玉米ZmCPK12基因真核表达载体的构建及转化

Construction and Transformation of Eukaryotic Express Vector of ZmCPK12 Gene from Maize

[目的]CDPK是一类依赖于Ca^(2+)而不依赖CaM及磷脂的蛋白激酶,或类钙调素结构域的蛋白激酶,是植物和低等动物所特有。研究为玉米CDPK基因家族在抗逆中的作用提供基础。[方法]构建玉米ZmCPK12基因真核表达载体是了解玉米中该基因功能的重要途径之一。实验利用RT-PCR技术扩增得到大小为1 533 bp的玉米ZmCPK12基因,经限制性内切酶SdI和NotI进行消化,将目的基因与真核表达质粒pREP-5N连接,获得重组真核表达质粒ZmCPK12-5N。[结果]通过菌落PCR、双酶切及测序等方法进行鉴定,重组真核表达质粒ZmCPK12-5N构建成功。制备酵母感受态,将重组质粒电击转入酵母中,酵母菌落PCR结果显示电击转化成功。[结论]成功构建了玉米ZmCPK12基因真核表达载体,并且成功转化了酵母。

[Objective]CDPK only found in plants and lower animals,is a protein kinase dependent on Ca^(2+) but not on CaM and the phospholipid,or a class of calmodulin domain protein kinase.The purpose of this research is to provide the basis for understanding the function of the CDPK family genes from maize in the resistance.[Method]Construction of ZmCPK12 eukaryotic expression vector is an important way to understand the gene function in maize.In this paper,ZmCPK12 of 1 533 bp was cloned by using RT - PCR, and was digested by the restriction of enzymes SalI and NotI,and then it was connected with pREP -5N to obtain the recombinant eukaryotic expression plasmid ZmCPK12 -5N.[Result]Restriction enzyme digestion and sequencing methods were identified by colony PCR,and the results showed that the recombinant eukaryotic expression the plasmid ZmCPK12-5N was successfully constructed.Preparation of yeast competent,by electric shock,the recombinant plasmid was transformed into yeast.Yeast drop - PCR results showed that electroporation was successful.[Conclusion]Construction of ZmCPK12 gene eukaryotic expression vector and transformation were successful.