新疆艾比湖沉积物中免培养放线菌的多样性

Actinobacterial diversity in sediments of Ebinur lake, Xinjiang, China

摘要:

[目的]采用免培养的方法研究新疆艾比湖湖底沉积物放线菌组成及多样性。[方法]采集并混合5份艾比湖湖底沉积物样本，并提取其总DNA，采用放线菌通用引物对其16S rRNA基因序列进行Touchdown PCR，构建放线菌16S rRNA基因文库。蓝白斑筛选后随机挑选白色克隆分析，利用限制性内切酶Hha Ⅰ进行酶切分型，挑选具有独特限制性片段差异的阳性克隆进行测序分析。序列经ChimeraCheck检测，BLAST同源比对及构建16S rRNA基因序列系统发育树。[结果]随机挑选192个白色克隆，其中166个为阳性克隆，选取51个具有独特限制性片段差异的克隆进行序列分析。测序结果进行比对以及Chimera Check检测后，共获得36个可操作单元（Operational Taxonomic Units, OTUs），GenBank注册号为KR182090-KR182131。文库覆盖度结果表明克隆文库涵盖了本环境中90.4%的放线菌类群。聚类结果显示，艾比湖湖底沉积物中放线菌分为2个类群，第1个类群属于放线菌门（Actinobacteria），放线菌纲（Actinobacteria）中的放线菌目（Actinomycetales）、丙酸杆菌目（Propionibacteriales）、微球菌目（Micrococcales）和棒杆菌目（Corynebacteriales）4个目，该类群占克隆文库18.1%；另外1个类群属于Unclassified Actinobacteria的类群，分为3个不同的group，占整个克隆文库的81.9%。[结论]新疆艾比湖湖底沉积物中存在多种未知的放线菌类群。

关键词:盐湖;艾比湖;放线菌;多样性

Abstract:

[Objective] The goal of this study was to identify uncultured actinobacterial diversity in Xinjiang Ebinur salt lake sediment.[Methods] Total DNA was extracted from 5 sediments of Ebinur lake and the actinobacterial 16S rRNA gene clone libraries were constructed through touchdown PCR program with the common primers. White clones were randomly selected after blue-white spot screening and detected by PCR. Positive clones were analyzed by restriction fragment length polymorphism (RFLP) with restriction enzyme Hha Ⅰ, and clones with unique RFLP were selected to analyze their sequences. After Chimera Check analysis, the sequence homology was analyzed by BLAST and phylogenetic tree was constructed by MEGA 5.0 software.[Results] In total 192 white clones were randomly selected and 166 clones were positive clones. The sequences of 51 clones with unique RFLP were analyzed and detected with Check Chimera program. The results show that 36 OTUs were obtained and GenBank accession numbers were KR182090-KR182131. The library coverage C value was 90.4% and 36 OTUs could be divided into 2 clusters by phylogenetic analysis. The first cluster accounted for 18.1% of the library and belonged to one class Actinobacteria of phylum Actinobacteria, which contained 4 orders including Actinomycetales, Propionibacteriales, Micrococcales and Corynebacteriales. Another cluster was the unclassified actinobacteria, divided into 3 groups and accounted for 81.9% of the library.[Conclusion] Ebinur Lake has a lot of unknown actinobacteria that need to be further investigated.

Key words:salt lake;Ebinur lake;actinobacteria;diversity