小菜蛾APN1和APN2基因协同参与Bt毒素Cry1Ac抗性分子机理

Receptor-binding mechanism on insects have always been on the hotspots of insects resistance to Bacillus thuringiensis toxins(Bt toxins)，but the receptor of Bt toxins in Plutella xylostella is still poorly elucidate at present. Although our research has been pointed out that APN1 and APN2 are partly attended resistance to Bt toxin Cry1Ac in P. xylostella through gene expression and RNA interference, the relative trans-regulatory mechanism of resistance need further understand carefully how to coordiante with APN1 and APN2. Here genotype will be determined through linkage analysis of APN1 and APN2 allele genes with Cry1Ac resistance, while APN1 and APN2 alone or together may associate with Bt resistance will be profromed by RNA interference mediated APN1 or APN2 single gene silencing and both togther genes silencing, respectively. Stimulatnoesuly the affinity between expression APN1 and APN2 proteins in cell line sf9 and Bt toxin Cry1Ac will be done by ELISA, which reveals the differentiation to Bt toxin in target resistance and susceptible insects, Take overall, systematic study of APN family genes contribute to the molecular basic knownledge of resistance would be benefit to make correct strategy for sustainable control to Bt in P. xylostella.

氨肽酶（APN）是昆虫重要候选的Bt毒素受体之一，而小菜蛾中APN基因是否作为受体参与Bt毒素Cry1Ac抗性的形成及其成员之间的调控模式，目前未有明确定论。基于前期研究小菜蛾Cry1Ac抗敏品系的APN1和APN2基因序列比对、基因表达水平和RNA干扰分析，二者均与小菜蛾Cry1Ac抗性相关，并且在表达量上存在反式调控的现象。为此，本项目将通过基因分型研究APN1和APN2之间的遗传连锁；同时干扰APN1和APN2基因与分别干扰单一基因，探讨APN1和APN2单独及其共同作用与Bt抗性的相关性；应用酶联免疫吸附剂测定法比较Sf9细胞真核表达的APN1和APN2蛋白与Cry1Ac蛋白的亲和力，进一步验证二者与Bt抗性的相关性。旨在探讨小菜蛾APN1和APN2基因反式调控协同参与Bt毒素Cry1Ac抗性形成的分子机理，为阐明小菜蛾Bt抗性机制及其抗性治理策略的制定提供一定理论依据。

氨肽酶（APN）是昆虫重要候选的Bt毒素受体之一，而小菜蛾中APN基因是否作为受体参与Bt毒素Cry1Ac抗性的形成及其成员之间的调控模式，目前未有明确定论。主要研究内容：1.小菜蛾APN1和APN2基因遗传连锁分析：以抗敏品系为亲本杂交后得到的F1代而后与亲本进行回交得到了种群，检测APN1 和APN2 基因型分布情况，依据遗传学定律分析二者是否遗传连锁相关。2.小菜蛾APN1和APN2参与Bt抗性相关性：分别和同时对APN1和APN2 基因进行基因沉默，检测沉默其中单一基因对另一基因表达水平的影响，并检测基因沉默后小菜蛾种群对药剂的敏感性的变化情况判断与Bt抗性的相关性。主要结果及关键数据：1. 构建了小菜蛾Cry1Ac杂交种群及近等基因系，经过6代反汰选，抗性倍数能达到5000倍；2. 分析了APN1和APN2基因分布型及二者遗传连锁，APN1 R APN1 R APN2 RAPN2 S 和APN1 R APN1 S APN2 R APN2 R 基因型均在回交种群中出现，表示APN1和APN2基因在同一连锁群上。3. 克隆得到小菜蛾敏感、抗性品系（APN1和APN2）基因的全长。4.分析了小菜蛾敏感、抗性品系中APN1和APN2序列比对，比较抗敏品系小菜蛾APN1基因的碱基序列一致性为97.45%，而APN2为98.15%；5. 研究了小菜蛾敏感、抗性品系中APN1和APN2表达水平，APN1基因除1龄无显著差异外，其余各龄期敏感品系表达量均低于抗性品系，并且差异达显著水平（P＜0.05）；APN2基因除1龄外，其余各龄期和中肠组织中敏感品系表达量均高于抗性品系，并且差异达显著水平（P＜0.05）；6. 进行了抗性、敏感品系小菜蛾中肠APN2在Sf9细胞系中的表达与鉴定；7. 分析了小菜蛾中肠APN2和APN4基因RNAi研究，表明表明APN2单基因对小菜蛾Cr1Ac影响抗性作用较大。8. APN2重组蛋白与Cry1Ac毒素的免疫荧光定位分析和Cry1Ac毒素对Sf9细胞的毒性分析，表达抗、敏小菜蛾中肠APN2蛋白的Sf9细胞死亡数孵育Cry1Ac蛋白后相对于孵育前存在极显著差异。本研究探讨了小菜蛾APN1和APN2基因协同参与Bt毒素Cry1Ac抗性形成的分子机理，为阐明小菜蛾Bt抗性机制及其抗性治理策略的制定提供一定理论依据。