苏云金芽胞杆菌新型非3-Domian类晶体蛋白Cry6Aa 作用线虫的穿孔模式研究

Bacillus thuringiensis is the most important insecticidal bacterial, it’s main active products is the insecticidal crystal protein (Abbreviated as Cry). At present, there are 74-class and more than 780 Cry proteins have been found in B. thuringiensis, most of which has a 3-Domain structure. After more than 20 years of research, we obtained the detailed action mechanism of the 3-Domain-Structure like Cry proteins. However, the action model of the non-3-Domain structure like Cry protein is still not clear. The Cry6Aa (one kind of important non-3-Domain structure like Cry protein) has high activity against several important plant parasite nematodes, which could induce the cell pore forming in nematode, and has similar structure to one kind of ɑ-pore forming toxin (PFTs) from E. coli. However, the membrane receptors and the detailed action mechanism of Cry6Aa-mediated cell pore forming are unclear. In this project, we will use a free living nematode Caenorhabditis elegans as model, to screen and identify the membrane receptors (we have confirmed it as membrane lipids) that mediate Cry6Aa target nematode as a ɑ-PFTs, then to reveal the detailed biochemical and cytological mechanisms of the specific membrane lipid receptor during Cry6Aa form the pore in nematode cell, as well as the specific signals pathways during this process. This project will reveal a novel action mechanism for non - 3 - Domain structure like Cry protein target its host, and will also provide a theoretical basis for the design of novel B. thuringiensis pesticide and transgenic plants based on nematicidal Cry proteins to control plant parasite-nematodes.

苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)是最成功的杀虫微生物，其杀虫的主要活性成分为杀虫晶体蛋白(Cry)。目前Bt中已发现74大类780余个Cry蛋白，大部分具有3-Domain结构。经过20多年研究， 3-Domain结构类Cry蛋白作用机制已经明确，但非3-Domain结构类的作用机制并不清楚。Cry6Aa是一种重要的非3-Domain结构杀虫蛋白，对多种植物线虫具有高活性，具有与ɑ-穿孔素类似结构并可导致线虫细胞穿孔。但介导Cry6Aa细胞穿孔的受体和具体的穿孔方式都不清楚。本项目拟以秀丽小杆线虫为模型，鉴定介导Cry6Aa在细胞上穿孔的受体（已鉴定为膜脂类），并解析膜脂受体介导Cry6Aa穿孔的生化和细胞学机制。本项目将揭示一种全新的非3-Domian结构类Cry蛋白作用宿主机制，并可为新型杀线虫Bt农药创制和转基因抗线虫植物设计提供理论依据。

苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)是最成功的杀虫微生物，其杀虫的主要活性成分为杀虫晶体蛋白(Cry)。目前Bt中已发现74大类780余个Cry蛋白，大部分具有3-Domain结构。经过20多年研究，3-Domain结构类Cry蛋白作用机制已经明确，但非3-Domain结构类的作用机制仍并不清楚。Cry6Aa是一种结构新颖的重要非3-Domain结构杀虫蛋白，对多种植物寄生线虫具有高活性，具有与ɑ穿孔毒素类似结构并可导致线虫细胞穿孔。但介导Cry6Aa细胞穿孔的受体和具体的穿孔机制都不清楚。本项目以秀丽隐杆线虫为模型，鉴定到了介导Cry6Aa在细胞上穿孔的膜脂受体组分；并解析了Cry6Aa蛋白结合膜脂受体后多聚成12聚状态的分子机制；鉴定了Cry6Aa蛋白中关键穿孔活性位点，通过改在该活性区域可提高蛋白杀虫活性；最后明确了Cry6Aa蛋白穿孔后导致的细胞生理学表型变化及其机制，同时鉴定到Cry6Aa使用线虫膜蛋白RBT-1作为功能受体帮助毒素杀虫的分子机制。此外，本项目解析了产Cry6Aa的高效杀线虫Bt菌株YBT1518通过负调控另一种杀虫晶体蛋白Cry5B高效杀虫的分子机制；证明了Cry5B毒素靶向线虫CDH-8受体杀虫同时受到宿主蛋白酶体系统抵御，而该受体及防御信号通路均不参与Cry6Aa杀虫过程；明确了高毒力Bt菌株靶向肠道连接快速杀虫的机制及不同毒力水平的Bt菌株进化规律。本项目揭示了Bt产生的3-Domain 类Cry蛋白作用宿主的分子机制，并比较了其与非3-Domain 类Cry蛋白高效杀虫机制的异同，为新型杀线虫Bt农药创制和转基因抗线虫植物设计提供理论依据。