菵草抗精噁唑禾草灵相关代谢酶基因的挖掘及其机理解析

Wheat is an important crop in China. Beckmannia syzigachne is a serious gramineous weed in wheat fields in China. Fenoxaprop-P-ethyl is one of the most widely used aryloxyphenoxypropionate herbicides (AOPPs) in China to control annual gramineous weeds in wheat fields. However, its controlling efficiency has been decreasing with the contiuous application in fields. We collected several B. syzigachne populations with high resistance to fenoxaprop-P-ethyl. Mechanisms of target site resistance have been clarified, while those of metabolic resistance have been confirmed in these populations and are still unclear. In order to reveal the effects and mechanisms about eight metabolizing enzyme families (cytochrome P450s, esterases, hydrolases, oxidases, peroxidases, glutathione-S-transferases, glycosyltransferases and transporter proteins) in fenoxaprop-P-ethyl-resistant B. syzigachne, we propose to (1) build the gene bank of the metabolizing enzyme families, by isolating the genes of the eight metabolizing enzyme families with transcriptome sequencing and de novo assembly, combined with RT-PCR and RACE; (2) illuminate the expression patterns of these genes in response to fenoxaprop-P-ethyl in fenoxaprop-P-ethyl-resistant B. syzigachne populations employing Real-time PCR; (3) identity the differences in genetic structures of these genes between the sensitive and resistant B. syzigachne, by cloning and comparing relative gene sequences. Then, the metabolizing enzyme genes related to fenoxaprop-P-ethyl-resistance will be confirmed and metabolic resistance mechanisms will be analyzed. The results of this project will help us understand the mechanism of metabolic herbicide-resistance, provide key information to the management of weeds with metabolic resistance, and thus are significant for increasing agricultural production and farmer incomes.

小麦是我国重要粮食作物，菵草是我国小麦田恶性杂草，精噁唑禾草灵是防除小麦田禾本科杂草的重要药剂，目前抗性严重。本课题组已在多地发现抗精噁唑禾草灵菵草种群，抗性水平达到高抗，靶标抗性机理已明确，且存在代谢抗性。为了明确细胞色素P450氧化酶、氧化酶、酯酶、水解酶、过氧化氢酶、谷胱甘肽硫转移酶、糖基转移酶及转运蛋白八大代谢酶家族在抗性中的作用及机理，本课题拟通过转录组测序结合RT-PCR和RACE技术，分离菵草八大代谢酶家族的基因，建立菵草代谢酶家族基因库，研究基因库中基因的mRNA表达水平，明确抗性菵草对精噁唑禾草灵胁迫响应的表达模式，克隆基因库中基因，比对基因序列，确定抗性与敏感菵草基因结构差异，挖掘出与抗性相关的代谢酶基因，解析菵草抗精噁唑禾草灵的代谢酶分子机理。本研究既能补充杂草代谢抗性理论，又能指导抗性菵草的科学防除，延缓抗性发展，有利于实现农业增产、农民增收的根本目的。

截止2019年12月，本项目组承担的国家自然科学资金项目“菵草抗精噁唑禾草灵相关代谢酶基因的挖掘及其机理解析”已全部顺利完成，实现了研究目标。1、成功建立菵草的代谢酶家族基因库。结合转录组测序的分析结果及与已知植物中的代谢酶基因的比对结果，菵草中所有可能的代谢酶基因均被鉴定出来。再根据转录组测序获得的每个代谢酶基因的EST序列，结合RACE和RT-PCR技术，分别得到每个代谢酶基因的序列全长，最终成功建立菵草的代谢酶家族基因库。该代谢酶家族基因库总计有333个菵草代谢酶基因，涉及到杂草代谢精噁唑禾草灵三个阶段的8大代谢酶家族。2、菵草代谢酶家族基因的基因表达模式是菵草抗精噁唑禾草灵的重要原因。对菵草代谢酶家族基因库中的333个基因的表达模式进行了分析，发现333个基因中共有15个基因在三个抗性种群中的表达水平显著高于敏感种群中的表达，还有5个基因的表达水平则是在抗性种群中显著低于敏感种群中，其余基因的表达水平则在抗敏种群之间无显著性差异。在15个上调表达的基因中，有4个基因是除草剂诱导后才上调表达的，包括Esterase PIR7B，GST-U1，GST-U6和UDP-glycosyltransferase 73C1。3、菵草代谢酶家族基因的结构变化在抗性产生中发挥了部分作用。分别从三个抗性种群和敏感菵草中扩增出其代谢酶家族基因库中的基因，并测序进行比对。在阶段I中，发现三个基因CYP87A3，Peroxidase 1，Esterase PIR7B有氨基酸变化。在阶段II中，2个基因GST-U6，UDP-glycosyltransferase 85A2在抗敏种群间有氨基酸变化。此外，GST-U1基因中，发现在258位碱基由C变化为T，但并未导致氨基酸变化。4、菵草代谢抗性相关基因的进一步验证。成功分离并得到了三对分别具有相同遗传背景的抗性种群与敏感种群。以敏感种群作为对照，用这些种群对本节发现的代谢抗性相关基因进行初步验证。结果发现：与4个敏感种群相比，3个抗性种群中15个上调表达的代谢酶基因仍然上调表达，5个下调表达的代谢酶基因在抗性种群中也依然表达较低；而抗性与敏感菵草种群之间也依然存在6个导致氨基酸变化的突变。