Bt新型营养期杀虫蛋白Vip7Aa的受体蛋白鉴定及抗性机制研究

目前转基因作物在全球范围内大面积种植，商业化的抗虫转基因作物的杀虫基因大多数来自于苏云金芽孢杆菌（Bt）的Cry杀虫蛋白基因。但长期大面积使用单一的杀虫基因来控制农业害虫，存在靶标害虫容易产生抗性的风险。将多个不同类型的杀虫基因同时转入作物不仅可以拓宽其杀虫谱、同时基因之间也不容易产生交互抗性，甚至还存在协同增效作用，是延缓害虫产生抗性的有力措施。vip7Aa基因是从海南热带原始雨林鉴定克隆的具有完全自主知识产权的Bt新型营养期杀虫蛋白基因，生物测定表明其表达产物对鳞翅目昆虫有很高的杀虫活性（专利申请号：201410128118.2）。本项目将重点鉴定Vip7Aa杀虫蛋白的靶标昆虫肠道上特异的受体蛋白，并进一步探索新型Vip7Aa蛋白的杀虫与抗性机制。本研究为下一步应用vip7Aa基因进行生物防治奠定基础，并为vip7Aa基因作为抗虫转基因作物培育的候选基因提供科学依据。

Genetically modified（GM）crops are planted worldwide with large-scale at present and the insecticidal crystalline protein genes from Bacillus thuringiensis (Bt) act as the key source for most commercialization transgenic crops. But it was fully recognized that the main threat for the long-term and large area success of such crops with single insecticidal crystal protein gene is the potential of insects to develop resistance or cross-resistance. The use of combinations of insecticidal proteins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that it`s necessary to screen the novel toxins with effectiveness against agricultural insect pest and we attemptted a large-scale screening of Bt isolates from the Tropical Rainforest in Hainan Island to characterize the new insecticidal protein genes for transgenic expression and resistant management. As the result that the new vegetative insecticidal protein(vip) genes of vip7Aa with the target insects of Lepidoptera order were identified and cloned from Bt strain HS66(Patent application No: 201410128118.2). In this study, ligand blotting assay, GST pull down, Co-immunoprecipitation assay and mass spectrometry would be carried out to determine the receptor to the novel Vip7Aa toxins in target insect midguts. Simultaneously, the mode of action of Vip7Aa would be conduced. The most cases of cross-resistance are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMVs) prepared from the target insect larvae. Another goal of the project aims to understand insect resistance mechanism of the new Vip7Aa toxins. The research data would support the use of these novel vip7Aa gene for development of GM crops to increase target range and delay the onset of heliothine resistance.