| Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry |
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| 作者姓名： | HOU Jianjun LAI Hongyan HUANG Bangqin CHEN Jixin |
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| 作者单位： | HOU Jianjun(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;College of Life Sciences,Hubei Normal University,Huangshi 435002,China;State Key Laboratory of Marine Environmental Science,Environmental Science Research Center,Xiamen University.Xiamen 361005.China;Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology,Yangtse River Fisheries Research Institute,Jinzhou 434000.China;State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China);LAI Hongyan(College of Life Sciences,Hubei Normal University,Huangshi 435002,China;Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology,Yangtse River Fisheries Research Institute,Jinzhou 434000.China);HUANG Bangqin,CHEN Jixin(State Key Laboratory of Marine Environmental Science,Environmental Science Research Center,Xiamen University.Xiamen 361005.China)
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| 基金项目： | The Fujian Provincial Government of China，the Xiamen Municipal Government of China，the China Postdoctoral Science Foundation，the Open Fund of the State Key Laboratory of Freshwater Ecology and Biotechnology;Chinese Academy of Sciences，高等学校博士学科点专项科研基金，the Open Fund of the Key Laboratory of Freshwater Fish Germplasm and Biotechnology of Ministry of Agriculture;Chinese Academy of Fishery Sciences，国家自然科学基金 |
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| 摘 要： | Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02＞PM28S02＞PM28S01＞PM18S01,and that of the probes specific to T. pulchella was TP18S02＞TP28S01＞TP28S02＞TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.
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| 关 键 词： | 荧光原位杂交 微小原甲藻 流式细胞仪 荧光显微镜 高山 识别 DNA探针 寡核苷酸探针 |
| 收稿时间： | 2008/2/27 0:00:00 |
| 修稿时间： | 2008/9/16 0:00:00 |
Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry |
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| HOU Jianjun,LAI Hongyan,HUANG Bangqin,CHEN Jixin.Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry[J].Acta Oceanologica Sinica,2009,28(2):103-114. |
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| Authors: | HOU Jianjun LAI Hongyan HUANG Bangqin and CHEN Jixin |
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| Institution: | 1
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
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College of Life Sciences, Hubei Normal University, Huangshi 435002, China
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State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005, China
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Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology, Yangtse River Fisheries Research Institute, Jinzhou 434000, China
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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China |
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| Abstract: | Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were ampli.ed, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a .uorescence in situ hybridization method using .ow cytometry. Epi.uorescence micrographs showed that these speci.c probes labeled with .uorescein isothiocyanate entered the algal cells and bound to target sequences, and the .uorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were speci.c and e.ective for P. minimum and T. pulchella, without any speci.c binding to other algal species. The hybridization e.ciency of di.erent probes speci.c to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes speci.c to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The di.erent hybridization e.ciency of the DNA probes could also be shown in the .uorescent signals between the labeled and unlabeled cells demonstrated using .ow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples. |
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| Keywords: | oligonucleotide DNA probes Prorocentrum minimum Takayama pulchella fluorescence in situ hybridization flow cytometry |
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