稗乙酰乳酸合成酶206位氨基酸突变与五氟磺草胺抗性关系的研究

Rice is an important crop in China. Barnyardgrass (Echinochloa crus-galli) is a serious gramineous weed in paddy field in China. Penoxsulam is one of the most widely used herbicides in China to control annual gramineous weeds, such as barnyardgrass. However, its controlling efficiency has been decreasing with the contiuous application in rice fields. We have collected several barnyardgrass populations that are highly resistant to penoxsulam in Jiangsu and Anhui provinces. Mechanisms of target site resistance (mainly mutations of acetolactate synthase) have been clarified in these populations. Interestingly, no reported mutation of the ALS gene was found in resistant AXXZ-8 population. However, a novel mutation phenylalanine-206-leucine (Phe-206-Leu) of the ALS gene was found in AXXZ-8 population for the first time in weeds in nature. In order to reveal whether this mutation is related to penoxsulam resistance in barnyardgrass, we propose to (1) study the bioinformatics molecular modeling of ALS gene with Phe-206-Leu mutation, (2) express the ALS protein with Phe-206-Leu mutation in yeast , and (3) transfer ALS Phe-206-Leu mutation in Arabidopsis thaliana. The results of this study can reveal a novel target-site-mutation that confers resistance to ALS-inhibitors, and provide the key information to the management of barnyardgrass.

水稻是我国三大粮食作物之一，稗是我国稻田危害最严重的恶性杂草。五氟磺草胺是防除稗等杂草的最重要药剂，但近几年来已有研究发现我国长江中下游地区水稻田稗抗性严重。本实验室已在江苏、安徽等省发现多个对五氟磺草胺高抗的稗种群，其抗性机理主要是抗性种群靶标乙酰乳酸合成酶（Acetolactate synthase，ALS）发生氨基酸突变。但在抗性AXXZ-8种群中未发现已报道的ALS靶标突变，而是发现ALS基因206位苯丙氨酸突变成亮氨酸，这在自然界杂草中为首次发现。为明确这一突变是否导致了稗对五氟磺草胺的抗性，本项目拟通过生物信息学层面的ALS基因分子模拟、酵母蛋白表达、拟南芥的转基因等研究以明确ALS基因206位突变与五氟磺草胺抗性的关系。此研究结果可发现ALS基因新靶标突变，丰富杂草靶标抗性机理，还能为该抗性稗的治理提供有效的技术指导。

首先利用同源建模的方法得到高质量的稗ALS蛋白模型并与五氟磺草胺进行分子对接。从对接结果来看敏感型ALS蛋白的Phe-206与五氟磺草胺之间会形成π-π堆积效应；而当该位点变成Leu-206时，ALS与五氟磺草胺之间的π-π堆积效应消失。更直观的对接打分显示当206位氨基酸发生突变后，五氟磺草胺与稗ALS蛋白的对接打分下降了9.78%，说明206位氨基酸突变会使二者的亲和力下降。综合相互作用及对接打分来看，206位苯丙氨酸在五氟磺草胺与稗ALS蛋白的稳定结合过程中起到重要作用，而当该位点发生Phe-206-Leu突变后会导致稗ALS对五氟磺草胺敏感性下降进而导致稗对五氟磺草胺产生抗性。这些结果在理论上证明了206位氨基酸突变会导致稗对五氟磺草胺产生抗性。.借助昆虫细胞SF9进行ALS蛋白表达并完成纯化。Western-bolt检测结果显示在70 kDa附近有单一条带，说明突变及敏感重组蛋白特异性表达成功。蛋白纯化后的离体敏感性测定显示突变型ALS蛋白的催化活性比敏感型ALS蛋白的活性低，表明206位氨基酸突变会导致ALS自身的催化活性有一定程度的下降；而在多个五氟磺草胺剂量的处理下得到突变型及敏感型重组蛋白的IC50值分别为6.56和0.81 μM，表明突变型ALS蛋白相较于敏感型ALS蛋白对五氟磺草胺的敏感性下降了8倍左右。这些结果在蛋白水平证明了206位氨基酸突变会导致ALS对五氟磺草胺敏感性降低，从而导致稗对五氟磺草胺产生抗性。.采用沾花法将敏感型及突变型稗ALS基因分别转入拟南芥，筛选鉴定后进行敏感性验证，以野生型拟南芥为对照。培养皿法结果表明突变型拟南芥的抗性强于敏感型拟南芥，而敏感型拟南芥强于野生型拟南芥。这表明外源稗ALS基因的转入会提高拟南芥对五氟磺草胺的抗性，更重要的是突变型拟南芥比敏感型拟南芥表现出更强的五氟磺草胺抗性；通过土壤培养法进行多剂量下的整株生物测定，进一步得出突变型拟南芥对敏感型拟南芥产生4倍左右的抗性。这些结果在整株水平证明了206位氨基酸突变会导致五氟磺草胺抗性，新突变是稗AXXZ-8种群对五氟磺草胺产生抗性的靶标酶机理。.综合分子对接、重组蛋白敏感性测定以及拟南芥转基因的结果，可以确定206位氨基酸突变（Phe-206-Leu）能够导致稗对五氟磺草胺产生抗性，成功证明Phe-206是杂草中与ALS类除草剂抗性相关的第九个氨基酸位