论文部分内容阅读
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。
Bursaphelenchus xylophilus is the pathogen of pine wood nematode and interacts with its associated bacteria. In order to study the interaction between Bursaphelenchus xylophilus and its associated bacteria, Nycodenz media centrifugation and SDS-lysis were used to enrich the associated bacteria of Bursaphelenchus xylophilus. DNA was extracted by phenol / chloroform extraction and the fosmid of Bursaphelenchus xylophilus Metagenomic library. The size of the inserted fragment of library cloning was 30 ~ 45kb with the average length of 40kb. This library contains 19200 clones and contains a total of 7680000 kb of DNA. Library stability test showed that the inserted DNA fragments can stably inherit in the fosmid plasmid, and no insert was found to be missing or rearranged. A total of 96 clones were randomly selected and sequenced. BLAST analysis showed that the sequences of B. xylophilus were 5.2%, bacterial sequences accounted for 64.6%, and no homologous sequences were 14.6%. Analysis of the diversity of associated bacteria showed that Stenotrophomonas was the dominant species and Sphingomonas, Cupriavidus and Pseudomonas were the dominant species. The establishment of this library laid the foundation for revealing the interaction between Bursaphelenchus xylophilus and its associated bacteria and the ecological functions of accompanying bacteria.