二化螟对氯虫苯甲酰胺抗性相关长链非编码RNA的鉴定和功能研究

The rice stem borer, Chilo suppressalis (Walker) is one of the most serious rice pests. The pest has had developed severe resistance against chlorantraniliprole which is the main insecticide in chemical control. In order to prolong the life time of chlorantraniliprole, the mechanism of resistance formation from the angle of long non-coding RNAs (lncRNA) will be studied in this project. The laboratory C.suppressalis strain was selected, showing high resistance to chlorantraniliprole. Resistance tightly involved protein coding genes were obtained. In the present proposal, the important lncRNAs associated with the chlorantraniliprole resistance in C. suppressalis will be found by using RNA-seq, and lncRNA-mRNA pairs will be obtained by co-expression analysis. The lncRNA-mRNA-miRNA network will be discoveryed through target prediction of miRNAs. CRISPR/Cas9 technology is used to construct the mutant lines of important lncRNAs. The mutant lines will be treated by chlorantraniliprole, the mortality, the RNA and protein expression of lncRNA and its target genes will be tested. The research results will provide evidences of molecular mechanisms for chlorantraniliprole resistance, which will be useful for prolonging the use cycle of chlorantraniliprole and improving the effect of chemical control.

二化螟是水稻重要害虫，目前已对防治该虫的当家品种之一的氯虫苯甲酰胺产生严重抗性。本项目拟从长链非编码RNA (lncRNA)角度开展研究，进一步阐释抗性形成机理，针对性地设计抗性治理措施，从而有效地缓解抗性的发展，达到延长使用寿命的目的。本课题组已筛选获得氯虫苯甲酰胺抗性品系及抗药性相关基因，拟进一步采用RNA-Seq，发掘二化螟中与氯虫苯甲酰胺抗性相关的lncRNA，采用共表达分析得到lncRNA-mRNA作用对；通过靶标预测，得到lncRNA-miRNA-mRNA互作网络。利用CRISPR/Cas9技术构建重要lncRNA的突变系，检测受到氯虫苯甲酰胺胁迫后突变品系的死亡率，并从核酸、蛋白水平检测lncRNA及其靶标基因的表达。本项目的实施可阐明lncRNA在二化螟对氯虫苯甲酰胺抗性形成中的作用，有助于拓宽对害虫抗药性机理的认识，为延长氯虫苯甲酰胺的使用周期、提高防治效果提供理论基础。

二化螟是危害水稻的重要害虫，目前已对防治该虫的当家品种之一的氯虫苯甲酰胺产生严重抗性。本项目拟从长链非编码RNA (lncRNA)角度开展研究，进一步阐释抗性形成机理，针对性地设计抗性治理措施，从而有效地缓解抗性的发展，达到延长使用寿命的目的。本课题组结合二化螟基因组，利用RNA-Seq技术，从链特异性文库中获得40,573个转录本，进一步鉴定得到3,470个lncRNA转录本。在氯虫苯甲酰胺抗、感品系中分别获得335和297个差异表达的lncRNA；RT-PCR验证了其中的20个基因间区lncRNA（lincRNA），这些lincRNA的差异表达倍数（log2FC）排名在前40。qPCR验证它们的表达量情况，结果显示，MSTRG.25315.3, 25316.8和7482.1这3个lincRNA在抗性品系中差异高表达，MSTRG.3932.19, 3932.15, 17788.5, 29805.1, 和22483.6这5个lincRNA在敏感品系中差异高表达。通过RNAi方法敲低MSTRG.7482.1的mRNA水平后，二化螟在氯虫苯甲酰胺胁迫下死亡率上升，表明该lincRNA与二化螟对氯虫苯甲酰胺抗性机制形成有关。另一方面，克隆了lincRNA MSTRG.25316.8，6875.40和25315.3的全长转录本，它们的最终长度分别为12,596, 11,800和6,795 bp。基于此，募集到了5个二化螟miRNA，即miRNA-236、259、158、243和091；进一步，收集公共数据库中含有3`UTR的ABC，P450和Esterase基因序列，使用前述5个miRNA进行靶标预测，最终得到了ceRNA网络。这将为进一步验证（体外和体内）“lncRNA-miRNA-mRNA”提供基础。