牛繁缕抗苯磺隆相关代谢酶基因的发掘及功能分析

Water chickweed(Myosoton aquaticum L.) is one of the main problematic weeds infesting wheat fields, especially regions following a wheat-rice rotation in China. The development of herbicide resistance in water chickweed is a serious threat for wheat yield. Previous study indicated that P450s and/or GSTs mediated enhenced tribenuron-methyl metabolism is a powerful mechanism for herbicide resistance in water chickweed. However, P450s and GSTs are two enzyme superfamilies, which members confer herbicide resistance in water chickweed remain unknown. In previous work, we have obtained the sensitive biotype and the resistant biotype which has evolved metabolism resistance to tribenuron-methyl. With these valuable materials, the RNA-seq will be utilized to get the different transcribed genes between resistant and susceptible biotypes. The expression of different transcribed genes that relevant to herbicide metabolism will be analyzed by qRT-PCR to screen key genes involved in metabolism resistance, and to reveal the contribution of tribenuron-methy induction to the expression of key genes. Then, transgenic technology will be used to verify the function of key genes to confirm the resistant gene and characterize the resistance patterns to different herbicides. This sutdy will reveal the resistance genes associated with metabolic resistance and be helpful to preliminarily clarify the molecular basis for metabolism resistance to tribenuron-methyl in water chickweed. The results have important theoretical and practical significance for the monitoring and controlling of herbicide-resistant water chickweed. In addition, the resistant genes obtained in this study will lay the foundation for the future in-depth studies on resistance evolution and making new strategy for weed management.

牛繁缕是我国麦田常见危害杂草，稻麦轮作田发生尤为严重，其对除草剂抗性的发展严重威胁小麦生产安全。前期研究发现代谢酶P450s、GSTs活性增强是牛繁缕对苯磺隆产生抗性的重要原因之一，但P450s、GSTs酶系哪些家族成员赋予牛繁缕抗药性尚不明确。项目拟以苯磺隆代谢抗性生物型和敏感生物型牛繁缕为材料，首先通过转录组测序和基因信息分析筛选抗性和敏感牛繁缕间差异表达的代谢酶基因，以发掘潜在抗性基因；经qRT-PCR分析潜在抗性基因在除草剂胁迫前后不同时期的表达量差异，以确定关键基因并揭示其表达量与苯磺隆诱导的关联性；最后利用转基因技术验证分析关键基因功能，以明确代谢抗性基因及其导致的抗性模式。研究结果可发掘具有重要应用价值的抗性基因，初步探索牛繁缕对苯磺隆代谢抗性的分子机理，对牛繁缕的抗性监测及治理具有重要理论和实践意义。发掘的抗性基因可为今后深入研究抗性遗传，制定农田杂草绿色防控策略奠定基础。

杂草抗药性严重威胁杂草可持续治理及作物生产安全，代谢抗性是一类重要的杂草抗药性机理，但目前对其分子机制仍知之甚少。本项目以对苯磺隆存在明显代谢抗性表型的HR、MR牛繁缕种群为材料，利用 RNA-Seq 技术筛选了与苯磺隆代谢抗性相关的差异基因。通过基因注释、表达差异基因富集分析、代谢通路分析等生物信息学分析，确定了 67 个可能与苯磺隆代谢相关且存在显著表达差异的基因，进一步通过 qRT-PCR对 67个差异表达基因在 RNA-Seq测序样品以及 HR/MR种群的平行样品中的表达量均进行了验证，筛选到在抗性植株中均持续高表达的基因23个：8条CYP基因、2 条GST 基因、3条GT 基因、7 条ABC transporter基因、1条POD基因以及1条氧化酶基因、1条水解酶基因，标记为潜在代谢抗性基因。通过转基因拟南芥对HR植株c36251_g1（注释到CYP86A206）、c46086_g1（注释到GST-DHAR2）和c40142_g1（注释到Peroxidase 70）进行了基因功能验证。转基因纯合拟南芥植株对除草剂的抗性表型鉴定结果表明，在苯磺隆0.5μM浓度下，转CYP86A206及GST-DHAR2拟南芥与WT相比表现出了较强的苯磺隆耐受性；同时，转CYP86A206基因植株对0.01μM浓度的双氟磺草胺表现出一定程度的耐药性，而对嘧草硫醚、氟唑磺隆、啶磺草胺、吡氟酰草胺未表现出明显耐药性。本项目初步明确了牛繁缕对苯磺隆代谢抗性的分子机理，对牛繁缕的抗性监测及治理具有重要理论和实践意义。发掘的抗性基因可为今后深入研究抗性遗传，制定农田杂草绿色防控策略奠定基础。