CRISPR/Cas9介导的斜纹夜蛾抗拟除虫菊酯候选基因功能研究

The common cutworm, Spodoptera litura (Fabricius), which is a polyphagous agricultural pest, caused serious damage to crops and had developed serious resistance to pyrethroid insecticides. However, the resistance mechanism is still not clear nowadays and needs to be further studied. CRISPR/Cas9 system is an efficient gene edit tool and has been used to study the insecticides resistance mechanism in several pests. In our previous study, eight resistance-related candidate genes (CYP3, CYP6, CYP13, CYP16, EST1, GST3, UGT5 and ABC6) had been identified from pyrethroid resistant and susceptible populations of S. litura by RNA-Sequence. This project aims at studying the function of these candidate genes. The coding sequence of these candidate genes will be cloned and analyzed via bio-informatics, then the relative expression level in different life stage and tissues will be detected, which is intended to complete the sequence information and expression characterization of the candidate genes. CRISPR/Cas9 system will be employed to edit the candidate genes. Bioassay will be conducted to detect the change of sensibility to pyrethroid insecticides on the off-springs of candidate gene knocked out strains, which is to evaluate the role of candidate genes in pyrethroid resistance. The results will facilitate to clarify the molecular mechanism of pyrethroid resistance in S. litura.

斜纹夜蛾是一种危害严重的杂食性农业害虫，对拟除虫菊酯类（简称菊酯类）杀虫剂抗性严重，而抗性机制尚不明确。CRISPR/Cas9系统作为一种高效的基因编辑技术，已应用在昆虫抗药性机制研究。在前期的研究中，申请人已通过转录组测序技术，从菊酯类高抗和敏感的斜纹夜蛾种群中筛选出CYP3、CYP6、CYP13、CYP16、EST1、GST3、UGT5和ABC6等8个抗性相关候选基因。本项目拟针对这些候选基因进行全长的克隆和生物信息学分析，并检测候选基因的时空表达水平，从而完善候选基因的序列信息和表达特征；利用CRISPR/Cas9系统编辑各候选基因，测定编辑成功的后代对菊酯类杀虫剂的敏感性变化，以甄别候选基因在抗性发展中的作用。研究结果将有助于阐明斜纹夜蛾对菊酯类杀虫剂产生抗性的分子机制。

为了阐明斜纹夜蛾对拟除虫菊酯和有机磷类杀虫剂的抗性机制，本研究采用CRISPR/Cas9技术对在抗性种群中显著过量表达的细胞色素P450基因进行了功能研究。通过合成并注射CYP304A1、CYP6B2-like、CYP6B5-like的sgRNA和Cas9蛋白，建立了一套能够成功用于斜纹夜蛾的基因编辑技术体系，并获得了CYP304A1成功敲除的纯合种群，生测结果表明CYP304A1的敲除能显著增加斜纹夜蛾对毒死蜱和高效氯氰菊酯的敏感性。此外，将在抗性种群中显著过量表达的GST基因进行异源表达，发现重组蛋白GSTE9可以显著代谢毒死蜱和功夫菊酯，GSTE12可以显著代谢氰戊菊酯、功夫菊酯、辛硫磷、毒死蜱，GSTS4和GSTO2只对功夫菊酯稍有代谢活性。RNAi结果表明GSTE9的沉默可以显著增加对毒死蜱和功夫菊酯的敏感性；GSTE12的沉默可以显著增加对氰戊菊酯、功夫菊酯和毒死蜱的敏感性。以上结果说明斜纹夜蛾对拟除虫菊酯和有机磷类杀虫剂的抗性由多家族多基因共同参与，可为斜纹夜蛾的抗性治理提供理论基础。