油菜乙酰乳酸合酶突变体S638N的酶学特性及其对ALS类除草剂的抗性

摘要

Acetolactate synthase (ALS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. ABnALS1R gene from herbicide-resistant mutant line M9 inB. napus, was previously isolated and demonstrated to be resistant to the imidazolinone (IMI) herbicides. This research was to reveal the differences of enzymatic characteristics and its resistance to ALS inhibitor herbicides between the mutant S638N and the wild-type enzyme. The BnALS1R gene was constructed and expressed inEscherichia coli along with the wild-type. The target recombinant proteins with the pre-dicted molecular weight (74 kD) were successively expressed inEscherichia coliand purified by SDS-PAGE. The enzymatic activity of the purified S638N and wild-type was then measured in enzyme reaction systems under different temperatures and pH values. Results showed that the S638N resembled the wild-type in their enzymatic activity, showing maximum activity at 37°C and pH 7.0, and no significant difference in theKm andVmax between the S638N and wild-type. The activation of the S638N by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined and generated similar results to that of the wild-type. The mutant enzyme was inactive when one of three cofactors was omitted. However, the S638N was more resistant to IMI herbicides than the wild-type in contrast to Su herbicides that inhibited the S638N as well as the wild-type. Therefore, the S638N has resistance spe-cific to IMI herbicides with unalteration of the enzymatic reaction characteristics.%在对油菜抗咪唑啉酮类除草剂基因BnALS1R克隆与功能验证基础上，为比较抗性基因编码的乙酰乳酸合酶突变体 S638N 酶学特性及其对 ALS 类除草剂抗性与野生型的差异，构建基因原核表达载体，在大肠杆菌中表达S638N和野生型的重组融合蛋白。SDS-PAGE和Western blot分析表明， S638N和野生型均能表达出约74 kD的特异性重组蛋白。纯化目的蛋白，在不同温度和pH条件下，测定S638N和野生型的酶活性。结果显示，温度和pH对突变酶活性的影响与野生型相同，表现为先升后降，在37℃、pH 7.0条件下催化活性均最高。同时，该突变酶的酶学动力学参数Km和Vmax与野生型没有显著差异，其对3个辅助因子的响应曲线也与野生型类似，缺少其中任何一个辅助因子均使突变酶 S638N基本都没有活性。然而，突变酶 S638N对 IMI类除草剂抗性显著高于野生型，而对 Su类除草剂敏感性和野生型相同。因此，突变酶S638N具有对IMI类除草剂的专一抗性，但未改变酶学反应特征。