Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum  

作　　者：唐祥海 于仁成 周名江 于志刚 

机构地区：[1]Key Laboratory of Marine Chemistry Theory and Technology,Ministry of Education,Ocean University of China [2]Key Laboratory of Marine Ecology and Environmental Sciences,Institute of Oceanology,Chinese Academy of Sciences

出　　处：《Chinese Journal of Oceanology and Limnology》2012年第2期256-263,共8页中国海洋湖沼学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China(No.41006082);the Postdoctoral Science Foundation(No.20090461272);the National Natural Science Foundation of China for Creative Research Groups(No.40821004)

摘　　要：The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms （HABs）. This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization （FISH） to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences （GenBank accession numbers JF906989-JF906999） of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade （＂Global clade＂） of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP （paralytic shellfish poisoning）-producing PC clade （＂Pacific clade＂）. The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol （M-GC-1, M-PC-3）, or just nucleolus （M-PC-2）. Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

关 键 词：fuorescence in situ hybridization （FISH） Alexandrium minutum rRNA probe 

分 类 号：X835[环境科学与工程—环境工程]