利用DNA shuffling技术鉴定草甘膦N-乙酰转移酶抗性相关位点

›› 2007, Vol. 9 ›› Issue (5): 110-114.

利用DNA shuffling技术鉴定草甘膦N-乙酰转移酶抗性相关位点

金丹[1,2] 陈明[3] 马瑞强[4] 杨志荣[1] 陈建[5]

[1]四川大学生命科学学院,教育部生物资源与环境重点实验室,成都610064 [2]西南大学生物技术中心,重庆400716 [3]中国农业科学院生物技术研究所,北京100081 [4]中国农业大学生物学院,北京100094 [5]第三军医大学高原军事医学系病理生理教研室,重庆400038

收稿日期:2007-04-23 修回日期:2007-09-28 出版日期:2007-10-15 发布日期:2009-10-19
通讯作者: 陈建，讲师，博士。Tel：023-68752340—8204；E—mail：jchenone @hotmail．con。杨志荣，教授，博士，研究方向为微生物遗传学。
作者简介:金丹|博士研究生|研究方向为微生物遗传学
基金资助:
国家自然科学基金项目“苯胺双加氧酶诱导型启动子和调控蛋白互作的分子机理”（30470047）资助.

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

JIN Dan, CHEN Ming, MA Rui-qiang, YANG Zhi-rong| CHEN Jian

1. College of Life Sciences, Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, |Sichuan University, Chengdu 610064 |2. Biotechnology Research Center, Southwest University, Chongqing 400716 |3. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081|4. College of Biology Science |China Agricultural University, Beijing 100094;5. Department of Patho-physiology and Institute of High Altitude Medicine, |Third Military Medical University, Chongqing 400038, China

Received:2007-04-23 Revised:2007-09-28 Online:2007-10-15 Published:2009-10-19

摘要/Abstract

摘要：

为研究高抗草甘膦N-乙酰转移酶（glyphosateN—acetyhransferase，GAT）的草甘膦抗性相关位点，以高抗草甘膦的gatl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DNAshuffling，获得了抗性较野生型gatl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（$52P）、MTGATT6（153M）、MTGAT7（F56L）、MTGAT8（R82G）、MTGAT11（119T／1722L）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。

关键词: 草甘膦草甘膦N-乙酰转移酶 DNA shuffling 点突变

Abstract:

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 glyyphosate-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 andd its function.

Key words: glyphosate, glyphosate N-acetyltransferase , DNA shuffling , site mutant

中图分类号:

Q555.3 S188

金丹[1,2] 陈明[3] 马瑞强[4] 杨志荣[1] 陈建[5]. 利用DNA shuffling技术鉴定草甘膦N-乙酰转移酶抗性相关位点[J]. , 2007, 9(5): 110-114.

JIN Dan, CHEN Ming, MA Rui-qiang, YANG Zhi-rong| CHEN Jian. To Identify Sites Related to Glyphosate-tolerance in Glyphosate |N-acetyltransferase by DNA Shuffling Technology[J]. , 2007, 9(5): 110-114.