【作者】 李玉凤；

【导师】 冯纪年；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2007， 硕士

【摘要】 昆虫病原线虫共生菌是共生于昆虫病原线虫(entomopathogeic nematodes, EPNs)肠道内的一类细菌,在EPNs中对昆虫的致病力起关键性作用,与线虫之间的关系可以概括为:线虫携带共生菌进入寄主昆虫体内,并释放到昆虫的血腔中;共生菌在昆虫血腔内繁殖,产生杀虫活性极高的蛋白毒素,使昆虫患败血症,一般在48h内死亡。由于这类杀虫毒素蛋白具有高效、杀虫谱广和杀虫迅速等诸多优点,从共生菌中克隆杀虫毒性基因用于开发新型生物源杀虫物质,以延缓害虫对目前使用的杀虫物质产生的抗性,已成为当今的研究热点。昆虫病原线虫共生菌已成为一类有待开发的生物资源,基于此,昆虫病原线虫共生菌的分类鉴定是开展相关研究的基础。分子鉴定:昆虫病原线虫共生菌YL001是西北农林科技大学无公害农药研究中心分离得到的一株有较强杀虫活性的共生菌,其分子鉴定是根据目前已知16S rDNA基因,设计引物对P1P2,以YL001菌株的总DNA为模板进行PCR扩增,产物大小为568bp,GenBank的接收号为EF206655,此序列与GenBank登录号为XN16RNA0的序列相似性达99.5%。构建系统进化树,显示与Xenorhabdus nematophilus的亲缘关系最近。昆虫病原线虫共生菌YL001与GenBank中现有的昆虫病原线虫共生菌同源序列进行比对,与Xenorhabdus nematophilus的相似形高达99.5%,遗传距离为0.5,进一步证实了该菌株就是Xenorhabdus nematophilus。杀虫毒性基因的克隆:以YL001为材料,根据已报道的杀虫毒素基因-菌毛蛋白亚基序列,设计引物对P3P4,以YL001菌株的总DNA为模板进行PCR扩增,获得特异性片段,并将此片断克隆到T-easy载体上,获得了重组子pGEM-PSYL001,序列测定和分析结果表明,PSYL001阅读框全长537bp,编码179个氨基酸,预测分子质量和等电点分别为18.9ku和6.94,GenBank的接收号为DQ537944。序列比较结果表明,与菌毛蛋白亚基类基因高度同源(同源性达99%)。更多还原

【Abstract】 Symbiotic bacteria of entomopathogenic nematodes are symbiose in intestines of entomopathogenic nematodes. Symbiotic bacteria play an important role in the pathogenicity of insect. The relationship of symbiotic bacteria and nematode can be summed up as: nematodes carry symbiotic bacteria into insect hosts and release to haemocoel of insect; Symbiotic bacteria reproduces and produces insecticidal toxin protein with high activity which leads to die from septicemia within 48h. The insecticidal toxin protein has the advantage of highly active, broad spectrum, high speed and so on. Cloned the gene of insecticidal toxin from symbiotic bacteria is used to develop the new biological insecticidal resources in order to prevent the resistant. Symbiotic bacteria have become the biological resources which remainto be developd.So accuracy of classification and identification is the basis to carry out the relevant studies.Molecular identification: Steinernema sp. YL001 was separated by research and development center of biorational pesticide Northwest Agriculture and Forestry University which is high-drug strain. Based on the sequence of 16S rDNA reported previously a pair of primers P1P2 was designed. A specific band(568bp in length)was amplified from DNA of Steinernema sp. YL001. The accession number in Genbank is EF206655 which had 99.5% similarity on nucleotides level to XN16RNA0. Construct the phylogenetic trees showed the relationship with Xenorhabdus nematophilus was the nearest. Compared the 16S rDNA from Steinernema sp. YL001 with other symbiotic bacteria showed that Steinernema sp.YL001 had 99.5% similarity on nucleotides level to Xenorhabdus nematophilus and its divergence was 0.5. So the Steinernema sp. YL001 was identified as Xenorhabdus nematophilus.Clone the gene of insecticidal toxin: Based on the sequence of Pilin Subunit reported previously a pair of primers P3P4 was designed. A specific band was amplified from DNA of Xenorhabdus nematophilus YL001.The segment was cloned into T-easy vector. Analyzed the results of sequencing showed that the length of PSYL001 was 537bp, 179 amino acids were encoded. The predicted MW and pI are 18.9ku and 6.94.The accession number in Genbank is DQ537944.Comparison showed that this gene has 99% similarity on nucleotides level to other Pilin Subunit gene.更多还原