荧光原位杂交(fluorescence in situ hybridization,FISH)技术在染色体定位、基因克隆、遗传标记以及染色体畸变研究中得到了广泛的应用。随着分子生物学的飞速发展,用于FISH技术的染色体特异重复序列探针的开发和利用有了巨大进展。就...荧光原位杂交(fluorescence in situ hybridization,FISH)技术在染色体定位、基因克隆、遗传标记以及染色体畸变研究中得到了广泛的应用。随着分子生物学的飞速发展,用于FISH技术的染色体特异重复序列探针的开发和利用有了巨大进展。就近年来小麦、黑麦等FISH技术中染色体特异重复序列探针的研究进展及应用作一综述。展开更多
Chinese shrimp （Fenneropenaeus chinensis） is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to i...Chinese shrimp （Fenneropenaeus chinensis） is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization （FISH） was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene （rDNA） of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in E chinensis. The successful application ofFISH in E chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes ofpenaeid shrimps.展开更多
Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellat...Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.展开更多
文摘荧光原位杂交(fluorescence in situ hybridization,FISH)技术在染色体定位、基因克隆、遗传标记以及染色体畸变研究中得到了广泛的应用。随着分子生物学的飞速发展,用于FISH技术的染色体特异重复序列探针的开发和利用有了巨大进展。就近年来小麦、黑麦等FISH技术中染色体特异重复序列探针的研究进展及应用作一综述。
基金Supported by the Key Program of the National Natural ScienceFoundation of China(No.30730071)the National High-Tech Researchand Development Program of China(863 program)(No.2007AA09Z430)the General Program of National Natural Science Foundation of China(Nos.30471347&40706048)
文摘Chinese shrimp （Fenneropenaeus chinensis） is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization （FISH） was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene （rDNA） of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in E chinensis. The successful application ofFISH in E chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes ofpenaeid shrimps.
基金supported by the National Basic Research Program of China (973 Program) (No.2011CB403602)the National High Technology Research and Development Program of China (863 Program) (No.2007AA09200111)the National Marine Public Welfare Research Project (201205031-02)
文摘Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.
