二化螟对氯虫苯甲酰胺抗性相关microRNA的鉴定与功能分析

Chilo suppressalis is one of the most important rice pests. At present, the control of C. suppressalis is primarily dependent on the application of diamides insecticides—chlorantraniliprole. C. suppressalis have developed resistance to chlorantraniliprole due to long-term use of single insecticide, which threatening the security of rice production. MicroRNAs are a group of small RNAs involved in various biological processes through negative regulation of mRNAs at post-transcriptional level. Some studies indicated that miRNAs could help pests developing resistance to insecticides by regulating the expression of their target genes. In this study, chlorantraniliprole-susceptible and -resistant C. suppressalis were used and following experiments were conducted for clarifying the mechanism of C. suppressalis resistant to chlorantraniliprole. 1) constructing the miRNA library and identify the species of miRNAs in C. suppressalis; 2) analyzing the differences between susceptible and resistant strains and screening the miRNAs probably involved in the resistance regulation; 3) up- and down-expressing the selected miRNAs in vivo, and then clarify the relation between miRNAs and resistance by analyzing the insect susceptibility to chlorantraniliprole; 4) predicting the target genes of miRNAs and constructing the relation between miRNAs and their target genes, and then analyzing the function of miRNAs related to resistance. The results of this study would be helpful for illuminating the molecular mechanism of resistance to diamides insecticides, and providing a theoretical basis for delaying the resistance development of C. suppressalis in field and developing a more effective C. suppressalis management strategy.

二化螟是水稻的重要害虫之一，近年来，它的防治主要依赖于氯虫苯甲酰胺等双酰胺类杀虫剂，单一药剂的长期使用致使二化螟产生抗性，给水稻的安全生产带来重大隐患。MicroRNA是一类通过基因转录后调控参与生命过程的小分子RNA，研究表明miRNA能够通过调控其靶基因表达使害虫产生抗药性。本项目以二化螟的敏感品系和氯虫苯甲酰胺抗性品系为材料，构建miRNA文库，鉴定其种类；比较品系之间miRNA的差异性，筛选参与抗性调控的miRNA；活体提高或抑制相关miRNA表达，测定二化螟对氯虫苯甲酰胺等双酰胺类药剂敏感度变化，初步明确差异表达显著的miRNA与抗性关系；预测差异表达显著的miRNA靶基因，构建miRNA与其靶基因之间的调控网络关系，明确其与抗性关系及调控机制。研究结果将有利于全面阐明二化螟对双酰胺类杀虫药剂抗性的分子机制，为延缓二化螟抗药性产生及制定合理有效的二化螟防治策略提供理论依据。

二化螟是水稻的重要害虫之一。近年来，二化螟的防治主要依赖于氯虫苯甲酰胺等双酰胺类杀虫剂，使得田间二化螟很快对其产生了高水平抗性。本研究以室内选育的二化螟对氯虫苯甲酰胺敏感和抗性品系以及田间抗性种群为试验材料，利用miRNA测序分析、定量PCR、显微注射改变二化螟体内miRNA表达量等方法，揭示了二化螟对氯虫苯甲酰胺产生抗性的分子机制。研究结果共发表相关论文9篇，其中SCI论文5篇，中文核心期刊论文4篇。培养硕士研究生2名。通过4年的研究，顺利完成了研究计划内容：1）明确了14种miRNA在二化螟对氯虫苯甲酰胺敏感和抗性品系中的表达量变化；2）筛选了11种二化螟对氯虫苯甲酰胺抗性相关的miRNA，为进一步研究miRNA调控的二化螟对氯虫苯甲酰胺抗性的分子机理奠定了坚实基础；3）揭示了2种miRNA：PC-3p-184144_5和bmo-miR-375-3p_R-2表达量的变化显著影响了其靶基因鱼尼丁受体基因CsRyR的表达，从而改变了二化螟对双酰胺类药剂氯虫苯甲酰胺和氟苯虫酰胺的敏感度。因此PC-3p-184144_5和bmo-miR-375-3p_R-2这2种miRNA表达量的降低导致其靶基因CsRyR表达量提高是二化螟对氯虫苯甲酰胺产生的重要机制之一。本项目研究结果为阐明其他害虫对双酰胺类杀虫剂抗性的分子机制，以及双酰胺类杀虫剂抗性害虫的科学治理提供了重要的理论基础。