易错PCR定向进化技术提高蒙古黄芪病程相关蛋白AmPR-10核酸酶活性的研究  

Improvement in Nuclease Activity of Astragalus membranaceus Pathogenesis-Related Protein-10(AmPR-10)by Error Prone PCR Directed Evolution

作　　者：王珏 吴娜 张育敏 闫海洋 胡丽丽 WANG Jue;WU Na;ZHANG Yumin;YAN Haiyang;HU Lili(Department of Basic Medicine,Shanxi University of Chinese Medicine,Jinzhong 030600,China)

机构地区：[1]山西中医药大学基础医学院,山西晋中030600

出　　处：《化学与生物工程》2022年第2期23-27,51,共6页Chemistry & Bioengineering

基　　金：山西省自然科学基金面上项目(201901D111339);山西省高等学校科技创新项目(2020L0407,2020L0420);山西中医药大学科技创新能力培育计划(2020PY-JC-06,2021PY-JC-06)。

摘　　要：以分子伴侣skp修饰的蒙古黄芪病程相关蛋白AmPR-10(Astragalus membranaceus pathogenesis-related protein-10)全基因为模板,通过易错PCR定向进化技术构建突变体文库,以酵母tRNA为底物建立高通量筛选方法,获得了核酸酶活性提高的AmPR-10突变体。经一轮定向进化后,突变体A4的核酸酶活性比野生型提高45%;测序结果显示,突变体A4基因序列在465位增加了1个碱基A,进而由移码突变引起了多肽序列的提前终止,使得突变体A4 C末端缺失了3个氨基酸;从空间上看,AmPR-10基因的突变减小了其C末端α螺旋对空腔的遮蔽作用,有利于目的蛋白与配体或底物的结合。该研究结果对AmPR-10核酸酶活性机制的探讨具有重要意义,为抗病抗逆黄芪植株的培育奠定了理论基础。The full gene of Astragalus membranaceus pathogenesis-related protein-10(AmPR-10)modified by molecular chaperone skp is adopted as a template,the mutant library is constructed via error prone PCR directed evolution,and the high-throughput screening method is established by using yeast tRNA as a substrate,so that we obtain the mutant AmPR-10 with increased nuclease activity.After a round of directed evolution,the nuclease activity of mutant A4 is 45%higher than that of the wild type.Sequencing results show that a base A is added at the site 465 of the mutant A4 gene,which leads to the premature termination of the polypeptide sequence due to frameshift mutation,resulting in the deletion of three amino acids at C-terminal of the mutant A4.Spatially,the mutation of AmPR-10 gene reduces the shielding effect of the C-terminalαhelix of AmPR-10 on the cavity,which is conducive to the binding of the target protein to the ligand or substrate.The study is of great significance to the investigation of the mechanism of AmPR-10 nuclease activity,and lays a theoretical foundation for the cultivation of disease and stress resistant Astragalus plants.

关 键 词：易错PCR 定向进化 AmPR-10 核酸酶活性 

分 类 号：Q51[生物学—生物化学] Q814