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

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.

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。

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。目前，亟待将多个不同类型的杀虫基因同时转入作物，这样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，其氨基酸序列与现有的Cry和Cyt 蛋白没有任何同源性，与已发现的Vip蛋白同源性很低（27%-33%）。生物测定表明Vip7Aa蛋白对鳞翅目昆虫小菜蛾、棉铃虫、尤其是甜菜夜蛾、斜纹夜蛾和草地贪夜蛾有很高的杀虫活性。令人欣喜，我们意外的发现Vip7Aa蛋白对致倦库蚊、埃及伊蚊和秀丽隐杆线虫也表现出较好的杀虫活性。目前，尚未有人报道Vip3类蛋白对双翅目昆虫和线虫有杀虫活性，这将改变大家习惯的认为Vip3类蛋白杀虫活性仅限于鳞翅目夜蛾类昆虫。本项目重点利用GST pull down、Western blotting、蛋白质质谱和竞争结合等技术，鉴定Vip7Aa蛋白在甜菜夜中肠道BBMV的受体蛋白，我们发现Vip7Aa与Cry蛋白在甜菜夜蛾中肠上存在不同的结合蛋白，这样昆虫很难产生抗性和交互抗性。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。