基于靶标ALS基因突变的反枝苋对噻吩磺隆抗性差异的分子机理

Amaranthus retroflexus is a troublesome weed in autumn ripe crop fields, such as maize, soybean and cotton. Previous study showed many A. retroflexus populations with different resistant level to thifensulfuron-methyl was mainly conferred by ALS site mutation. However, the resistance differentiation mechanism of different ALS site mutation to thifensulfuron-methyl is still unclear. This study is Amaranthus retroflexus is a troublesome weed in autumn ripe crop fields, such as maize, soybean and cotton. Previous study showed many A. retroflexus populations with different resistant level to thifensulfuron-methyl was mainly conferred by ALS site mutation. However, the resistance differentiation mechanism of different ALS site mutation to thifensulfuron-methyl is still unclear. This study is conducted with 7 A. retroflexus biotypes with different ALS site mutation, to detect the resistant level and cross resistance of different ALS mutation to thifensulfuron-methyl from whole plant level, target enzyme activity and transformant using physiology, molecular biology and GM technology; to identify the interaction mechanism of ALS protein and thifensulfuron-methyl using homology modeling, size exclusion chromatography and crystal X-ray diffraction technology. The expected results are to clarify the resistance differentiation mechanism of different ALS site mutation to thifensulfuron-methyl, and to elucidate the genetic mechanism between diversity of target gene mutation and herbicide resistance in weeds. It can also provide theoretical basis for the new herbicide creation based on ALS and sustainable management of herbicide resistant weeds.

反枝苋是我国玉米、大豆和棉花等秋熟作物田的恶性杂草。前期研究发现多个反枝苋种群对主控除草剂噻吩磺隆具有不同水平的抗性，初步证实靶标ALS基因位点突变是产生抗性的主要原因，但不同位点突变对噻吩磺隆的抗性差异机理尚不明确。本项目基于前期获得的7个ALS不同位点突变的抗性反枝苋生物型，拟采用生理生化、分子生物学和转基因技术，从整株抗性、靶标酶活性和转化体抗性等多方面探究ALS不同位点突变对噻吩磺隆抗性水平和交互抗性谱的差异机理；并进一步采用同源建模、凝胶排阻色谱和X射线晶体衍射等技术研究ALS蛋白空间结构及其与噻吩磺隆的分子识别，揭示ALS蛋白与噻吩磺隆的互作机制。研究结果有望从多层次深入揭示ALS基因不同位点突变导致对噻吩磺隆抗性差异的分子机理，阐明靶标基因突变多样性与杂草抗药性产生的内在遗传机制，为基于ALS靶标的新农药创制和抗药性杂草的可持续治理提供重要依据。

抗噻吩磺隆反枝苋不断发生和扩散，给农业生产造成严重威胁。本项目以不同ALS位点突变的反枝苋为对象，研究不同ALS位点突变导致对噻吩磺隆抗性差异机理。.研究不同反枝苋生物型对噻吩磺隆抗性水平表明，7个抗性反枝苋生物型对噻吩磺隆均具有不同程度的抗药性。其中，Asp-376-Glu、Trp-574-Leu，Gly-654-Tyr突变的反枝苋生物型对噻吩磺隆表现出高抗性，相较敏感种群抗性倍数均在10倍以上；Ala-122-Thr、Ala-205-Val、Ser-653-Thr生物型对噻吩磺隆表现出低抗性，抗性水平在10倍以下。.研究不同反枝苋生物型对ALS抑制剂交互抗性谱表明:1）Asp-376-Glu，Trp-574-Leu和Gly-654-Tyr突变生物型对烟嘧磺隆的抗性水平最高，而Ser-653-Thr表现出低抗水平。2）7个ALS突变反枝苋生物型对IMI类除草剂咪唑乙烟酸均表现出高抗性。其中Trp-574-Leu反枝苋生物型对咪唑乙烟酸抗性最高，达40倍左右。3）测定了不同反枝苋生物型对TP类除草剂唑嘧磺草胺的抗药性，结果表明Asp-376-Glu，Trp-574-Leu和Gly-654-Tyr的反枝苋生物型表现出高抗性，而Ala-122-Thr、Ala-205-Val和Ser-653-Thr生物型表现为低水平抗性。4）测定了不同反枝苋生物型对SCT类除草剂氟唑磺隆的抗性水平。发现Asp-376-Glu和Trp-574-Leu对氟唑磺隆抗性最高，其次是Gly-654-Tyr和Ser-653-Thr，Ala-205-Val和Ala-122-Thr突变生物型表现为敏感。5）比较不同反枝苋生物型对PTB类除草剂双草醚的抗药性发现，Trp-574-Leu生物型表现出高抗性。Asp-376-Glu和Gly-654-Tyr表现出低抗性，而Ala-122-Thr表现为敏感。.通过体外重组过表达ALS基因，提取和纯化了抗性和敏感ALS蛋白。对抗性和敏感ALS蛋白与噻吩磺隆的亲和力研究发现，ALSr与噻吩磺隆的亲和力显著高于ALSs。通过晶体培养、纯化分别得到了敏感型（S）和574突变型（Trp-574-Leu）蛋白质晶体，为开展除草剂与靶标蛋白的互作提供了支撑。