【作者】 李明华；

【导师】 王中康； 简恒；

【作者基本信息】 西南农业大学 ， 微生物学， 2003， 硕士

【摘要】 昆虫病原线虫共生菌是寄生于昆虫病原线虫肠道内的一类细菌，与线虫互惠共生。该类细菌分致病杆菌属（Xenorhabdus）和光杆状菌属（Photorhabdus）两个属，属下共有6个种。共生菌对昆虫具有极强的致病力，大部分菌株具有血腔活性，LD₅₀仅为1—10个活细胞。国内外科研工作者已分离到了具有杀虫活性的脂多糖、蛋白质等化合物，并发现共生菌杀虫蛋白杀虫谱宽、杀虫活性强，是极具应用潜力的生物杀虫剂。昆虫病原线虫共生菌杀虫蛋白已成为国内外研究的热点。 本研究选取了11个菌株为实验材料，通过对菌悬液的生物测定，筛选得到了一株高毒力的菌株，为采自北京郊区的嗜线虫致病杆菌（X.nematophila）CB6菌株。论文确定了本菌株具有杀虫活性的物质为蛋白质，测定了其生物活性，最后分离纯化得到杀虫蛋白纯品，现将主要研究结果总结如下： 1． 比较了11个菌株菌悬液的杀虫活性。结果表明，不同菌株对棉铃虫初孵幼虫的活性不同，其中CB6、A54、Dan和4-Ps菌株的活性较高，生长抑制率分别为99.48％、89.49％、83.80％和73.54％。而NC34、Wh03和E-6-7等菌株活性较低，生长抑制率分别为-9.57％、26.23％和28.64％，各菌株对棉铃虫幼虫的致死率都很低。 2． 确定了CB6菌株发酵液中具有杀虫活性的物质为蛋白质。菌体细胞60℃加热10min致死后，对棉铃虫的生长抑制率与未处理样品无差别，100℃处理后活性则完全丧失。发酵上清液经过沸水浴加热或胃蛋白酶降解后，活性大大降低，在40μl／g饲料的浓度时，生长抑制率分别降低74.51％和67.01％。细胞经洗涤、超声波破碎后提取上清液，用同样的方法处理，活性同样明显下降，在40μl／g饲料的浓度，下降87.13％和59.14％。由此可见，CB6菌株杀虫物质对热和胃蛋白酶敏感，说明这种物质为蛋白质。 3． 测定了杀虫粗蛋白对棉铃虫生长发育和取食的影响。杀虫粗蛋白对棉铃虫致死率较低，但却有极高的生长抑制率，并明显延长幼虫历期，严重影响幼虫的化蛹及蛹的羽化。叶碟法试验表明，该杀虫蛋白对棉铃虫高龄幼虫拒食效果明显，选择 嗜线虫致病杆菌CB6菌株杀虫齿白的活性及其分离纯化 性拒食率和非选择性拒食率都较高。4．分离纯化了cB6菌株胞外杀虫蛋白。发酵液离心除去菌体后，在上清液加入 （NH。）。SO。分段盐析，然后上 DEAE－Sepharose Fast FIOw离于交换柱层析，得主 3个蛋白峰，将具有活性的蛋白峰浓缩后再经过 Sephadex G上00过滤层析，得到 了分子量大于670kDa的蛋白质纯品，在284urn的波长下具有最大吸收峰。生测 结果表明，杀虫蛋白对热敏感，活性受酸碱度的影响不大，在州七时活性较高， 对胃蛋白酶和蛋白酶K较敏感，而活性却不受胰蛋白酶的影响。 研究结果表明，CB6菌株发酵液中具有杀虫活性的物质为蛋白质，该种蛋白质严重影响棉铃虫幼虫的取食和生长发育，具有较高的生物活性。本文为进一步研究该菌株杀虫蛋白的理化性质及该杀虫蛋白基因的克隆提供了依据和基础。更多还原

【Abstract】 Xenorhabdus and Photorhabdus are entomopathogenic bacteria symbiotically associated with entomopathogenic nematodes, six species included. They are highly virulent to insects. LD50 is generally 1-10 live cells when injected into the hemocoels of insects. Researchers in the world have purified lipopolysaccharide and protein from cells and supernatant of fermentation culture, which had insecticidal activity. The results of bioassay showed that the insecticidal protein was highly virulent against several orders of insects and potential as a biocontroll agent.In this paper, insecticidal activity of eleven strains was examined by means of oral bioassay, and a high virulent strain isolated from the suburb of Beijing was obtained, named Xenorhabdus nematophila CB6. Experiments showed that the substance of insecticidal activity was protein. And the insecticidal protein was isolated and purified from the fermentation culture. The main results gained in the study were as follows:1. Insecticidal activities of fermentation suspension of eleven strains were compared. It was found that the virulence of the eleven strains was different, of which four strains, namely strains CB6, A54,Dan and 4-Ps, were with higher activity of growth inhibition than the others against neonate of H.armigera. However, the mortality of H.armigera was very low after feeding on the diet treated with the fermentation suspension of each strain.2. The bioassay indicated that the substance with growth inhibition activity in X.nematophila CB6 fermentation suspension was protein.This activity didn’t depend on the survival of cell, but on the supernatant of either fermentation culture or celllysate solution. Analyzed by enzymatic hydrolysis or heat teatment, the activity of the supernatant decreased greatly. On the concentration of 40 l/g diet, the growth inhibition rates of fermentation supernatant were reduced by 74.51% and 67.01% after treated with 100 C for 10 minites and hydrolyzed with pepsin lor 4 hours, and by 87.13% and 59.14% of cell lysate supernatant. All these properties were proteinous and it colud be concluded that the insecticidal substance was protein.3. The insecticidal protein had high effect on the growth and development of larvae of H.armigera, and had significant antifeedant activity. Compared to the CK, the treatment with insecticidal protein obviously decreased larvae weight, prolonged larvae stage, decreased the rate of pupation and eclosion. In choice antifeedant experiment and no-choice antifeedant experiment, only a little proportion of treated leaves was eaten by insect compared to untreated leaves, and the antifeedant effect was quite notable.4. The insecticidal protein of strain CB6 was isolated and purified. Through centifugation, the cell was removed ,then crude protein was precipitated with ammonium sulfate. After column chromatography on DEAE-Sepharose Fast Flow and Sephadex G-200, several protein were obtained. The bioassay with neonate of H.armigera showed that only one proteir had growth inhibition activity. Analyzed by native PAGE, the molecular weight of the insecticidal protein was more than 670kDa. The protein was sensitive to pepsin and proteinase K, not to trypsin. Toxity of the protein was stable under the temperature of 30 C-60 C and the pH of 5-8. There was only one absorbant peak from 220nm to 400nm, and the maxmun absorbant peak was at 284nm.The experiments indicated that the insecticidal substance in the X.nematophila CB6 culture broth was protein. The insecticidal protein had great effect on feeding, growth and development of H.armigera. The results in the paper provided a base for studying on the properties of the insecticidal protein and the cloning of the insecticidal gene.更多还原