重金属胁迫下河蚬内脏中GST mRNA表达及酶活性分析
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摘要
采用RACE技术克隆河蚬(Corbicula fluminea)谷胱甘肽-S-转移酶(glutathione S-transferase,GST)基因全长序列,利用DNAMAN 6.0软件序列进行比对,Smart分析保守域和功能位点,SignalIP 4.0预测分析该基因信号肽,MEGA 5.0绘制系统进化树,利用荧光定量PCR和酶活性技术检测Cd~(2+)、Cu~(2+)和Pb~(2+)胁迫后GST在河蚬肝胰腺中的表达特征,为河蚬内脏GST mRNA和酶生物标志物评价指标的建立提供参考。结果表明,GST基因全长952 bp(GenBank登录号:KX211963),编码一个由217个氨基酸组成的多肽,理论等电点PI为5.98,分子量为25.169kD。氨基酸序列中含有GST基因家族特有的GST-N结构域和GST-C结构域,系统进化分析表明,河蚬GST基因与菲律宾帘蛤(Rudiraps philippinarum)同源性最高,为90.32%。重金属胁迫结果显示:GST mRNA表达量和酶活性均高于相同时间点的对照组,并随着胁迫时间的延长呈先升高后降低趋势,每个实验组峰值出现时间略有差异,且峰值结果均显著高于相应的对照组(P<0.05)。Cd~(2+)胁迫后GST mRNA表达和酶活性峰值均出现在24 h,且m RNA在48 h的表达量也显著高于对照组(P<0.05);Cu~(2+)胁迫后GST mRNA表达峰值和酶活性峰值分别出现在12 h和48 h,且在24h胁迫组酶活性也显著高于对照组(P<0.05);Pb~(2+)胁迫后GST mRNA表达和酶活性峰值均出现在48 h,且24 h的mRNA表达量也显著高于对照组(P<0.05)。综上,河蚬GST对重金属刺激敏感,说明河蚬内脏中GST可以作为水体环境重金属污染的指示分子。
To lay a foundation for the establishment of glutathione S-transferase(GST) m RNA and enzyme biomarker evaluation indexes of Corbicula fluminatum viscera, the complete mRNA sequence of GST gene was obtained by RACE, sequence alignment was performed using DNAMAN 6.0 software, conserved domains and functional sites were predicted by Smart program, signal peptide was predicted with signalip 4.0 program. phylogenetic tree was drawn using MEGA 5.0 software, and the mRNA expression changes of GST in hepatopancreas of C. fluminea after Cd~(2+), Cu~(2+) and Pb~(2+) stress were detected by fluorescence quantitative PCR.Results showed that the full-length mRNA sequence of C. fluminea GST gene was 952 bp(GenBank accession number: KX211963),and encoded a protein of 217 amino acids with molecular weight of 25.169 kD and PI of 5.98, containing a GST-N domain and a GST-C domain. The amino acid sequences of GSTs between C. fluminea and Rudirapes philippinarum showed a similarity of more than 90.32%. The results indicated that the m RNA expression and enzymatic activities of GST in groups exposed to heavy metal were higher than those in the control group at each time point and increased first and then decreased with the prolongation of stress time, but the peaking time of each experimental group was slightly different and significantly higher than that in the control group(P<0.05). Under Cd~(2+) stress, both the m RNA expression level and enzymatic activities of GST reached to peak at 24 h, and the m RNA expression levels at 48 h was that significantly higher than that in the control group(P<0.05); Under Cu~(2+) stress, the mRNA expression level and enzymatic activities of GST respectively reached to peak at 12 h and 48 h, and the enzymatic activities at 24 h was significantly higher than that in the control group(P<0.05). Under Pb~(2+) stress, both the mRNA expression level and enzymatic activities of GST were peaking at 48 h, and the m RNA expression levels at 24 h was significantly higher than that in the control group(P<0.05). This experiment shows that GST is sensitive to heavy metal stimuli, indicating that GST in viscera of C. fluminea can be used as an indicator molecule to detect heavy metal pollution in water environment.
To lay a foundation for the establishment of glutathione S-transferase(GST) m RNA and enzyme biomarker evaluation indexes of Corbicula fluminatum viscera, the complete mRNA sequence of GST gene was obtained by RACE, sequence alignment was performed using DNAMAN 6.0 software, conserved domains and functional sites were predicted by Smart program, signal peptide was predicted with signalip 4.0 program. phylogenetic tree was drawn using MEGA 5.0 software, and the mRNA expression changes of GST in hepatopancreas of C. fluminea after Cd~(2+), Cu~(2+) and Pb~(2+) stress were detected by fluorescence quantitative PCR.Results showed that the full-length mRNA sequence of C. fluminea GST gene was 952 bp(GenBank accession number: KX211963),and encoded a protein of 217 amino acids with molecular weight of 25.169 kD and PI of 5.98, containing a GST-N domain and a GST-C domain. The amino acid sequences of GSTs between C. fluminea and Rudirapes philippinarum showed a similarity of more than 90.32%. The results indicated that the m RNA expression and enzymatic activities of GST in groups exposed to heavy metal were higher than those in the control group at each time point and increased first and then decreased with the prolongation of stress time, but the peaking time of each experimental group was slightly different and significantly higher than that in the control group(P<0.05). Under Cd~(2+) stress, both the m RNA expression level and enzymatic activities of GST reached to peak at 24 h, and the m RNA expression levels at 48 h was that significantly higher than that in the control group(P<0.05); Under Cu~(2+) stress, the mRNA expression level and enzymatic activities of GST respectively reached to peak at 12 h and 48 h, and the enzymatic activities at 24 h was significantly higher than that in the control group(P<0.05). Under Pb~(2+) stress, both the mRNA expression level and enzymatic activities of GST were peaking at 48 h, and the m RNA expression levels at 24 h was significantly higher than that in the control group(P<0.05). This experiment shows that GST is sensitive to heavy metal stimuli, indicating that GST in viscera of C. fluminea can be used as an indicator molecule to detect heavy metal pollution in water environment.
引文
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BLANCO-PENEDO I,CRUZ J M,LOPEZ-ALONSEl M,et al.,2006.Influence of copper status on the accumulation of toxic and essential metals in cattle[J].Environment International,32(7):901-906.
CHALE F M M,2002.Trace metal concentrations in water,sediments and fish tissue from lake Tanganyika[J].Science of the Total Environment,299(1-3):115-121.
CHEMALE G,PERALLY S,LACOURSE E J,et al.,2010.Comparative proteomic analysis of triclabendazole response in the liver fluck Fasciola hepatica[J].Journal of Proteome Research,9(10):4940-4951.
HARMS L,FRICKENHAUS S,SCHIff ER M,et al.,2014.Gene expression pro?ling in gills of the great spider crab Hyas araneus in response to ocean acidi?cation and warming[J].BMC Genomics 15:789.
LAURITANO C,ROMANO G,RONCALLI V,et al.,2016.New oxylipins produced at the end of a diatom bloom and their effects on copepod reproductive success and gene expression levels[J].Harmful Algae,55:221-229.
LENARTOVA V,HOLOVSKA K,JAVORSK Y,2000.The influence of environmental pollution on the SOD and GST-isoenzyme patterns[J].Water Science and Technology,42(12):209-214.
LU X,WANG C,LIU B Z,2015.The role of cu/Zn-SOD and Mn-SOD in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretix[J].Fish&Shell?sh Immunol,42(1):58-65.
PRETTO A,LORO V L,MORSCH V M,et al.,2010.Acetylcholinesterase activity,lipid peroxidation,and bioaccumulation in silver catfish(Rhamdia quelen)exposed to cadmium[J].Arch Environ Contam Toxicol,58(4):1008-1014.
ROSSI F,PALOMBELLA S,PIRRONE C,et al.,2016.Evaluation of tissue morphology and gene expression as biomarkers of pollution mussel Mytilus galloprovincialis caging experiment[J].Aquatic Toxicology,181:57-66.
SHEEHAN D,MEADE G,FOLEY V M,et al.,2001.Structure,Function and Evolution of Glutathione Transferases:Implications for Classification of Non-Mammalian Members of an Ancient Enzyme Superfamily[J].Biochemical Journal,360(Pt 1):1-16.
XIE Y H,2017.Molecular characterization of the HSP70 and HSP90 genes in Asian clam(Corbicula fluminea)and their expression analysis during heavy exposure[J].Gene Reports,7:18-24.
程炜轩,梁旭方,李观贵,等,2009.鳜鱼两种谷胱甘肽S-转移酶基因c DNA的克隆与分析[J].生态毒理学报,4(4):537-543.CHEN W X,LIANG X F,LI G G,et al.,2009.Molecular cloning and sequence analysis of alpha-and rho-classesof Glutathione S-Transferases Perch(Siniperca chuatsi)[J].Asian Journal of Ecotoxicology,4(4):537-543.
胡影,王志刚,徐伟慧,等,2016.两种典型细菌的生长和氧化应激酶对领苯二甲酸二丁酯的响应[J].生态环境学报,25(7):1225-1229.HU Y,WANG Z G,XU W H,et al.,2016.Response of bacterial growth and the activities of oxidative enzymes to di-n-butyl phthalate contamination[J].Ecology and Environmental Sciences,25(7):1225-1229.
蒋玫,李磊,沈新强,等,2013.底泥浸出液对脊尾白虾抗氧化解毒酶及相关基因表达的影响[J].生态环境学报,22(7):1182-1186.JIANG M,LI L,SHEN X Q,et al.,2013.Effects of lixivium from ooze mud exposure on activity of antioxidant enzymes and related genes expression of Exopalaemon carinicauda mysis larvae[J].Ecology and Environmental Sciences,22(7):1182-1186.
李雅琴,马晓国,张燕萍,等,2013.三聚氰胺在罗非鱼肝脏内的积累及对EROD和GST活性的影响[J].生态环境学报,22(12):1951-1955.LI Y Q,MA X G,ZHANG Y P,et al.,2013.Accumulation of melamine in tilapia liver and its effect on EROD and GST activities[J].Ecology and Environmental Sciences,22(12):1951-1955.
刘慧慧,何建瑜,赵荣涛,等,2014.重金属胁迫下厚壳贻贝谷胱甘肽S-转移酶基因表达分析[J].海洋与湖沼,45(2):274-280.LIU H H,HE J Y,ZHAO R T,et al.,2014.Molecular expression pattern of Glutathione S-transferases gene in mytilus coruscus exposed to heavy metals[J].Oceanologia et limnologia sinica,45(2):274-280.
罗凯娅,刘欣欣,葛端阳,等,2012.鳗弧菌(Vibrio anguillarum)侵染对青蛤(Cyclina sinensis)谷胱甘肽硫转移酶及其基因表达的影响[J].海洋与湖沼,43(4):735-740.LUO K Y,LIU X X,GE D Y,et al.,2012.Effect of Vibrio anguillarum on activity and gene expression of Glutathione S-Transferases in Cyclina sinensis[J].Oceanologia et limnologia sinica,43(4):735-740.
苗晶晶,2010.苯并[a]芘对栉孔扇贝分子毒理学机制的研究[D].青岛:中国海洋大学.MIAO J J,2010.Molecular Mechanism behind the Toxic effects of Benzo(a)Pyrene on Scallop Chlamys farreri[D].Qingdao,Ocean University of China Qingdao.
聂湘平,陈菊芳,王翔,等,2008.环丙沙星在异育银鲫(Allo-gynogenetic crucian)体内的积累分布及其毒性效应[J].生态学报,28(1):246-252.NIE X P,CHEN J F,WANG X,et al.,2008.Bioaccumulation of ciprofloxacin in Allogynogenetic crucian carp and ite toxic effects[J].Acta ecologica sinica,28(1):246-252.
曾丽璇,陈桂珠,余日清,等,2004.水环境中Cd和Cu污染对监测生物河蚬累积效应的影响[J].农业环境科学学报,23(50):964-967.ZENG L X,CHEN G Z,YU R Q,et al.,2004.Accumalation effecte of biomonitoring indicator Asian clam(Coebicula fluminea)under different Cd and Cu pollution condition[J].Journal of Agro-Environment Science,23(50):964-967.
张永国,亢晋勇,2006.谷胱甘肽硫转移酶的生理功能述评[J].忻州师范学院学报,22(3):125-128.ZHANG Y G,KANG J Y,2006.Review of physiology roles of Glutathione S-Transferases[J].Journal of Xin Zhou Teacher University,22(3):125-128.
左伟东,余泉友,李斌,等,2007.家蚕谷胱甘肽转移酶基因的克隆、序列分析及其表达模式[J].农业生物技术学报,15(4):595-601.ZUO W D,YU Q Y,LI B,et al.,2007.Cloning,sequence analysis and expression pattern of Glutathione S-transferase gene in bombyx mori[J].Journal of Agricultural Biotechnology,15(4):595-601.
BLANCHETTE B N,SINGH B R,2002.Induction of glutathione S-transferase in the Northern Quahog Mercenaria mercenaria after exposure to the polychlorinated biphenyl(PCB)mixture aroclor1248[J].Journal of Protein Chemistry,21(8):489-494.
BLANCO-PENEDO I,CRUZ J M,LOPEZ-ALONSEl M,et al.,2006.Influence of copper status on the accumulation of toxic and essential metals in cattle[J].Environment International,32(7):901-906.
CHALE F M M,2002.Trace metal concentrations in water,sediments and fish tissue from lake Tanganyika[J].Science of the Total Environment,299(1-3):115-121.
CHEMALE G,PERALLY S,LACOURSE E J,et al.,2010.Comparative proteomic analysis of triclabendazole response in the liver fluck Fasciola hepatica[J].Journal of Proteome Research,9(10):4940-4951.
HARMS L,FRICKENHAUS S,SCHIff ER M,et al.,2014.Gene expression pro?ling in gills of the great spider crab Hyas araneus in response to ocean acidi?cation and warming[J].BMC Genomics 15:789.
LAURITANO C,ROMANO G,RONCALLI V,et al.,2016.New oxylipins produced at the end of a diatom bloom and their effects on copepod reproductive success and gene expression levels[J].Harmful Algae,55:221-229.
LENARTOVA V,HOLOVSKA K,JAVORSK Y,2000.The influence of environmental pollution on the SOD and GST-isoenzyme patterns[J].Water Science and Technology,42(12):209-214.
LU X,WANG C,LIU B Z,2015.The role of cu/Zn-SOD and Mn-SOD in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretix[J].Fish&Shell?sh Immunol,42(1):58-65.
PRETTO A,LORO V L,MORSCH V M,et al.,2010.Acetylcholinesterase activity,lipid peroxidation,and bioaccumulation in silver catfish(Rhamdia quelen)exposed to cadmium[J].Arch Environ Contam Toxicol,58(4):1008-1014.
ROSSI F,PALOMBELLA S,PIRRONE C,et al.,2016.Evaluation of tissue morphology and gene expression as biomarkers of pollution mussel Mytilus galloprovincialis caging experiment[J].Aquatic Toxicology,181:57-66.
SHEEHAN D,MEADE G,FOLEY V M,et al.,2001.Structure,Function and Evolution of Glutathione Transferases:Implications for Classification of Non-Mammalian Members of an Ancient Enzyme Superfamily[J].Biochemical Journal,360(Pt 1):1-16.
XIE Y H,2017.Molecular characterization of the HSP70 and HSP90 genes in Asian clam(Corbicula fluminea)and their expression analysis during heavy exposure[J].Gene Reports,7:18-24.
程炜轩,梁旭方,李观贵,等,2009.鳜鱼两种谷胱甘肽S-转移酶基因c DNA的克隆与分析[J].生态毒理学报,4(4):537-543.CHEN W X,LIANG X F,LI G G,et al.,2009.Molecular cloning and sequence analysis of alpha-and rho-classesof Glutathione S-Transferases Perch(Siniperca chuatsi)[J].Asian Journal of Ecotoxicology,4(4):537-543.
胡影,王志刚,徐伟慧,等,2016.两种典型细菌的生长和氧化应激酶对领苯二甲酸二丁酯的响应[J].生态环境学报,25(7):1225-1229.HU Y,WANG Z G,XU W H,et al.,2016.Response of bacterial growth and the activities of oxidative enzymes to di-n-butyl phthalate contamination[J].Ecology and Environmental Sciences,25(7):1225-1229.
蒋玫,李磊,沈新强,等,2013.底泥浸出液对脊尾白虾抗氧化解毒酶及相关基因表达的影响[J].生态环境学报,22(7):1182-1186.JIANG M,LI L,SHEN X Q,et al.,2013.Effects of lixivium from ooze mud exposure on activity of antioxidant enzymes and related genes expression of Exopalaemon carinicauda mysis larvae[J].Ecology and Environmental Sciences,22(7):1182-1186.
李雅琴,马晓国,张燕萍,等,2013.三聚氰胺在罗非鱼肝脏内的积累及对EROD和GST活性的影响[J].生态环境学报,22(12):1951-1955.LI Y Q,MA X G,ZHANG Y P,et al.,2013.Accumulation of melamine in tilapia liver and its effect on EROD and GST activities[J].Ecology and Environmental Sciences,22(12):1951-1955.
刘慧慧,何建瑜,赵荣涛,等,2014.重金属胁迫下厚壳贻贝谷胱甘肽S-转移酶基因表达分析[J].海洋与湖沼,45(2):274-280.LIU H H,HE J Y,ZHAO R T,et al.,2014.Molecular expression pattern of Glutathione S-transferases gene in mytilus coruscus exposed to heavy metals[J].Oceanologia et limnologia sinica,45(2):274-280.
罗凯娅,刘欣欣,葛端阳,等,2012.鳗弧菌(Vibrio anguillarum)侵染对青蛤(Cyclina sinensis)谷胱甘肽硫转移酶及其基因表达的影响[J].海洋与湖沼,43(4):735-740.LUO K Y,LIU X X,GE D Y,et al.,2012.Effect of Vibrio anguillarum on activity and gene expression of Glutathione S-Transferases in Cyclina sinensis[J].Oceanologia et limnologia sinica,43(4):735-740.
苗晶晶,2010.苯并[a]芘对栉孔扇贝分子毒理学机制的研究[D].青岛:中国海洋大学.MIAO J J,2010.Molecular Mechanism behind the Toxic effects of Benzo(a)Pyrene on Scallop Chlamys farreri[D].Qingdao,Ocean University of China Qingdao.
聂湘平,陈菊芳,王翔,等,2008.环丙沙星在异育银鲫(Allo-gynogenetic crucian)体内的积累分布及其毒性效应[J].生态学报,28(1):246-252.NIE X P,CHEN J F,WANG X,et al.,2008.Bioaccumulation of ciprofloxacin in Allogynogenetic crucian carp and ite toxic effects[J].Acta ecologica sinica,28(1):246-252.
曾丽璇,陈桂珠,余日清,等,2004.水环境中Cd和Cu污染对监测生物河蚬累积效应的影响[J].农业环境科学学报,23(50):964-967.ZENG L X,CHEN G Z,YU R Q,et al.,2004.Accumalation effecte of biomonitoring indicator Asian clam(Coebicula fluminea)under different Cd and Cu pollution condition[J].Journal of Agro-Environment Science,23(50):964-967.
张永国,亢晋勇,2006.谷胱甘肽硫转移酶的生理功能述评[J].忻州师范学院学报,22(3):125-128.ZHANG Y G,KANG J Y,2006.Review of physiology roles of Glutathione S-Transferases[J].Journal of Xin Zhou Teacher University,22(3):125-128.
左伟东,余泉友,李斌,等,2007.家蚕谷胱甘肽转移酶基因的克隆、序列分析及其表达模式[J].农业生物技术学报,15(4):595-601.ZUO W D,YU Q Y,LI B,et al.,2007.Cloning,sequence analysis and expression pattern of Glutathione S-transferase gene in bombyx mori[J].Journal of Agricultural Biotechnology,15(4):595-601.