松材线虫伴生细菌宏基因组fosmid文库构建及特征分析

Construction and analysis of the metagenomic fosmid library for the bacteria carried by the pine wood nematode

松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作。为研究松材线虫和伴生细菌的互作关系,本研究用Nycodenz介质离心和SDS裂解法富集松材线虫的伴生细菌,以酚/氯仿抽提法提取DNA,构建了松材线虫伴生细菌fosmid宏基因组文库。文库克隆的插入片段大小分布在30~45kb,平均长度为40kb。该文库包含19200个克隆,共计包含7680000kb DNA。文库稳定性检测表明插入的DNA片段能够在fosmid质粒中稳定遗传,没有发现插入片段丢失或重排。随机挑选96个克隆进行末端测序,BLAST分析表明：该文库中松材线虫序列占5.2%,细菌序列占64.6%,无同源序列14.6%。对伴生细菌多样性分析表明：Stenotrophomonas为优势种群,Sphingomonas、Cupriavidus和Pseudomonas为次优势种群。该文库的建立为揭示松材线虫与其伴生细菌的互作关系及伴生细菌的生态功能奠定了基础。

The pine wood nematode,Bursaphelenchus xylophilus is the pathogen of pine wilt disease and interacts with its carried bacteria.To analyze the interaction between B.xylophilus and the carried bacteria,the metagenomic fosmid library of the bacteria associated with B.xylophilus was constructed.The bacteria were enriched by Nycodenz centrifugation and sodium dodecyl sulfate（SDS）lysis,and the genomic DNA was extracted by the phenol-chloroform method.The size of the insert fragment of the clones was range from 30 kb to 45 kb.The library consisted of 19 200 clones with an average insert fragment size of 40 kb,thus the total size of the fosmid library was estimated at 7 680 000 kb（=19 200×40 kb）.Fosmid stability assays indicated that the insert DNA was stable during propagation in the fosmid system.The end sequencing results of randomly selected 96 clones was compared in NCBI with BLAST program.It was showed that the percentage of B.xylophilus,bacteria and other no significant similarity sequences was 5.2%,64.6% and 14.6%,respectively.The analysis of the bacterial diversity revealed that Stenotrophomonas was the dominant group.Sphingomonas,Cupriavidus and Pseudomonas were the secondary dominant communities.The library supplied the foundation for analysis of ecological role of bacteria and its interaction with B.xylophilus.