抗磺酰脲类除草剂油菜不同突变体的抗性效应及机理研究

Weed infestation is a main biological disaster in rapeseed (Brassica napus L) production, and also is one of the important factors restricting the development of mechanized production and crop cultivation manners of lightened and simplified. Chemical herbicides are extremely effective tools for weed control. However, chemical control of weed for rapeseed in China is quite limited due to a shortage of commercialized herbicide-resistant rapeseed. Sulfonylurea (SU) herbicides have been widely used for controlling weeds because of their wide spectrums for weed control, low usage rates, and residual soil activity. Different SU herbicide-resistant mutants in Brassica napus were screened from M2 seeds mutagenized with ethyl methane sulfonate (EMS). The objective of this program is to determine the relative resistance levels, the mode of inheritances, and biochemical and molecular mechanisms of herbicide resistance in these mutants. Genes encoding acetohydroxyacid synthase (AHAS, the site of action for SU herbicides) are isolated from the mutants. Molecular mechanism for resistance to SU herbicides is revealed by the complementation experiments and prokaryotic expression. At last, molecular markers and detection procedure are developed on the base of DNA sequence. It aims to pyramiding SU herbicide-resistant genes to develop high herbicide-resistant rapeseed by molecular marker-assisted selection. Therefore, this research provides the theoretical base and technical support for breeding the SU herbicide-resistant cultivars in China.

农田杂草是油菜生产主要生物灾害，也是制约油菜机械化、轻简化栽培的重要因素之一。化除是控制田间草害的有效手段，但我国油菜化除面积非常有限，主要原因是缺少抗除草剂油菜品种。磺酰脲类（SU）除草剂具有高效低量、杀草谱广、土壤残留期短等特点，是应用范围最广的一类除草剂。然而，由于缺少实用的抗SU类除草剂油菜种质或基因，截止目前国内还没有培育出商业化的抗SU油菜。本项目以我们通过EMS诱变获得的抗SU类除草剂油菜不同突变体为材料，研究其抗性效应、遗传规律和生化机制；克隆除草剂靶标乙酰羟基酸合酶（AHAS）基因，明确不同突变发生的基因及位点，并对抗性基因进行原核表达和功能互补验证，解析不同抗性突变分子机理；建立抗性基因分子标记选择体系，聚合抗性基因创制高抗SU油菜新材料。通过以上研究为我国抗SU类除草剂油菜新品种选育提供理论基础和技术支撑。

我国油菜抗磺酰脲（SU）类除草剂种质（基因）资源相对缺乏，抗性品种选育进展缓慢。本课题利用前期获得的M342、DS3和5N等抗SU类除草剂突变体开展抗性评价、靶基因克隆与功能验证、标记开发和基因聚合研究，明确了抗SU油菜抗性产生的生化、分子和遗传机理。主要结果如下：①M342对SU类除草剂的抗性是除草剂推荐使用杀草浓度3-4倍，PN19是1-2倍，M196是2-3倍，DS3和5N是12-16倍。②抗性材料中的AHAS酶对除草剂的敏感性降低是抗性产生的生化基础，其中M342、PN19和M196是单基因突变位点抗性，M342抗性基因为BnAHAS3R（G1667T，W574L），PN19抗性基因为BnAHAS1-2R（G1676T，W574L），M196抗性基因为BnAHAS1-3R（C545T，P197L），拟南芥转基因验证了抗性基因功能。DS3和5N为双基因突变位点抗性，DS3含抗性基因BnAHAS1-3R和BnAHAS3R，5N含抗性基因BnAHAS1-2R和BnAHAS3R，抗性基因间具有协同作用。③除草剂处理可诱导AHAS基因表达量的上调，但突变体的上调水平高于野生型。④BnAHAS3定位在叶绿体中，酶最适反应pH值为7.0，最佳反应温度为37℃，酶学常数Km为6.55 mmol.L-1和Vmax为6.40μmol.mg-1.h-1。⑤开发抗除草剂基因功能标记3个。以上研究为我国抗SU油菜品种选育提供了技术支撑和重大应用价值的基因资源。目前M342、DS3和5N已被全国34家育种单位引种应用，这对解决我国油菜生产杂草防控难题，推动油菜产业发展具有重要意义。