致倦库蚊对球形芽胞杆菌和化学杀虫剂的抗性和抗性机理

Bacillus sphaericus is a naturally occuring Gram-positive, spore-forming aerobic bacterium. Some strains produce binary toxins against mosquito larvae, which have been widely applied to the biological control of mosquitoes. However, the binary toxin can easily induce target mosquito to develop high-level of resistance both in laboratory and under field conditions, and the frequent occurrence of high-level resistance to binary toxin in mosquitoes has hindered the further application of B. sphaericus as microbial control agents. In previous studies, a laboratory and a filed-collected B. sphaericus resistant Culex quiinquefasciatus colonies were characterizated and their resistance stabilities were investigated. In this study, 1) three resistant C. quinquefasciatus to organophosphorus, carbamate and pyrethroidthe insecticides were selected respectivelyin at the laboratory; 2) the resistance and cross-resistance of both B. sphaericus and chemical pesticide resistant colonies to chemical pesticides and B. sphaericus will be evaluated; 3) the activities of esterase, mixed-function oxidase, acetylcholinesterase, glutathione transferase among susceptible (SLCq) and resistant colonies will be compared, and their correction with resistance will be determined; 4) the binding and inhibition dynamic of receptors from brush border membrane fractions (BBMF) with the binary toxin and chemical pesticides will be determined, and 5) the effects of the site mutation of key resistance related genes on enzyme activity and resistance development will be unraveled. The result will provide an insight into the understanding the inheritance and mechanism of resistance to Bs and chemical pesticides in moaquitoes, and developing approaches for the resistance management and resistant mosquito control in field.

球形芽胞杆菌（简称Bs）是一种在自然界中广泛分布的好气芽胞杆菌。由于其在芽胞形成过程中产生的二元毒素对蚊幼虫具有特异性毒杀作用,广泛应用于蚊虫的综合防治中。但长期大量使用Bs制剂导致目标蚊虫产生高水平的抗性，野外抗性蚊虫品系的出现影响了Bs作为生物杀虫剂的应用。本项目是在前期Bs野外和实验室致倦库蚊抗性蚊虫品系的抗性稳定性和抗性遗传研究的基础上，实验室筛选获得对有机磷类、氨基甲酸酯类和拟除虫菊酯类化学杀虫剂高抗性的蚊虫品系，确定不同抗性蚊虫品系对Bs和化学杀虫剂的交叉抗性，分析抗性和敏感蚊虫品系酯酶、多功能氧化酶、乙酰胆碱酯酶和谷胱甘肽转移酶的活性，比较抗性和敏感蚊虫毒素结合蛋白差异，探讨蚊虫体内抗性功能基因突变，酶活性变化和毒素结合位点改变与抗性产生的关系，阐明致倦库蚊对Bs和化学杀虫剂产生抗性的机理，为Bs杀虫剂在蚊虫综合防治中的持续应用及抗性蚊虫的控制提供理论依据。

球形芽胞杆菌产生杀蚊毒素，对蚊幼虫具有特异性毒杀作用，在世界范围内被成功地应用于疾病媒介蚊虫的生物防治。本项目对敏感(CqSL)和抗性致倦库蚊(CqRL/C3-41)的二元毒素受体基因cqm1进行克隆及序列比对，发现无论在cDNA和基因组水平，抗性库蚊受体基因cqm1R均在445 bp处缺失了一个碱基C，该缺失导致翻译提前终止，产生截短表达的受体蛋白。大肠杆菌中表达的敏感库蚊受体Cqm1S蛋白具有α-葡萄糖苷酶活性，能和BinB亚基特异性结合，而Cqm1R由于缺失了受体蛋白的3/4 C-末端，丧失了α-葡萄糖苷酶活性，不能和BinB亚基特异性结合。免疫印迹实验表明，尽管单个核苷酸缺失不影响cqm1基因在抗性致倦库蚊中的转录，但却检测不到Cqm1蛋白的表达。因此cqm1R是一个新的抗性等位基因，功能性Bin毒素受体的缺乏是致倦库蚊对球形芽胞杆菌C3-41产生高水平抗性的主要原因。.另外本项目对cry48Aa和cry49Aa基因进行克隆并在B.t无晶体突变株中表达，并与Bin毒素敏感和抗性致倦库蚊BBMF进行特异性和竞争结合。结果发现Cry48Aa和Cry49Aa毒素与Bin毒素敏感和抗性致倦库蚊BBMF都具有较高的结合特异性，结合能力无明显差异。异源竞争试验结果表明Cry49Aa不能竞争封闭Cry48Aa毒素与Bin毒素敏感和抗性致倦库蚊虫BBMF蛋白的结合位点，其IC50均 >17 μM，相反，Cry48Aa毒素可以有效地和Cry49Aa毒素竞争结合BBMF蛋白上的结合位点，其IC50分别为22.1 ± 3.7和15.4 ± 2.6 nM。Cry48Aa 和其它3-D毒素一样，在Cry49Aa辅助蛋白存在的情况下，可以与BBMF相互作用形成大于250kDa的寡聚体。对Cry49Aa毒素蛋白进行N-端和C-端截短表达，Far-western斑点杂交分析显示Cry49Aa蛋白的N-端区域负责和Cry48Aa相互作用。与蚊幼虫BBMF蛋白的竞争结合分析发现Cry49Aa的C-端负责和BBMF结合。生物测定结果显示截短表达的Cry49Aa蛋白不能联合Cry48Aa毒素杀死蚊幼虫，说明Cry49Aa的完整性对毒素毒力的发挥是必不可少的。