Xenorhabdus nematophilus BP品系杀虫毒素基因的克隆与鉴别  

Cloning and Characterization of Insecticidal Toxin Genes from Xenorhabdus nematophilus BP

作　　者：崔龙[1] 邱礼鸿[1] 房媛媛[1] 庞义[1] 李国勋[2] 

机构地区：[1]中山大学生命科学学院生物防治国家重点实验室,广东广州510275 [2]河北农业大学植物保护学院,河北保定071001

出　　处：《中山大学学报（自然科学版）》2003年第3期47-50,63,共5页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家自然科学基金资助项目 ( 30 170 143);广东省自然科学基金团队资助项目 ( 0 0 0 5 942 32 172 )

摘　　要：构建了昆虫病原线虫共生菌XenorhabdusnematophilusBP品系的粘粒文库并用生物测定的方法从中筛选到 2个对棉铃虫初孵幼虫有口服抑杀作用的克隆cos83和cos76。为初步确定这两个克隆的毒素基因与已报道基因的差异程度 ,根据已发表的共生菌毒素基因序列资料设计了 7对引物对这两个克隆进行PCR扩增并对扩增产物进行测序和分析。从毒力较强的cos83中 ,7对引物均扩增出与目标片段大小一致的产物 ,而从cos76中只有 5对扩增到目标片段。测序结果显示 ,cos76的 5个PCR扩增产物与cos83的对应扩增产物DAN序列同源性为 10 0 %。以cos83的 7个PCR扩增产物所编码氨基酸序列进行BLAST分析 ,它们与X .nematophilusPMF12 96和PhotorhabdusluminencencW14毒素相应区间的平均同源性分别为 94%和 5 8% ,说明cos83毒素是共生菌口服毒素家族的一员但与同类的其它毒素有一定的差异 ,值得对其杀虫谱 ,作用机理等进行进一步的研究。A cosmid library of Xenorhabdus nematophilus BP, the symbiotic bacteri a of entomopathogenic nematode (EN) Steinernema carpocapsae, was constructed and two clones of the resulting library, cos76 and cos83 were found possess str ong and moderate oral toxicity against neonate of cotton ball worm Helicoverpa arm igera, respectively. To identify the toxin genes presented in these two clones , seven pairs of primers were designed based on the published sequences and used for PCR amplification with both clones. All seven pairs of primers yielded prod ucts from cos83 while only five did from cos76. All PCR products were sequenced. The results showed that each sequence of the five PCR products obtained from c os76 was identical with that from cos83, indicating that the toxin genes in cos 76 were part of those in cos83. The BLAST results showed that the amino acid seq uences deduced from seven PCR products from cos83 had mean homology of 95% and 65% with those from insecticidal toxin genes of X.nematophilus PMF1296 and P.luminescens W14, respectively. The results indicate that toxin produced by cos83 is a member of the oral toxin family of EN symbiotic bacteria but it diff er substantially from other members of the family therefore worthy of further i nvestigation.

关 键 词：XENORHABDUS nematophilus BP 粘粒文库 杀虫阳性克隆 筛选 

分 类 号：Q93[生物学—微生物学]