| CHEN Guofu,WANG Quanfu,ZHANG Chunyun,ZHANG Baoyu,WANG Guangce,LU Douding,XU Zhong,YAN Peishen. 2013. Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization. Acta Oceanologica Sinica, 32(2):66－75 |
| Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization |
| Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization |
| Received:May 19, 2011 Revised:November 05, 2011 |
| DOI：10.1007/s13131-013-0278-4 |
| Key words:Prorocentrum minimum Karenia mikimotoi fluorescence in situ hybridization taxonomic probe |
| 中文关键词: Prorocentrum minimum Karenia mikimotoi fluorescence in situ hybridization taxonomic probe |
| 基金项目:Shandong Province Young and Middle-Aged Scientists Research Awards Fund under contract No. BS2010HZ002; the National Natural Science Foundation of China under contract Nos 41106082 and 41176141; Key Laboratory of Marine Ecology and Environmental Science and Engineering, SOA under contract No. MESE-2011-06; the Basic Research of Harbin Institute of Technology Outstanding Talents Cultivation Plan of Class III. |
| Author Name | Affiliation | E-mail | | CHEN Guofu | Harbin Institute of Technology(Weihai), Weihai 264209, China
The First Institute of Oceanography, State Ocenic Administration, Qingdao 266061, China | | | WANG Quanfu | Harbin Institute of Technology(Weihai), Weihai 264209, China | wangquanfu2000@126.com | | ZHANG Chunyun | Harbin Institute of Technology(Weihai), Weihai 264209, China | linmeng23@sohu.com | | ZHANG Baoyu | Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China | | | WANG Guangce | Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China | | | LU Douding | The Second Institute of Oceanography, State Ocenic Administration, Hangzhou 310012, China | | | XU Zhong | Harbin Institute of Technology(Weihai), Weihai 264209, China | | | YAN Peishen | Harbin Institute of Technology(Weihai), Weihai 264209, China | |
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| Abstract: |
| Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p >0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae. |
| 中文摘要: |
| Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p >0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae. |
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