microRNA参与稗草对二氯喹啉酸和五氟磺草胺产生抗性的调控机理

Barnyardgrass (Echinochloa crus-galli (L.) Beauv) with herbicide resistance threatens the rice yield and quality seriously in China. Previous studies indicated that the resistance of barnyardgrass in paddy fields tended to evolve from single-resistance to multi-resistance. However, the molecular regulation mechanism of barnyardgrass resistance evolution remains unclear. In this study, four barnyardgrass biotypes (multi-resistant biotype, single-resistant biotypes and susceptible biotype) with similar ecological and genetic backgrounds were segregated from the same barnyardgrass line which develop resistance to both quinclorac and penoxsulam. On this basis, microRNA-mRNA combined analysis and microRNA-RNA targeting relationship validation will be carried out to screen the key microRNAs and target genes regulating barnyardgrass evolution from single- to multi-resistance. Gene expression characteristics analysis and transgenic function verification will be used to excavate the signaling pathways and functional characteristics of microRNAs regulating barnyardgrass resistance to quinclorac and penoxsulam. The results will be helpful to reveal the new mechanism of barnyardgrass evolution from single-resistance to multi-resistance in paddy fields. It will aslo provide scientific theoretical basis for making a new strategy of green prevention and control of weeds in crop fields and has broad application prospects.

抗药性稗草种群严重影响我国水稻的产量和品质。前期研究发现，我国稻田抗性稗草呈现单药抗性向多抗性发展的趋势，但是对于稗草由单抗向多抗性进化的分子调控机制，现阶段知之甚少。申请者拟以来自同一株兼抗二氯喹啉酸和五氟磺草胺的多抗性稗草分离获得并可稳定遗传的稗草多抗性（兼抗二氯喹啉酸和五氟磺草胺）、单抗性（仅抗二氯喹啉酸或五氟磺草胺）和敏感生物型为供试材料。采用组学分析和分子生物学等多学科的研究手段，通过microRNA-mRNA联合分析、microRNA-mRNA靶向关系验证，寻找出调控稗草由单抗向多抗进化的关键microRNA与靶基因；通过供试材料间基因表达特征差异性分析、转基因功能验证，最终挖掘出microRNA参与调控稗草抗二氯喹啉酸和五氟磺草胺的信号通路及其功能特征。研究结果有助于发现稻田稗草种群由单抗向多抗性进化的新机理，为制定我国作物田杂草的绿色防控新策略提供理论依据，应用前景广阔。

稗草（Echinochloa crus-galli）作为稻田最主要的恶性杂草之一，严重影响水稻的产量和品质。目前，我国稻田抗性稗草呈现单药抗性向多抗性发展的趋势，但是对于稗草对除草剂产生多抗性的分子调控机制，现阶段知之甚少。本研究以生态遗传背景一致的稗草多抗性和敏感生物型为研究材料，首先通过剂量-反应曲线测定稗草多抗性生物型对二氯喹啉酸和五氟磺草胺均产生了高水平的抗药性，抗性指数分别为164.25和13.60倍；并且其对水稻田常用除草剂氰氟草酯、噁唑酰草胺、双草醚和氯氟吡啶酯均产生了不同程度的抗药性，尤其对氰氟草酯产生了10.0倍的高水平抗药性，具有很高的抗性风险。其次明确稗草多抗性生物型对五氟磺草胺的抗性机制为靶标基因ALS 发生Trp-574-Leu突变引起的靶标抗性机制，并且供试稗草对二氯喹啉酸的抗药性与其对二氯喹啉酸的吸收转运无关。进一步利用液相色谱-串联质谱联用（LC-MS/MS）方法检测多抗性和敏感稗草生物型间氰氟草酯及其代谢物氰氟草酸含量的差异性变化。结果表明，药剂处理后24 h，抗性生物型中活性代谢物氰氟草酯含量比敏感生物型降低60%，说明细胞色素P450介导的代谢酶活性增强导致稗草对氰氟草酯产生了代谢抗性。MicroRNA和mRNA联合分析明确了多抗性稗草的microRNA及其靶基因代谢通路主要集中在类黄酮和类胡萝卜素等次生代谢物的生物合成途径。通过代谢途径响应分析和荧光定量测定关键基因表达量，阐明miR1848-x通过靶向负调控稗草中细胞色素P450酶CYP75B3的表达提高稗草对除草剂的抗药性，并获得转稗草细胞色素P450基因CYP75B3拟南芥T2代株系。研究结果为深入揭示细胞色素P450介导的稗草多抗性调控机制提供了新思路，为挖掘农田杂草绿色防控的新途径提供理论依据。