基于易错PCR技术的α-淀粉酶基因的定向进化研究  

Directed evolution in vitro of α-amylase gene by error-prone PCR

作　　者：张建云[1] 谷立坤[1] 陈红歌[2] 

机构地区：[1]河南工程学院资源与环境工程系,河南郑州451191 [2]河南农业大学生命科学学院,河南郑州450002

出　　处：《中国酿造》2010年第2期94-97,共4页China Brewing

基　　金：河南省科技厅科技攻关项目(092102210212)

摘　　要：采用基因体外定向进化策略中易错PCR技术,用碱基类似物5-溴脱氧尿苷三磷酸(5-BrdUTP)部分取代脱氧胸苷三磷酸(dTTP),对野油菜黄单胞菌的α-淀粉酶基因进行了体外诱变。通过鉴别培养基进行筛选,得到5个具有高酶活的诱变基因,利用生物学软件DNAstar和出发基因序列进行比较,分析了突变位点和酶功能变化的相应关系。结果显示:基因诱变后酶活的改变主要是由淀粉酶基因空间结构的改变而引起的,而不是由启动子的变化引起的。Random mutagenesis on α-amylase gene in vitro was conducted based on error-prone PCR strategy, 5-BrdUTP partly replace dTTP when replication started. By using selective medium, we screened five mutated genes with higher enzyme activity. The obtained sequences were aligned with the the original gene by the biological software DNAstar. The results showed that the changes of enzyme activity after mutation mainly caused by the changes of structure of a-amylase gene, not caused by the changes of the promoter.

关 键 词：定向进化 碱基类似物 诱变 Α-淀粉酶基因 

分 类 号：Q556[生物学—生物化学]