应用DGGE技术分析流清河湾扇贝养殖海区细菌群落结构的季节变化

细菌是海区生态环境的重要组成部分,为了解扇贝养殖海区细菌群落结构的季节性变化,连续11个月(2009年5月—2010年4月,2月除外)定期采集青岛流清河湾扇贝养殖海区水深0.5 m处水样,过滤收集粒径0.22～3.00μm的微生物,采用CTAB法提取样品总DNA,PCR扩增细菌16S rDNA V3-V5可变区序列,并通过DGGE技术对所得序列进行分离,结果共得到36条不同位置的条带,1月份水样细菌群落条带数最为丰富,3月份水样的细菌群落条带数最少。基于各月份DGGE条带数目和相对光密度,对11个月份样品的细菌群落多样性进行UPGMA聚类分析,结果表明,3、4和5月份细菌群落多样性具有较高的相似性,优先聚为一支,其余月份中,7月和8月,9月和12月,10月和11月分别优先聚为一支,而1月份和6月份均单独分为一支。剪切DGGE图谱中的20条主要谱带重扩增、测序,并进行BLAST比对。结果表明,20条序列与已知序列的相似性均在94%以上,这20条序列所代表的细菌分属于α-变形菌亚门(α-Proteobacteria)、β-变形菌亚门(β-Proteobacteria)、γ-变形菌亚门(γ-Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和疣微菌门(Verrucomicrobia)。

Seasonal changes of bacterial community composition in the scallop culture area of Liuqinghe Bay studied by PCR-DGGE

The seasonal changes of bacterial community composition in the scallop culture area of Liuqinghe Bay in Qingdao were analyzed in this study.Five liters of water samples from the 0.5 m depth were collected each time every month from May 2009 to April 2010,except February,and the one liter water samples were filtered by sterilized micro-filtration membranes to collect the micro-organisms within the size of 0.22 μm to 3.00 μm.Total DNA of the samples were extracted by CTAB method,the bacterial 16S rDNA V3-V5 variable sequence was amplified by PCR,the amplified sequences were separated by DGGE(denaturing gradient gel electrophoresis)techniques,and the DGGE fingerprint was analyzed by software Quantity One.Totally,36 bands with different positions were showed.The most abundant bacterial composition was found in January,and the least was found in March.Based on the the UPGMA cluster analysis,the monthly samples were grouped into 2 seasonal types:March to May,June to January.A total of 20 dominant DGGE bands were successfully cloned and sequenced.Blast results showed that the similarities between these cloned sequences and the sequences in the GenBank were all over 94%.The 20 bands belonged to α-Proteobacteria,β-Proteobacteria,γ-Proteobacteria,Bacteroidetes,Actinobacteria,and Verrucomicrobia,respectively.