利用DNA shuffling技术鉴定草甘膦N-乙酰转移酶抗性相关位点

To Identify Sites Related to Glyphosate-tolerance in Glyphosate N-acetyltransferase by DNA Shuffling Technology

为研究高抗草甘膦N-乙酰转移酶(glyphosate N-acetyltransferase,GAT)的草甘膦抗性相关位点,以高抗草甘膦的gat1基因和不具草甘膦抗性的gat2基因为出发基因,通过一轮双基因DNA shuffling,获得了抗性较野生型gat1下降的突变体14个:10个单位点突变,3个双位点突变,1个3位点突变。其中,MTGAT3(H41Q)、MTGAT5(S52P)、MTGAT6(I53M)、MTGAT7(F56L)、MTGAT8(R82G)、MTGAT11(I19T/P22L)和MTGAT14(Y70H/L98P/K119E)7个突变体的草甘膦抗性几乎丧失。本工作为深入研究GAT1蛋白分子结构与功能之间的关系奠定了基础。

To study on some sites related to glyphosate-tolerance in glyphosate N-acetyhransferase （GAT） with high glyphosate-tolerance, the high glyphosate-tolerant gatl gene and the gat2 gene nearly without glyphosate-tolerance were used to directed evolution by DNA shuffling. As a result, fourteen gatl mutants containing lower glyphosatetolerance than wild-type gatl were obtained by selection with different concentrations of glyphosate （5 mmol · L^-1 to 50 mmol · L^- 1 ）. Ten of them were single site mutated, three ones were double sites mutated, while one was three sites mutated. Among them, the glyphosate-tolerance of mutants MTGAT3 （ H41Q ）, MTGAT5 （ S52P ）, MTGAT6 （I53M） ,MTGAT7 （F56L） ,MTGAT8 （R82G） ,MTGAT11 （I19T/P22L） and MTGAT14（Y70H/L98P/K119E） were almost lost. This research work has laid a foundation for further studying the relationship between GAT1 protein structure and its function.