应用DGGE技术研究扇贝养殖海域微型真核浮游生物多样性

Application of denaturing gradient gel electrophoresis(DGGE)to study the diversity of eukarytic nanoplankton in the sea area for scallop culture

摘要:

为了研究扇贝养殖海区微型真核浮游生物群落多样性,明确养殖扇贝发病时期高丰度微型真核浮游生物种类,探讨微型真核浮游生物与栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)水平传播的可能关系。于2009年和2010年从青岛流清河湾扇贝养殖海区采集了9个月份的海水样品,经25和3 μm的滤膜过滤收集海水中3~25 μm的浮游生物,扩增18S rDNA可变区序列,并利用变性梯度凝胶电泳(denaturing gradient gelelectrophoresis,DGGE)技术对扩增序列进行分离以分析微型真核浮游生物多样性。结果表明,该养殖海区微型真核生物包括甲藻、纤毛虫、眼虫、定鞭藻、硅藻、盘蜷虫、隐藻、领鞭毛虫、变形虫和Cercozoan,其中甲藻类和纤毛类生物的最高相对丰度分别达41.0%和38.2%,是海区的优势种类。各月份DGGE谱带聚类分析结果表明,2009年6、7、8、9月份浮游生物群落组成较为相似。中肋骨条藻在扇贝发病前后均有分布。结合相关扇贝AVNV已有的研究结果,研究认为中肋骨条藻是AVNV水平传播的参与者之一,但海区中广泛分布的甲藻和纤毛虫与AVNV传播的关系还有必要进一步研究。

关键词:浮游生物; 栉孔扇贝; 急性病毒性坏死病毒; 变性梯度凝胶电泳

Abstract:

In order to study the diversity of eukarytic nanoplankton in the sea area for scallop culture,and clarify the relationship between eukaryotic nanoplankton and transmission of zhikong scallop (Chlamys farreri) acute viral necrosis virus(AVNV),seawater samples were collected monthly in 2009 and 2010 from Qingdao Liuqing River Bay’s sea area.3-25 μm plankton components were filtered and amplified 18S rDNA variable region sequences from filtered samples were analyzed by DGGE technique.The results show that the main eukaryotic nanoplankton in the sea area included Dinozoa,Ciliophora,Euglenozoa,Haptophyta,Ochrophyta,Labyrinthulomycota,Cryptista,Choanozoa,Amoebozoa and Cercozoan.The amount of Dinozoa and Ciliophora was the highest at 41% and 38.2%,respectively.The cluster analysis based on mensal DGGE bands shows that the plankton community from May to November 2009 is similar in the diversity of eukarytic nanoplankton.According to the studied results on scallop AVNV,Skeletonema costatum was taken to be a vector of transmission of AVNV,and it distributed during the period of scallop massive death.However,the role of Dinozoa and Ciliophora during the transmission of AVNV has yet to be determined.

Key words:plankton; Chlamys farreri; acute viral necrosis virus(AVNV); DGGE