Nanchung基因R504Q突变参与褐飞虱对吡蚜酮的抗性

Pymetrozine (PM) is a recommended insecticide for the pest control of rice brown planthopper (BPH), which has developed moderate to high level of resistance according to recent resistance monitoring. However, the molecular mechanisms underlying the resistance remain elusive. PM was reported to target a Transient Receptor Potential (TRP) cation channel complex comprised of Nanchung (Nan) and Inactive (Iav), which are expressed in insect chordotonal stretch receptor cells. A field BPH population was collected and selected with PM in our laboratory. This strain (Nl-PMR) displayed high-level resistance (Resistance ratio = 243.3) to PM comparing to the susceptible strain (Nl-PMS). We have identified a point mutation (R504Q) located in the coding region of Nan from the Nl-PMR. Synergism tests indicated that metabolic resistance contributed little to this high level of resistance. As a result, we propose that this mutation might confer target-site resistance to PM. In this project, we will verified this hypothesis in behavioral, cellular and molecular levels on the basis of behavioural studies, immunohistochemistry, RNA interference, heterogeneous expression of transgenic fly, eukaryotic expression and calcium imaging methods. We believe this research will enrich the contents of the insecticide toxicology and provide valuable information for integrated resistance management of BPH.

吡蚜酮是防治褐飞虱的主推杀虫剂，室内监测结果表明褐飞虱田间种群已对其产生中到高水平抗性，但抗性机制尚不明确。吡蚜酮的分子靶标为昆虫弦音感受器上瞬时电位感受器家族中Nanchung和Inactive基因形成的蛋白复合物。我们以室内筛选得到对吡蚜酮高抗（抗性倍数为243.3倍）的褐飞虱品系为研究对象，比较抗、敏褐飞虱品系中Nanchung和Inactive基因序列，发现抗性品系Nanchung基因存在R504Q的点突变，且增效剂实验表明代谢抗性贡献不大，预示该突变可能与吡蚜酮高水平抗性有关。据此我们推测“Nanchung基因R504Q突变介导褐飞虱对吡蚜酮抗性”。本研究拟采用行为学测定、免疫组织化学、RNAi技术、转基因果蝇异源表达、细胞体外真核表达以及钙离子成像等实验技术，从行为、细胞和分子层面证明我们的假说。本项研究可丰富杀虫剂毒理学研究内容，为褐飞虱的抗性治理提供帮助。

吡蚜酮不仅抑制刺吸式口器害虫的取食行为，还能干扰昆虫的运动能力与生殖行为。鉴于吡蚜酮具有诸多独特的毒理机制，在褐飞虱对吡虫啉产生抗性后成为褐飞虱防控的当家品种。随着吡蚜酮的大规模使用，许多田间褐飞虱种群已经对吡蚜酮产生了不同水平的抗性，然而其抗性机制至今不详。首先，我们对靶标抗性机制进行了探究。通过作用靶标突变检测，我们发现了NlNan基因中存在一个氨基酸替换（NlNan-R504Q），电生理与转基因果蝇爬管结果均证实该突变位点并不介导褐飞虱对吡蚜酮的抗性，而仅是一个单核苷酸多态性。在转录组数据分析过程中，多个解毒酶基因（P450、ABC转运蛋白、溶质载体等）在抗吡蚜酮褐飞虱种群中高表达，表明解毒酶可能参与褐飞虱对吡蚜酮的抗性。随后，我们对褐飞虱敏感品系与吡蚜酮抗性品系进行了增效剂作用评估和解毒酶活性检测，结果表明胡椒基丁醚（PBO）对吡蚜酮抗性品系具有2.83倍的增效作用；吡蚜酮抗性品系的P450解毒酶活性也是敏感品系的1.7倍；定量PCR结果显示CYP6CS1和CYP301B1在吡蚜酮抗性品系中显著上调。当利用GAL4/UAS二元表达系统将CYP6CS1在黑腹果蝇中过表达后，其对吡蚜酮的敏感性显著下降；当利用RNAi技术沉默CYP6CS1的表达后，褐飞虱对吡蚜酮的抗性显著上升。本研究不仅证实CYP6CS1高表达赋予褐飞虱对吡蚜酮的抗性，还为褐飞虱的抗性治理提供了理论依据。