米氏凯伦藻细胞表面膜蛋白质组及其对温度变化的响应研究
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摘要
米氏凯伦藻(Karenia mikimotoi)近年在我国福建、浙江和广东沿海经常形成赤潮,其赤潮不仅影响到海洋生态系统的稳定,也严重威胁到水产养殖以及人类生命健康安全。本论文以米氏凯伦藻为研究对象,建立了米氏凯伦藻细胞表面膜蛋白质荧光标记技术和细胞膜蛋白质提取方法,运用荧光差异凝胶电泳技术(2-DDIGE)对膜蛋白质进行了分析,并研究了米氏凯伦藻的膜蛋白质组及其对环境温度变动的响应。实验共鉴定到44个细胞表面膜蛋白,其中有效注释27个,主要为转运蛋白、HSP70蛋白家族和捕光蛋白等。米氏凯伦藻在20°C条件下的细胞生长和光合作用要明显好于16°C和12°C,但16°C和12°C条件下的差别不大,表明低温限制了米氏凯伦藻的生长。当米氏凯伦藻从12°C快速转移至16°C和20°C时,藻细胞密度和光合作用效率短时间迅速降低,但细胞很快即适应温度变化。细胞膜上的转运蛋白和光合作用蛋白在其适应温度变化中起着重要作用。
Karenia mikimotoi is a toxic harmful algal bloom species distributed in the temperate and tropical coastal waters, and it can produce hemolytic toxin and fish toxin. In recent years, it often causes blooms in the coastal areas of Fujian, Zhejiang, and Guangdong provinces of China, resulting in the destabilization of the marine ecosystem and serious harms to local aquaculture and human health or even life. In this paper, fluorescence labeling and sequential extraction of cell membrane proteins from K. mikimotoi were established. Cell surface membrane proteins were analyzed using fluorescence difference gel electrophoresis(2-D DIGE), and the response of membrane proteome of K. mikimotoi to a changing environmental temperature was studied. In total, 44 cell surface membrane proteins were identified, of which 27 were annotated effectively, including mainly transporters, HSP70 protein family, and light harvesting protein. Cell growth and photosynthesis of K. mikimotoi grown in 20°C were better than those in 16°C and 12°C but no difference was found between 16°C and 12°C, indicating that a low temperature could limit the cell growth of K. mikimotoi. When ambient temperature of K. mikimotoi cells boosted rapidly from 12°C to 16°C or 20°C, cell density and photosynthesis efficiency decreased quickly but the cells could rapidly adapt to the temperature change. Therefore, we believe that the transport and photosynthetic proteins of a cell membrane play an important role in the cells' adaptation to a quick ambient temperature change.
Karenia mikimotoi is a toxic harmful algal bloom species distributed in the temperate and tropical coastal waters, and it can produce hemolytic toxin and fish toxin. In recent years, it often causes blooms in the coastal areas of Fujian, Zhejiang, and Guangdong provinces of China, resulting in the destabilization of the marine ecosystem and serious harms to local aquaculture and human health or even life. In this paper, fluorescence labeling and sequential extraction of cell membrane proteins from K. mikimotoi were established. Cell surface membrane proteins were analyzed using fluorescence difference gel electrophoresis(2-D DIGE), and the response of membrane proteome of K. mikimotoi to a changing environmental temperature was studied. In total, 44 cell surface membrane proteins were identified, of which 27 were annotated effectively, including mainly transporters, HSP70 protein family, and light harvesting protein. Cell growth and photosynthesis of K. mikimotoi grown in 20°C were better than those in 16°C and 12°C but no difference was found between 16°C and 12°C, indicating that a low temperature could limit the cell growth of K. mikimotoi. When ambient temperature of K. mikimotoi cells boosted rapidly from 12°C to 16°C or 20°C, cell density and photosynthesis efficiency decreased quickly but the cells could rapidly adapt to the temperature change. Therefore, we believe that the transport and photosynthetic proteins of a cell membrane play an important role in the cells' adaptation to a quick ambient temperature change.
引文
李成,2011.海洋甲藻蛋白质组学研究:以亚历山大藻为例.厦门:厦门大学博士学位论文,36-96
沈盎绿,2014.东海原甲藻和米氏凯伦藻种间竞争对海水温度变化的响应机制.上海:华东师范大学博士学位论文,102-113
张传松,2008.长江口及邻近海域赤潮生消过程特征及其营养盐效应分析.青岛:中国海洋大学博士学位论文,96-125
郭伟其,沙伟,沈红梅等,2005.东海沿岸海水表层温度的变化特征及变化趋势.海洋学报,27(5):1-8
Anaya C C,Church N,Lewis J P,2007.Detection and identification of bacterial cell surface proteins by fluorescent labeling.Proteomics,7(2):215-219
Bertomeu T,Hastings J W,Morse D,2003.Vectorial labeling of dinoflagellate cell surface proteins.Journal of Phycology,39(6):1254-1260
Bivik C,Rosdahl I,?llinger K,2007.Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes.Carcinogenesis,28(3):537-544
Cui S X,Huang F,Wang J et al,2005.A proteomic analysis of cold stress responses in rice seedlings.Proteomics,5(12):3162-3172
Dan?ík V,Addona T A,Clauser K R et al,1999.De novo peptide sequencing via tandem mass spectrometry.Journal of Computational Biology,6(3-4):327-342
Heller K,Kadner R J,1985.Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.Journal of Bacteriology,161(3):904-908
Hicke L,Dunn R,2003.Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.Annual Review of Cell and Developmental Biology,19(1):141-172
Higgins C F,Linton K J,2004.The ATP switch model for ABCtransporters.Nature Structural&Molecular Biology,11(10):918-926
Hockin N L,Mock T,Mulholland F et al,2012.The response of diatom central carbon metabolism to nitrogen starvation is different from that of green algae and higher plants.Plant Physiology,158(1):299-312
Holmes A R,Keniya M V,Ivnitski-Steele I et al,2012.The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFStransporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.Antimicrobial Agents and Chemotherapy,56(3):1508-1515
Jang J H,Hanash S,2003.Profiling of the cell surface proteome.Proteomics,3(10):1947-1954
Kadiu I,Wang T,Schlautman J D et al,2009.HIV-1 transforms the monocyte plasma membrane proteome.Cellular Immunology,258(1):44-58
Keller M D,Selvin R C,Claus W et al,1987.Media for the culture of oceanic ultraphytoplankton.Journal of Phycology,23(4):633-638
Koedding J,Howard P,Kaufmann L et al,2004.Dimerization of TonB is not essential for its binding to the outer membrane siderophore receptor FhuA of Escherichia coli.Journal of Biological Chemistry,279(11):9978-9986
Kornilovs’ka I,Nilsson I,Utt M et al,2002.Immunogenic proteins of Helicobacter pullorum,Helicobacter bilis and Helicobacter hepaticus identified by two‐dimensional gel electrophoresis and immunoblotting.Proteomics,2(6):775-783
Li C,Wang D Z,Dong H P et al,2012.Proteomics of a toxic dinoflagellate Alexandrium catenella DH01:detection and identification of cell surface proteins using fluorescent labeling.Chinese Science Bulletin,57(25):3320-3327
Liu P Q,Liu C E,Ames G F L,1999.Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter,the histidine permease.Journal of Biological Chemistry,274(26):18310-18318
Luche S,Santoni V,Rabilloud T,2003.Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two‐dimensional electrophoresis.Proteomics,3(3):249-253
Lundrigan M D,Kadner R J,1986.Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli.Homology among outer membrane receptors that interact with TonB.Journal of Biological Chemistry,261(23):10797-10801
Maloney P C,1990.Microbes and membrane biology.FEMSMicrobiology Letters,87(1-2):91-102
Maloney P C,1992.The molecular and cell biology of anion transport by bacteria.BioEssays,14(11):757-762
Marouga R,David S,Hawkins E,2005.The development of the DIGE system:2D fluorescence difference gel analysis technology.Analytical and Bioanalytical Chemistry,382(3):669-678
Mayrhofer C,Krieger S,Allmaier G et al,2006.DIGEcompatible labelling of surface proteins on vital cells in vitro and in vivo.Proteomics,6(2):579-585
Mitra S K,Gantt J A,Ruby J F et al,2007.Clouse and Michael B.Goshe.Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques.Journal of Proteome Research,6(5):1933-1950
Molloy M P,Herbert B R,Walsh B J et al,1998.Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis.Electrophoresis,19(5):837-844
Musil L S,Cunningham B A,Edelman G M et al,1990.Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines.The Journal of Cell Biology,111(5):2077-2088
Nylandsted J,Gyrd-Hansen M,Danielewicz A et al,2004.Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.The Journal of Experimental Medicine,200(4):425-435
Palenik B,Koke J A,1995.Characterization of a nitrogenregulated protein identified by cell surface biotinylation of a marine phytoplankton.Applied and Environmental Microbiology,61(9):3311-3315
Pressler U,Staudenmaier H,Zimmermann L et al,1988.Genetics of the iron dicitrate transport system of Escherichia coli.Journal of Bacteriology,170(6):2716-2724
Rea P A,2007.Plant ATP-binding cassette transporters.Annual Review of Plant Biology,58:347-375
Shin B K,Wang H,Yim A M et al,2003.Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.Journal of Biological Chemistry,278(9):7607-7616
Snyders S,Kohorn B D,1999.TAKs,thylakoid membrane protein kinases associated with energy transduction.Journal of Biological Chemistry,274(14):9137-9140
Toulza E,Shin M S,Blanc G et al,2010.Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella(Dinophyceae).Applied and Environmental Microbiology,76(13):4521-4529
Varkey D,Mazard S,Ostrowski M et al,2015.Effects of low temperature on tropical and temperate isolates of marine Synechococcus.The ISME Journal,10(5):1252-1263
Verrier P J,Bird D,Burla B et al,2008.Plant ABC proteins-a unified nomenclature and updated inventory.Trends in Plant Science,13(4):151-159
Wallin E,von Heijne G,1998.Genome‐wide analysis of integral membrane proteins from eubacterial,archaean,and eukaryotic organisms.Protein Science,7(4):1029-1038
Wang D Z,Li C,Xie Z X et al,2011.Homology-driven proteomics of dinoflagellates with unsequenced genomes using MALDI-TOF/TOF and automated De novo sequencing.Evidence-Based Complementary and Alternative Medicine,2011:471020
Wang D Z,Zhang Y J,Zhang S F et al,2013.Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense(Dinophyceae).PLoS One,8(5):e63659
沈盎绿,2014.东海原甲藻和米氏凯伦藻种间竞争对海水温度变化的响应机制.上海:华东师范大学博士学位论文,102-113
张传松,2008.长江口及邻近海域赤潮生消过程特征及其营养盐效应分析.青岛:中国海洋大学博士学位论文,96-125
郭伟其,沙伟,沈红梅等,2005.东海沿岸海水表层温度的变化特征及变化趋势.海洋学报,27(5):1-8
Anaya C C,Church N,Lewis J P,2007.Detection and identification of bacterial cell surface proteins by fluorescent labeling.Proteomics,7(2):215-219
Bertomeu T,Hastings J W,Morse D,2003.Vectorial labeling of dinoflagellate cell surface proteins.Journal of Phycology,39(6):1254-1260
Bivik C,Rosdahl I,?llinger K,2007.Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes.Carcinogenesis,28(3):537-544
Cui S X,Huang F,Wang J et al,2005.A proteomic analysis of cold stress responses in rice seedlings.Proteomics,5(12):3162-3172
Dan?ík V,Addona T A,Clauser K R et al,1999.De novo peptide sequencing via tandem mass spectrometry.Journal of Computational Biology,6(3-4):327-342
Heller K,Kadner R J,1985.Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.Journal of Bacteriology,161(3):904-908
Hicke L,Dunn R,2003.Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.Annual Review of Cell and Developmental Biology,19(1):141-172
Higgins C F,Linton K J,2004.The ATP switch model for ABCtransporters.Nature Structural&Molecular Biology,11(10):918-926
Hockin N L,Mock T,Mulholland F et al,2012.The response of diatom central carbon metabolism to nitrogen starvation is different from that of green algae and higher plants.Plant Physiology,158(1):299-312
Holmes A R,Keniya M V,Ivnitski-Steele I et al,2012.The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFStransporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.Antimicrobial Agents and Chemotherapy,56(3):1508-1515
Jang J H,Hanash S,2003.Profiling of the cell surface proteome.Proteomics,3(10):1947-1954
Kadiu I,Wang T,Schlautman J D et al,2009.HIV-1 transforms the monocyte plasma membrane proteome.Cellular Immunology,258(1):44-58
Keller M D,Selvin R C,Claus W et al,1987.Media for the culture of oceanic ultraphytoplankton.Journal of Phycology,23(4):633-638
Koedding J,Howard P,Kaufmann L et al,2004.Dimerization of TonB is not essential for its binding to the outer membrane siderophore receptor FhuA of Escherichia coli.Journal of Biological Chemistry,279(11):9978-9986
Kornilovs’ka I,Nilsson I,Utt M et al,2002.Immunogenic proteins of Helicobacter pullorum,Helicobacter bilis and Helicobacter hepaticus identified by two‐dimensional gel electrophoresis and immunoblotting.Proteomics,2(6):775-783
Li C,Wang D Z,Dong H P et al,2012.Proteomics of a toxic dinoflagellate Alexandrium catenella DH01:detection and identification of cell surface proteins using fluorescent labeling.Chinese Science Bulletin,57(25):3320-3327
Liu P Q,Liu C E,Ames G F L,1999.Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter,the histidine permease.Journal of Biological Chemistry,274(26):18310-18318
Luche S,Santoni V,Rabilloud T,2003.Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two‐dimensional electrophoresis.Proteomics,3(3):249-253
Lundrigan M D,Kadner R J,1986.Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli.Homology among outer membrane receptors that interact with TonB.Journal of Biological Chemistry,261(23):10797-10801
Maloney P C,1990.Microbes and membrane biology.FEMSMicrobiology Letters,87(1-2):91-102
Maloney P C,1992.The molecular and cell biology of anion transport by bacteria.BioEssays,14(11):757-762
Marouga R,David S,Hawkins E,2005.The development of the DIGE system:2D fluorescence difference gel analysis technology.Analytical and Bioanalytical Chemistry,382(3):669-678
Mayrhofer C,Krieger S,Allmaier G et al,2006.DIGEcompatible labelling of surface proteins on vital cells in vitro and in vivo.Proteomics,6(2):579-585
Mitra S K,Gantt J A,Ruby J F et al,2007.Clouse and Michael B.Goshe.Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques.Journal of Proteome Research,6(5):1933-1950
Molloy M P,Herbert B R,Walsh B J et al,1998.Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis.Electrophoresis,19(5):837-844
Musil L S,Cunningham B A,Edelman G M et al,1990.Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines.The Journal of Cell Biology,111(5):2077-2088
Nylandsted J,Gyrd-Hansen M,Danielewicz A et al,2004.Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.The Journal of Experimental Medicine,200(4):425-435
Palenik B,Koke J A,1995.Characterization of a nitrogenregulated protein identified by cell surface biotinylation of a marine phytoplankton.Applied and Environmental Microbiology,61(9):3311-3315
Pressler U,Staudenmaier H,Zimmermann L et al,1988.Genetics of the iron dicitrate transport system of Escherichia coli.Journal of Bacteriology,170(6):2716-2724
Rea P A,2007.Plant ATP-binding cassette transporters.Annual Review of Plant Biology,58:347-375
Shin B K,Wang H,Yim A M et al,2003.Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.Journal of Biological Chemistry,278(9):7607-7616
Snyders S,Kohorn B D,1999.TAKs,thylakoid membrane protein kinases associated with energy transduction.Journal of Biological Chemistry,274(14):9137-9140
Toulza E,Shin M S,Blanc G et al,2010.Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella(Dinophyceae).Applied and Environmental Microbiology,76(13):4521-4529
Varkey D,Mazard S,Ostrowski M et al,2015.Effects of low temperature on tropical and temperate isolates of marine Synechococcus.The ISME Journal,10(5):1252-1263
Verrier P J,Bird D,Burla B et al,2008.Plant ABC proteins-a unified nomenclature and updated inventory.Trends in Plant Science,13(4):151-159
Wallin E,von Heijne G,1998.Genome‐wide analysis of integral membrane proteins from eubacterial,archaean,and eukaryotic organisms.Protein Science,7(4):1029-1038
Wang D Z,Li C,Xie Z X et al,2011.Homology-driven proteomics of dinoflagellates with unsequenced genomes using MALDI-TOF/TOF and automated De novo sequencing.Evidence-Based Complementary and Alternative Medicine,2011:471020
Wang D Z,Zhang Y J,Zhang S F et al,2013.Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense(Dinophyceae).PLoS One,8(5):e63659
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