栉孔扇贝消化盲囊细菌群落组成的季节变化分析

摘要

为分析栉孔扇贝消化盲囊内细菌群落组成及其季节变化,于2009年6月—2010年6月(2月份除外)定期从青岛沙子口流清河湾扇贝养殖海区采集养殖的2龄栉孔扇贝,采用SDS方法提取消化盲囊的总DNA,并通过引物358f和907r采用PCR扩增细菌16S rDNA的V3～V5区序列,利用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分离所得扩增序列.DGGE图谱显示,12个月份的样品共有不同位置的谱带11条,其中9条谱带作为共有谱带在所有月份的栉孔扇贝消化盲囊中均有分布,占总谱带数的81.82％,表明整个养殖季节栉孔扇贝消化盲囊内的细菌群落组成相对稳定.对11条DGGE条带进行切胶回收、PCR扩增、纯化、克隆后测序,其中9条谱带克隆测序成功.经BLAST比对搜索,结果显示9条序列与NCBI数据库中的序列相似性达97％～99％,所代表的细菌分别属于4大类群,变形菌门( Proteobacteria)、厚壁菌门(Firmicutes)、蓝细菌门(Cyanobacteria)和放线菌门(Actinobacteria)；NCBI数据库中与其相近的细菌主要为马赛菌属(Massilia)、芽孢杆菌属(Bacillus)、微小杆菌属(Exiguobacterium)、聚球藻属(Synechococcus)、红球菌属(Rhodococcus)和未培养菌.%The seasonal changes of bacteria community composition in digestive diverticula of 2 age scallops Chlamys farreri were studied. The scallop samples were collected every month since June 2009 to June 2010 (except Feb. ) from Shazikou Liuqinghe sea area of Qingdao, which was the scallop culture area. The genomic DNA was extracted from digestive diverticula of scallop using SDS schizolysis method. Bacterial V3-V5 region of 16S rDNA was amplified with the 358f and 907r primers. Then, denaturing gradient gel electrophoresis(DGGE) was used to analyze and discriminate the amplified fragments. The DGGE fingerprint was analyzed by software Quantity One. The results showed that DGGE profiles exhibited 11 distinguishable bands,and 9 bands existed in all 12 months, the proportion of co-existed bands was 81. 82% , the results indicated that the bacterial community composition was relatively stable in the digestive diverticula. Only 9 DGGE bands were successfully re-amplified, cloned, sequenced and aligned with BLAST. The result showed that the bacterial sequences shared high similarities (97% -99% )with sequences logged on NCBI. And the 9 bands belong to 4 major groups: Proteobacteria, Firmicute, Cyanobacteria and Actinobacteria. The predominant bacteria species in digestive diverticula of scallops were Massilia, Bacillus, Synechococcus, Exiguobacterium ,Rhodococcus and uncultured bacteria.