小菜蛾对氯虫苯甲酰胺抗性相关lncRNA的功能分析

The diamondback moth (DBM), Plutella xylostella, is one of the most economically important cruciferous pest insects in the world. Chlorantraniliprole is a totally novel insecticide targeting at the ryanodine receptor (RyR) of insects. However, field populations of DBM have developed high resistance to this insecticide. Except for the point mutations identified in RyR, the overexpression of target gene and detoxifying enzymes are also proved to be closely correlated to the chlorantraniliprole resistance in DBM, but we know very little about their regulation mechanisms. The importance of long-noncoding RNA (lncRNA) in regulation of gene expression has become the new hotspot. In present study, the theory and techniques of epigenetics will be used to 1) identify the lncRNAs in DBM by using the RNA-seq and bioinformatics techniques, 2) Screen and identify the lncRNAs that possibly involved in chlrantraniliprole resistance in DBM and analyze their functions by RNAi and bioassays, and 3) Identify the DNA, proteins and miRNAs that interact with lncRNA and understand their mechanisms in chlorantraniliprole resistance regulation in DBM. The objective of this project is to identify the chlorantraniliprole resistance related lncRNAs in DBM and characterize their functions. The results of this study would be very helpful for the improve of insecticide toxicology theory and management of chlorantraniliprole resistant DBM populations in field.

小菜蛾是十字花科蔬菜的重要害虫。氯虫苯甲酰胺是以昆虫鱼尼丁受体为靶标的具有全新作用机制的杀虫剂，但目前小菜蛾已对其产生极高抗性。除靶标基因突变外，靶标基因及主要解毒酶基因过量表达也与其抗性密切相关，但对其调控机制缺乏研究。长链非编码RNA(lncRNA)在基因表达调控中起着重要作用。本项目拟：1）利用RNA-seq以及生物信息学分析的方法测定小菜蛾的lncRNA；2) 分析不同氯虫苯甲酰胺抗性水平小菜蛾种群中lncRNA的表达差异，明确与小菜蛾对氯虫苯甲酰胺抗性相关的lncRNA，通过RNAi及生物测定研究其功能；3）鉴定与lncRNA互作的DNA、蛋白质及miRNA，揭示lncRNA调控小菜蛾对氯虫苯甲酰胺抗性的途径及机制。通过上述研究，揭示参与小蛾对氯虫苯甲酰胺抗性的lncRNA调控网络。研究结果对于完善昆虫毒理学理论及有效治理氯虫苯甲酰胺抗性小菜蛾具有重要意义。

本项目围绕小菜蛾对氯虫苯甲酰胺抗性相关的lncRNA的鉴定及功能分析进行了系统研究。（1）在小菜蛾中共鉴定lncRNA 1309条，包括877条基因间lncRNAs、190条内含子lncRNAs、76条反义lncRNAs和166条正义重合lncRNAs；（2）在氯虫苯甲酰胺敏感品系和抗性品系间筛选出了一批差异表达的lncRNA，其中 22个lncRNAs与小菜蛾对氯虫苯甲酰胺抗性的关系最密切；（3）利用RNAi和体外表达代谢的方法，证明谷胱甘肽S-转移酶GSTu1与小菜蛾对氯虫苯甲酰胺的抗性相关；（4）进一步证明反义lnc-PxGSTu1-AS通过与GSTu1互补结合，维持了GSTu1的稳定性，进而参与调控小菜蛾对氯虫苯甲酰胺的抗性。通过上述研究，揭示了lncRNA通过调控解毒酶基因的表达介导小菜蛾对氯虫苯甲酰胺抗性的分子机制。研究结果为深入揭示昆虫非编码RNA功能的及其调控害虫抗药性的分子机制提供了重要参考，对于完善昆虫毒理学理论具有重要的理论意义。