嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定  

Purification of insecticidal proteins from Xenorhabdus nematophila HB310 and detection of their insecticidal activity

作　　者：王勤英[1] 南宫自艳[1] 陆秀君[1] 李秀花[2] 李国勋[1] 崔龙[1] 

机构地区：[1]河北农业大学植物保护学院 [2]河北省农林科学院植物保护研究所,河北保定071000

出　　处：《昆虫学报》2005年第3期353-358,共6页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30400296);河北农业大学重点基金"9816"项目

摘　　要：嗜线虫致病杆菌XenorhabdusnematophilaHB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85%饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白(毒素Ⅰ、毒素Ⅱ和毒素Ⅲ),胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD50为0.18μg头。SDS_PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10min,其杀虫活性没有显著变化;70℃处理10min对毒素Ⅲ杀虫活性没有显著影响。The extracts of extracellular and intracellular insecticidal proteins from Xenorhabdus nematophila HB310 strain, an entomopathogenic bacterium symbiotically associated with entomopathogenic nematode Steinernema carpocapsae, appeared high toxic activity by oral or injectable infection against several species of insects. Two oral insecticidal proteins, toxin Ⅰ and toxin Ⅱ, and another haemocoel insecticidal protein, toxin Ⅲ with injectable activity were purified from the intracellular protein extract by native_PAGE. The same three protein toxins also were obtained from the extracellular protein extract. There was only a single band for any of toxins above, but the results of SDS_PAGE showed four bands for toxin Ⅰ, three bands for toxin Ⅱ and one band for toxin Ⅲ, respectively. Bioassay results indicated that the oral insecticidal activity of toxin Ⅱ was higher than that of toxin Ⅰ to Helicoverpa armigera neonates. The injectable hemocoelic potency (LD (50)) of toxin Ⅲ was 0.18 μg/larva against Galleria mellonella fifth_instar larvae. The toxicity of toxin Ⅱ was stable under 50℃ in 10 min. The toxin Ⅲ was heat_stable to 70℃ in 10 min.

关 键 词：嗜线虫致病杆菌 毒素蛋白 纯化 杀虫活性 

分 类 号：Q966[生物学—昆虫学]