准东油田萘降解菌群培养及菌群双加氧酶基因多态性分析.docxVIP

准东油田萘降解菌群培养及菌群双加氧酶基因多态性分析 　　摘要：采用无机盐培养基，以萘为惟一的碳源物质?ψ级?油田石油污染土壤中的萘降解菌进行液体富集培养。经过5次培养后，利用固体平板对获得菌群萘降解能力进行检测，同时利用HPLC对降解产物进行检测。离心收集富集培养基中的菌体，提取基因组DNA，基于Illumina Miseq测序平台对16S rRNA序列进行测序及生物信息学分析，同时采用萘双加酶基因通用引物对萘双加氧酶基因片段进行扩增、测序及分析。结果表明，含有萘的固体平板有清晰透明斑的出现；高效液相检测结果显示，萘被降解成小分子产物，证明液体培养基可富集培养获得萘降解菌群。萘降解菌群16S rRNA测序比对结果显示，富集培养获得菌群分属于5门9纲15目24科31属，共33种细菌。另外共获得萘双加氧酶基因片段11条，GenBank注册号为KY-KY。系统发育树结果显示，所获萘双加氧酶基因分为4个类群。通过HPLC及双加氧酶基因的分子检测，验证已从准东油田石油污染土壤当中富集培养获得萘降解菌群。　　关键词：萘降解菌；高效液相色谱；细菌菌群；萘双加氧酶基因；淮东油田　　中图分类号： 文献标识码：A 文章编号：0439--3726-05　　DOI：/　　Abstract： According to the literature， liquid enrichment medium supplemented with naphthalene as the sole carbon was used to culture the naphthalene degrading microbial consortia from the contaminated soil in Xinjiang Zhundong oilfield. After 5 repeated cultivation， the degradation ability of microbial consortia was detected by the clarity circle method on plate， and the degradation products were detected by HPLC. In addition， the enriched microbial consortia were collected by centrifugation. The genomic DNA were isolated from the microbial consortia and the 16S rRNA gene was amplified and sequenced based on Illumina Miseq sequencing platform. Besides， the the naphthalene dioxygenase genes were amplified with universal primers for sequencing and bioinformatics analysising. The 16S rRNA sequencing results showed that the enriched microbial consortia were divided into 5 phyla， 9 classes， 15 orders， 24 families， 31 genera and 33 species. In addition， a total of 11 naphthalene dioxygenase gene segments were obtained and GenBank accession numbers were KY-KY， which could be divided into 4 clusters by phylogenetic analysis. Through the HPLC and naphthalene dioxygenase gene molecular detection proved that naphthalene degrading microbial flora have been obtained through the liquid enrichment culture.　　Key words： naphthalene degrading bacteria；high performance liquid chromatography；microbial consortia；naphthalene dioxygenase gene；Zhundong Oilfield