豌豆植物多肽PA1b抗虫机制的研究

In recent years, chemical pesticides are employed increasingly around the world, and this situation causes more and more attention due to their persistence and toxicity to non-target organisms and the environment. A variety of secondary metabolites have been produced during the process of long period coevolution between the plants and insects. These biopesticides of plant origins have earned much interest not only because of their target, low toxicity to human and mammals, but also because of these molecules with less impact on the environment. PA1b is a peptide extract from pea plants with significant insecticidal activity. Biochemical studies showed the presence of a high-affinity binding site for PA1b on the sensitive insect membrane extracts, but the molecular mechanism of PA1b action is unclear. In this study PA1b expression vector is going to be constructed and transformed into Sf9 cells, the pathway of PA1b toxicity will be identified by immunoblotting and viability assay of Sf9 cells, mRNA differential display, 2D-DIGE and MS based proteome profiling are going to be applied to identify the genes and proteins affected by PA1b, and the interactions between the proteins will be analyzed. This research will help us to reveal the molecular mechanism of PA1b action and provide theoretical data for the application of this binsecticide.

近年来，全世界范围内化学杀虫剂的使用量不断攀升，但化学杀虫剂的残留量高、生物安全性低而不断的引起人们的忧虑和指责。植物在长期与昆虫协同进化过程中，产生了多种防御昆虫取食的次生代谢物质，这种植物源性的天然产物农药对人及哺乳动物的毒性低，对非靶标生物的安全性高，对环境压力小，因近逐渐得到人们的关注。PA1b是一种从豌豆种子中分离提取并鉴定的生物活性多肽，具有非常重要的生物杀虫活性，研究表明在敏感型昆虫细胞膜上存在其特异结合位点，但PA1b对昆虫的具体毒害机制仍不清楚。本研究通过细胞转染技术在昆虫细胞中表达PA1b多肽，通过免疫杂交及细胞活性检测确定PA1b的毒害作用途径，利用mRNA差异显示技术、差异蛋白组学等方法分析参与PA1b毒害作用的差异基因及蛋白质，并对PA1b与差异蛋白及各差异蛋白之间的作用关系进行分析，初步确定PA1b发生毒害作用的信号通路，为PA1b的推广应用提供理论及实践基础。

近年来，全世界范围内化学杀虫剂的使用量不断攀升，但化学杀虫剂的残留量高、生物安全性低而不断的引起人们的忧虑和指责。植物在长期与昆虫协同进化过程中，产生了多种防御昆虫取食的次生代谢物质，这种植物源性的天然产物农药对人及哺乳动物的毒性低，对非靶标生物的安全性高，对环境压力小，因近逐渐得到人们的关注。PA1b多肽是一种良好的生物杀虫剂，批量生产PA1b多肽对农业生产、环境保护及研究PA1b的毒害作用机理方面具有重要意义。. 本研究以豌豆植物为实验材料，绿色荧光蛋白为报告基因，建立了豌豆发芽种子真空侵染体系，实验选择真空压力、菌体工作液浓度及真空侵染时间三种因子对该体系进行了优化，实验结果表明菌体工作液浓度OD600 =0.9-1.5时在0.08MPa的压力下真空处理1分钟为最佳侵染条件，在此条件下侵染豌豆种子，侵染后第8-9天豌豆植株茎部开始表达GFP,随后GFP在植物茎、叶脉及叶肉中扩散，侵染后12-14天外源蛋白表达率最高，表达量最大,是收获外源蛋白的最佳时期。. 利用建立的生产平台批量生产PA1b多肽，以RT-PCR方法成功克隆了PA1b基因，构建了含有PA1b基因的豌豆植物表达载体，侵染豌豆植物后12-14天对蛋白表达情况进行Westernblot检测，结果表明在豌豆植物中成功表达带有His标签的PA1b多肽。. 为进一步研究PA1b毒害作用机理，构建了含有PA1b基因的昆虫表达载体，用以转染昆虫细胞进而研究PA1b毒害作用途径。