不同养殖时期刺参肠道内菌群结构的分析

Bacterial flora in gut contents of sea cucumber Apostichopus japonicus at different culture periods

为研究不同养殖时期刺参Apostichopus japonicus肠道内的细菌菌群结构,采用PCR-DGGE技术对DGGE凝胶中的主要条带进行切割、回收、克隆、测序。结果表明：3、4、5、6、9、10月,大连瓦房店海区养殖刺参肠道内细菌16S r DNA-V3可变区序列经PCR扩增及DGGE电泳后分别获得49、49、54、53、52、51个条带;3月与9月的菌群结构相似度最高,为67.9%,4月与10月次之,为63.8%;经条带序列分析得到43个主要条带对应的核酸序列,11个与未培养菌相似,32个与归属为7个细菌类群的细菌序列相似,包括α-变形菌纲、γ-变形菌纲、δ-变形菌纲、黄杆菌纲、蓝藻纲、绿弯菌门、疣微菌门;α-变形菌纲、γ-变形菌纲、黄杆菌纲是不同养殖时期刺参肠道内的主要细菌群落,其中γ-变形菌纲所占比例最高,达25.00%~35.71%,除3月和9月外,黄杆菌纲的相对含量均高于α-变形菌纲。研究表明,春秋两季刺参肠道内菌群结构呈现类似的演替变化规律,α-变形菌纲所占比例下降,而γ-变形菌纲与黄杆菌纲所占比例上升。

The bacterial flora in gut contents was studied in cultured sea cucumber Apostichopus japonicus collected on June, September and October of 2009, and March, April and May of 2010 by PCR-DGGE in which variable re- gional fragments of 16S rDNA-V3 were amplified by specific primers of bacteria. There were 49, 49, 54, 53, 52 and 51 bands in the samples collected on March, April, May, June, September and October, respectively, in the DGGE gels which were cut, recovered, cloned, and sequenced. The maximal similarity （67.9%） in bacterial flo- ra was observed in the samples collected on March and September, followed by the samples （63.8%） collected on between April and October. The sequencing analysis revealed that 11 nucleic acid sequences were similar to uncul- tured bacteria, and 32 sequences were similar to the sequences of 7 groups of bacterial including α -proteobacte- ria, γ-proteobaeteria, δ-proteobacteria, Flavobaeteria, Cyanobacteria, Chloroflexi and Verrueomicrobia in the 43 major bands. The dominant groups in the bacterial flora in different culture periods were found to be α-proteobacte- ria, γ-proteobaeteria and Flavobacteria. It was found that γ-proteobaeteria had the maximal proportion, varying from 25.00% to 35.71%. There was higher relative count in Flavobaeterium than α-proteobacteria in most sam- ples except that on March and September. The findings indicated that similar flora succession was observed in spring and autumn, the proportion of α-proteobacteria being declined while that of γ-proteobacteria and Flavobac-terium elevated.