论]构建的过量表达载体及筛选得到的过量表达植株为验证bZIP23基因功能奠定了基础.

Construction of Arabidopsis Gene bZIP23 Overexpression Vector and Screening of Expression Plant

Objective] Arabidopsis were used as material to clone bZIP23 gene,we construct gene bZIP23 overexpression vector and screen expression plant.Method] Total RNA was extracted from Arabidopsis seedlings,and cDNA fragments of bZIP23 gene were amplified by RT-PCR.Using the restriction enzymes and T4 DNA ligase,cDNA fragments were subsequently cloned into PART27 vectors,and then were transformed into Trans-T1 phage resistant chemically competent cells.Analysis of bacterial colony PCR and cDNA sequencing were performed to confirm that cDNA of the Arabidopsis thaiiana bZIP23 gene was successfully cloned.The recombinant plasmids were obtained and transformed into Agrobacterium GV3101 cells.Wild-type Arabidopsis thaliana was transformed by using floral-dip method.Result] Analysis of bacterial colony PCR and DNA sequencing were performed to confirm recombinant plasmids,complementary and overexpression positive plants were obtained through genetic screening and identification of genetically modified methods.Conclusion] Construction of Arabidopsis gene bZIP23 overexpression vector and screening of expression plant laid the foundation for the function of gene bZIP23.