中文摘要：

采用部分互补的引物扩增表达载体的全序列,通过DpnⅠ酶切PCR产物去除甲基化的模板DNA,利用大肠杆菌自身的酶系统环化质粒DNA,只需设计1对引物,进行1次PCR反应,就直接获得了突变的表达载体。利用该方法成功地对拟南芥的丝氨酸/苏氨酸蛋白激酶编码基因ATPK64进行了定点突变。这种改进的DNA定点突变方法,具有简便、快捷、经济、成功率高的特点,是值得推广的PCR定点突变方法。

英文摘要：

Partial-complementary primers were designed to amplify full length of liner expression vector,after DpnⅠ enzyme digestion of the PCR products,the methylated template DNA was removed and the purified PCR product was transformed to E.coli competent cells.Circular plasmid DNA was obtained using the self-repairing system of E.coli and the correctly mutated gene was confirmed by sequencing.Thus the expression vector can be directly obtained using one pair of PCR primers,and one time PCR amplification reaction.The result indicated that site-directed mutagenesis of the gene encoding a serine/theronine protein kinase ATPK64 of Arabidopsis thaliana is performed successfully.This method saves the expense of purchasing commercialized kit,and is free from the steps of complicated PCR primer designment and ligation experiments after PCR reaction.Comparing with the traditional methods,this method has the advantage of being simple,rapid,low cost and cost and highly efficient.