重叠延伸PCR技术在单碱基定点突变中的作用

氨基酸通透酶(amino acid permease,AAPs)是一种跨膜转运蛋白,广泛参与植物体内氨基酸的吸收与转运。本试验通过重叠延伸PCR技术对氨基酸通透酶基因进行单碱基定点突变,以便为今后研究植物对外源氮的吸收与利用率奠定基础。本研究根据Tair数据库中拟南芥AtAAP1基因序列,利用Primer Premier 5.0软件设计含有1个突变位点的2对互补的定点突变引物和1对带有pCAMBIA3301-GFP载体的多克隆位点的接头引物,以野生型拟南芥叶片的cDNA为模板,进行3次PCR扩增,将获得的突变基因片段连接到pCAMBIA3301-GFP载体上,转化DH5α感受态细胞。将筛选的阳性菌株送至公司测序,测序结果表明拟南芥AtAAP1基因的第908位碱基A突变为T,实现了单碱基定点突变,这与预计结果相同,说明成功依靠重叠延伸PCR技术做到目的基因的定点突变。该技术能方便快速地获得定点突变序列,为进一步研究AtAAP1基因的功能奠定了基础。

Effect of Overlapping Extension PCR on Single-base Site-directed Mutation

Amino acid permease(AAPs)is a transmembrane transport protein,which is widely involved in the absorption and transport of amino acids in plants.In this study,the single-base site-directed mutagenesis of the amino acid permease gene was carried out by overlapping extension PCR,so as to lay a foundation for the future study of the absorption and utilization of exogenous nitrogen in plants.Based on the AtAAP1 gene sequence of Arabidopsis thaliana in the Tair database,two pairs of complementary site-directed mutagenesis primers containing one mutation site and one pair of linker primers with multiple sites of pCAMBIA3301-GFP vector were designed using Primer Premier 5.0 software.Using the cDNA of the leaves of wild-type Arabidopsis thaliana as the template,PCR amplification was performed three times.The obtained mutant gene fragment was cloned into the pCAMBIA-3301-GFP vector,and then was transformed into DH5αcompetent cells.The screened positive strain was sent to the company for sequencing,and the sequencing results showed that the 908th base A of AtAAP1 gene in Arabidopsis thaliana was mutated to T,achieving single-base site-directed mutagenesis,which was consistent with the expected results,indicating that the site-directed mutagenesis of the target gene was successfully achieved by using overlapping extension PCR technology.This technique can obtain site-directed mutagenesis sequence conveniently and quickly,which lays a foundation for further research on the function of AtAAP1 gene.