小RNA病毒HaNV通过Dicer 2诱导棉铃虫抗杆状病毒HaNPV的分子机制研究

RNA interference (RNAi) is an important antiviral mechanism in insects in which Dicer 2 endoribonuclease plays vital roles. Our research team have recently discovered a new picorna-virus HaNV in Helicoverpa armigera which could decrease host susceptibility to the baculovirus pathogen Helicoverpa armigera nucleopolyhedrovirus (HaNPV). Hence, HaNV is one of the factors that can affect the pathogenicity and epidemics of HaNPV. Also, we showed that HaNV infection could up-regulate immune gene Dicer 2 which involves in the HaNV-induced resistance to HaNPV, however, the molecular mechanisms remain unclear. In the current project, we aim to study the mechanism of the picorna virus HaNV inducing Dicer 2-mediated resistance to baculovirus HaNPV in H. armigera. On the first step, we will construct the infectious cDNA clone of HaNV and recombinant eukaryotic expression vector containing each of the five open reading frames of HaNV in order to identify the specific genes that might stimulate the up-regulation of Dicer 2. Second step, we will study the function of Dicer 2 in HaNV-induced resistance to HaNPV by down-regulating (RNAi, gene editing) and up-regulating Dicer 2. These studies along with the obtained results will provide a basis for insect-virus interaction and understanding of the epidemics of baculovirus pathogen and open a new avenue for improving the efficiency of nucleopolyhedrovirus biopesticides in pest management.

RNA干扰（RNAi）是昆虫抵抗外源病毒感染的重要机制，Dicer 2是RNAi的关键因子。我们前期研究发现棉铃虫小RNA病毒HaNV可降低寄主对棉铃虫核型多角体病毒（HaNPV）的敏感性，是影响HaNPV致病力的因素之一。进一步研究发现Dicer 2在感染HaNV的棉铃虫个体中高表达并参与寄主对HaNPV的免疫，但其分子机制尚未阐明。本项目将围绕HaNV通过Dicer 2诱导棉铃虫抗HaNPV的分子机制开展研究。首先，构建HaNV侵染性克隆及病毒各编码基因真核表达载体并转染棉铃虫细胞系，探究诱导Dicer 2高表达的HaNV病毒关键基因。其次，通过RNAi、基因编辑、基因回补等确认Dicer 2在HaNV诱导棉铃虫抗HaNPV中的功能。研究内容可为探究昆虫-病毒互作机制及杆状病毒田间流行规律提供理论基础，对病毒杀虫剂的研发及应用也具有重要的实践价值。

通过室内筛选，构建了未感染小RNA病毒HaNV、浓核病毒HaDV2及核型多角体病毒HaNPV等已知病毒的棉铃虫品系（HaNV-），并接种获得了感染HaNV的棉铃虫品系（HaNV+）。完成了HaNV编码5个开放阅读框（ORF）基因真核表达载体的构建，并进行细胞转染。研究表明HaNV感染棉铃虫48h后导致寄主155个基因下调表达，318个基因上调表达，免疫基因如Dicer 2、Repat 35、C型凝集素（HaCTL5）等分别显著上调表达1.9、2.6及7.5倍。共感染HaNV及HaNPV的棉铃虫HaCTL5、Dicer 2及Repat 35基因表达量也显著升高。通过RNAi降低以上三种目标基因表达量并接种HaNPV，96h后HaNPV病毒滴度显著升高。经细胞转染超表达Dicer 2基因后，显著抑制了AcMNPV病毒复制。通过分析来源于未感染和感染HaNV棉铃虫的小RNA文库的高通量测序数据，鉴定了10个上调的miRNA和11个下调的miRNA，其中miR-2a和miR-276可能参与棉铃虫被HaNV感染后的免疫应答。以上结果均表明Dicer2等以上三种关键基因在棉铃虫免疫HaNPV中发挥重要作用。.于黏虫等多种夜蛾科害虫体内发现了与HaNV亲缘关系较近的小RNA病毒。在非洲粘虫体内亦发现3种partiti-like新病毒，该病毒可在非洲粘虫、草地贪夜蛾和棉贪夜蛾中通过显微注射水平传播和母系垂直传播。该病毒与非洲黏虫寄主为有条件互利共生关系，可提高对NPV的抗性，但对草地贪夜蛾和棉贪夜蛾有害。另研究发现新浓核病毒HaDV2通过可提高棉铃虫对苏云金芽孢杆菌(Bt)的抗性，Bt作物的种植作为选择压之一显著提高了田间浓核病毒的感染率。以上几种病毒均可通过调控免疫等相关通路基因来提高寄主对Bt和NPV的抗性水平。.以上研究成果解析了棉铃虫与小RNA病毒的互作关系，表明了自然生态系统中物种关系的复杂性，同时初步揭示了HaNV通过诱导免疫基因介导对NPV抗性的机制。研究成果在发展害虫防治的新理论和新方法中具有重要的科学意义。