摘要
对斯氏线虫（Steinernera feltiae）比利时品系的共生菌伯氏致病杆菌（Xenorhabdus bovienii BE）进行发酵培养，测定结果显示，该菌发酵液对供试昆虫大蜡螟老龄幼虫有很高的血腔毒性。对发酵液硫酸铵盐析和乙酸乙酯抽提物等衍生物进行测定，发现共生菌的杀虫物质主要为胞外和胞内蛋白成分，且胞内粗提蛋白活性明显高于胞外蛋白。对两种粗提蛋白进行Marker分子量范围为（1．43-9．72）×10^4的SDS-PAGE分析，发现胞外粗提蛋白在此分子量范围内没有明显的条带，而胞内粗提蛋白则有较明显的5个条带。研究表明胞内粗提蛋白具有较高的热稳定性，测定的盐析最佳硫酸铵饱和度为80％，此粗提蛋白在24h和48h后对昆虫血腔毒性的LD50值分别为4．7316g／L和1．5715g/L；其在5d后对二龄幼虫的口服LD50为253．7579μg／g。胞内粗提蛋白凝胶过滤后呈现4个峰值，分别对其进行SDS-PAGE分析，只有浓度较大的峰值1和4产生明显条带，分子量分别为6．64×10。和（2．01-2．90）×10^4的2种复合蛋白；分别对它们进行血腔和口服毒性生物活性测定，发现分子量为（2．01-2．90）×10^4的2种复合蛋白杀虫活性高于分子量为6．64×10^4蛋白的杀虫活性，其在24h和48h后对昆虫血腔毒性的LD50分别为0．3772g／L和0．1238g/L，其在5d后对二龄幼虫的口服LD50为93．0021μg／g。
The symbiotic bacterium Xenorhabdus bovienii BE strain was extracted from the Belgian population of entomopathogenic nematode Steinernera feltiae. High toxicity to Galleria mellonella fifth instar larvae was observed when the insect hemocoel cavities were injected with the fermentation solutions of the bacteria. Studies on the insecticidal activity of substances from fermentation solution indicated that the toxic substances were proteins existed in both extracellular and intracellular environment, and the toxicity of intracellular proteins was higher than those of extracellular. SDS-PAGE analysis demonstrated five bands from intracellular proteins and no band from extracellular proteins when running the gel with molecular weight marker ranging from 1.43 ×10^4 to 9.72 × 104. The heat-stable temperature of the intracellnlar crude proteins was 70 ℃. The 50% lethal dose （LD50） of intracellular crude proteins were 4. 731 6 g/L and 1. 571 5 g/L against insect larvae in 24 h and 48 h respectively, and was 253. 757 9 μg/g against insect larvae oral cavity in 5 d. The crude proteins showed four absorption peaks after sephadex gel filtration and only two peaks exhibited obvious bands in SDS-PAGE analysis. The active substances of one band had high toxicity to insect instar larvae. SDS-PAGE showed that the substances combined two proteins with the molecular weights ranging from 2.01 - 10^4 to 2. 90 x 10^4. The 50% lethal dose （LD50） of the substances against G. mellonella larvae in 24 h and 48 h were 0. 377 2 g/L and 0. 123 8 g/L respectively, and was 93. 002 1 μg/g against insect larvae oral cavity in 5 d.
出处
《江苏农业学报》
CSCD
北大核心
2009年第1期59-67,共9页
Jiangsu Journal of Agricultural Sciences
基金
高等学校博士学科点专项科研基金项目(20030307021)
关键词
斯氏线虫
大蜡螟
伯氏致病杆菌
血腔毒性
SDS-PAGE
口服毒性
Steinernera feltiae
Xenorhabdus bovienii BE
Galleria mellonella
hemocoelic toxicity
SDS-PAGE
oral toxicity
