基于中肠miRNA-mRNA双重表达解析小菜蛾抗Bt的分子调控机制

Bacillus thuringiensis (Bt) is one of the most successful applications of insecticide. Insufficient understanding of the resistance mechanism of Bt seriously restricts its resistance resistance management and the further exploitation and application of its novel insecticidal proteins, moreover, it also seriously threatens the future utilization of Bt transgenic crops. Our previous study showed that the alteration of multiple receptors led to the Bt resistance in the diamondback moth (Plutella xylostella), which were all regulated by MAPK pathway. This foundation aims to further explore and clarify the genetic regulatory network and molecular mechanism of the alteration of multiple receptors regulated by MAPK pathway by deep transcriptome and miRNome sequencing analysis in the larval midgut of a near-isogenic P.xylostella strain resistant to Cry1Ac toxin and the susceptible strain. The miRNA-mRNA expression regulation network were constructed by miRNA-mRNA integrated analysis. Then the real time-quantitative PCR for miRNA and mRNA, genetic linkage analysis and gain-of-function loss-of-function analysis of miRNA will be utilized comprehensively for evaluating the role of key miRNA and target genes in the regulation mechanism of the resistance to Cry1Ac in P. xylostella, which will clarify the molecular regulation cascade network and interaction between the key miRNA and target genes, and finally reveal the Bt resistance mechanism regulated by miRNA. The results of this study are of great theoretical and practical significance for further exploitation and application of novel Bt insecticidal proteins.

苏云金芽孢杆菌（Bt）是应用最为成功的生物杀虫剂，但对其抗性机制认识的不足，严重制约着Bt杀虫蛋白的进一步开发与应用，并威胁着转Bt基因作物的使用寿命。前期研究发现，小菜蛾对Bt Cry1Ac的抗性由多受体基因变化所致，由其上游的丝裂原活化蛋白激酶（MAPK）途径调控。本项目拟在此基础上，以抗Cry1Ac近等基因系小菜蛾幼虫中肠为研究材料，开展miRNA和mRNA高深度联合测序，通过miRNA-mRNA整合分析，构建抗Bt小菜蛾中肠差异表达的miRNA-mRNA互作调控网络；利用实时荧光定量PCR、遗传连锁分析以及miRNA过量/沉默表达实验，研究评价关键miRNA及靶基因对小菜蛾抗Cry1Ac的贡献，阐明MAPK途径控制多受体基因变化的遗传调控网络，从而揭示小菜蛾对Cry1Ac抗性的miRNA调控机制。研究结果对Bt杀虫蛋白的开发与可持续应用具有重要的理论和实践意义。

苏云金芽孢杆菌（Bt）是应用最为成功的生物杀虫剂，但对其抗性机制认识的不足，严重制约着Bt杀虫蛋白的进一步开发与应用，并威胁着转Bt基因作物的使用寿命。前期研究发现，小菜蛾对Bt Cry1Ac的抗性由多受体基因变化所致。本项目在此基础上，以抗Cry1Ac近等基因系小菜蛾幼虫中肠为研究材料，开展miRNA和mRNA高深度联合测序，通过miRNA-mRNA整合分析构建了抗Bt小菜蛾中肠差异表达的miRNA-mRNA互作调控网络；鉴定到了437个差异表达的基因，鉴定到了153个新的miRNA，其中鉴定到了13个在抗感品系间差异表达的miRNA，联合分析发现了大量可能的Bt受体和与抗性相关的蛋白质差异表达，其中包括6个ABC转运蛋白、4个氨肽酶N等。其中发现多个与丝裂原活化蛋白激酶(MAPK)代谢途径相关的基因，在两个种群中存在表达差异，这些基因或蛋白的表达差异可能跟与MAPK调控受体引起抗性有关。另外还发现其他基因或蛋白质表达量的改变，包括中肠蛋白质酶、肌动蛋白、线粒体相关蛋白质、以及核糖体蛋白质等，这些可能参与小菜蛾对Bt抗性的部分过程。同时对重要的靶标基因ABCB1开展实时荧光定量PCR、遗传连锁分析以及沉默表达实验，研究评价该基因对小菜蛾抗Cry1Ac的贡献，从而揭示小菜蛾对Cry1Ac抗性的分子机制。研究结果对Bt杀虫蛋白的开发与可持续应用具有重要的理论和实践意义。