六溴环十二烷胁迫下红树蚬荧光定量PCR内参基因稳定性分析
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摘要
【目的】筛选出不同浓度六溴环十二烷(HBCD)胁迫下红树蚬(Polymesoda erosa)实时荧光定量PCR(qRT-PCR)的最适内参基因,用以准确校准目的基因的表达,为进一步开展分子毒理研究和开发分子生物标志物奠定基础。【方法】通过荧光定量PCR技术监测候选内参18S核糖体RNA(18S rRNA)、β-肌动蛋白(β-actin)、甘油醛-3-磷酸脱氢酶(GAPDH)、α-微管蛋白(α-tubulin)等管家基因,在红树蚬各组织受不同浓度(0μg/L、0.86μg/L、8.6μg/L)六溴环十二烷(HBCD)胁迫后的表达量,利用qRT-PCR及geNorm、NormFinder、BestKeeper等分析软件对不同条件下的表达数据进行处理,从而筛选出适合荧光定量PCR的最佳内参基因。【结果】受不同浓度HBCD胁迫后,鳃及肝胰中各管家基因的表达稳定性排序整体为β-actin=α-tubulin>GAPDH>18S rRNA。【结论】可单独或共同使用两个内参基因(β-actin、α-tubulin)校准荧光定量结果。
【Objective】This experiment was to select the best reference gene for quantitative PCR under different concentrations of hexabromocyclododecane treatments,which will accurately calibrate the expression of objective genes and lay a foundation for further study on molecular toxicology research and development of molecular biomarkers.【Methods】The expression level of four candidate genes(18 S rRNA、β-actin、GAPDH 、α-tubulin)on different tissues of Polymesoda erosa under different concentrations of hexabromocyclododecane treatments were monitored by using qRT-PCR technology,and the expression data obtained from qRTPCR was analyzed with different software,such as BestKeeper,geNorm,NormFinder to select the best reference gene suitable for real-time PCR.【Results】The results showed that after being stressed by different concentrations of HBCD,the order of candidate genes expression in gill and hepatopancreas was:β-actin=α-tubulin>GAPDH >18 S rRNA.【Conclusion】The qRT-PCR results can be calibrated using two reference genes(β-actin,α-tubulin)alone or in combination.
【Objective】This experiment was to select the best reference gene for quantitative PCR under different concentrations of hexabromocyclododecane treatments,which will accurately calibrate the expression of objective genes and lay a foundation for further study on molecular toxicology research and development of molecular biomarkers.【Methods】The expression level of four candidate genes(18 S rRNA、β-actin、GAPDH 、α-tubulin)on different tissues of Polymesoda erosa under different concentrations of hexabromocyclododecane treatments were monitored by using qRT-PCR technology,and the expression data obtained from qRTPCR was analyzed with different software,such as BestKeeper,geNorm,NormFinder to select the best reference gene suitable for real-time PCR.【Results】The results showed that after being stressed by different concentrations of HBCD,the order of candidate genes expression in gill and hepatopancreas was:β-actin=α-tubulin>GAPDH >18 S rRNA.【Conclusion】The qRT-PCR results can be calibrated using two reference genes(β-actin,α-tubulin)alone or in combination.
引文
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[15]卢彤岩,杨雨辉,徐连伟,等.达氟沙星对施氏鲟的急性毒性及组织残留检测[J].中国水产科学,2004,11(6):542-547.LU T Y,YANG Y H,XU L W,et al.Acute toxicity of danofloxacin in Amur sturgeon and the body residue[J].Journal of Fishery Sciences of China,2004,11(6):542-547.
[16]吕晓华,古燕,宋艳.壬基酚对红鲫、草鱼和鲢鱼的毒性及组织蓄积研究[J].卫生研究,2012,41(5):785-789.LV X H,GU Y,SONG Y.Toxicity and tissue accumulation of nonylphenol in Carassius auratus red variety,Grass Carp and Sliver Carp[J].Journal of Hygiene Research,2012,41(5):785-789.
[17]许星鸿,阎斌伦,徐加涛,等.重金属镉胁迫对日本蟳抗氧化酶活力、丙二醛含量及组织蓄积的影响[J].水产科学,2014,33(9):551-555.XU X H,YAN B L,XU J T,et al.Effects of cadmium stress on activities of antioxidant enzymes,malonaldehyde content and cadmium accumulation in asian swimming crab Charybdis japonica[J].Fisheries Science,2014,33(9):551-555.
[18]ZHANG H,PAN L Q,TAO Y X,et al.Identification and expression of differentially expressed genes in clamVenerupis philippinarumin response to environmental pollutant hexabromocyclododecane(HB-CD)[J].Journal of Experimental Marine Biology and Ecology,2013,445(3):166-173.
[19]ZHANG H,PAN L Q,TAO Y X.Antioxidant responses in clamVenerupis philippinarumexposed to environmental pollutant hexabromocyclododecane[J].Environmental Science and Pollution Research,2014,21(13):8206-8215.
[20]ETSCHMANN B,WILCKEN B,STOEVESAND K,et al.Selection of reference genes for quantitative realtime PCR analysis in canine mammary tumors using the geNorm algorithm[J].Veterinary Pathology,2006,43(6):934-942.
[21]张艳君,朱志峰,陆融,等.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展,2007,3(5):546-550.ZHANG Y J,ZHU Z F,LU R,et al.Selection of control genes in transcription analysis of gene expression[J].Progress in Biochemistry and Biophysics,2007,3(5):546-550.
[22]马飞.极大节旋藻实时荧光定量PCR内参基因的选择[D].青岛:中国海洋大学,2012.MA F.Reference genes for Arthrospira maxima when using quantitative real-time PCR[D].Qingdao:Ocean University of China,2012.
[23]BUSTIN S A,BENES V,GARSON J A,et al.The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments[J].Clinical Chemistry,2009,55(4):611-622.
[24]MALLONA I,LISCHEWSKI S,WEISS J,et al.Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J].BMC Plant Biology,2010,10:4.
[25]车志群,莫祖英,孙仁杰,等.荧光定量PCR方法及分子标志物在海洋污染监测中的应用进展[J].生态科学,2015,34(5):233-240.CHE Z Q,MO Z Y,SUN R J,et al.Review on application of methods of realtime fluorescent quantitative PCR and molecular marker in marine pollution monitoring[J].Ecological Science,2015,34(5):233-240.
[26]BAI Z Y,LIN J Y,MA K Y,et al.Identification of housekeeping genes suitable for gene expression analysis in the pearl mussel,Hyriopsis cumingii,during biomineralization[J].Molecular Genetics and Genomics,2014,289(4):717-725.
[27]FENG L Y,YU Q,LI X,et al.Identification of reference genes for qRT-PCR analysis in Yesso Scallop patinopecten yessoensis[J].PLoS ONE,2013,8(9):e75609.
[28]CUBERO-LEON E,CIOCAN C M,MINIER C,et al.Reference gene selection for qPCR in mussel,Mytilus edulis,during gametoge nesis and exogenous estrogen exposure[J].Environmental Science and Pollution Research,2012,19(7):2728-2733.
[29]MORGA B,ARZUL I,FAURY N,et al.Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR[J].Fish&Shellfish Immunology,2010,29(6):937-945.
[30]MAURIZ O,MANEIRO V,PEREZ-PARALLE M,et al.Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.[J].Aquaculture,2012,370-371(2):158-165.
[31]LLERA-HERRERA R,GARCIA-GASCA A,HU-VET A,et al.Identification of a tubulin?αgene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus[J].Marine Genomics,2012,6:33-44.
[32]DU Y S,ZHANG L L,XU F,et al.Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster(Crassostrea gigas)development by quantitative real-time PCR[J].Fish&Shellfish Immunology,2013,34(3):939-945.
[33]王琦,何毛贤.合浦珠母贝实时定量PCR内参基因的稳定性比较[J].南方水产科学,2013,9(6):33-40.WANG Q,HE M X.Stability comparison of reference genes of Pinctada fucata by real-time qPCR[J].South China Fisheries Science,2013,9(6):33-40.
[34]刘颖,王双耀,安立会,等.栉孔扇贝内参基因稳定性研究[J].生态毒理学报,2013,8(4):616-622.LIU Y,WANG S Y,AN L H,et al.Study on the expression stability of reference genes in Chlamys farreri[J].Asian Journal of Ecotoxicology,2013,8(4):616-622.
[35]鲍相渤,刘卫东,姜冰,等.内参基因在虾夷扇贝定量PCR中表达稳定性的比较[J].水产科学,2011,30(10):603-608.BAO X B,LIU W D,JIANG B,et al.Expression stability of reference genes for quantitative PCR in Japanese Scallop Patinopecten yessoensis[J].Fisheries Science,2011,30(10):603-608.
[2]PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[3]VANDESOMPELE J,DE P K,PATTYN F,et al.Accurate normalization of real-time quantitative RT-PCRdata by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):1-12.
[4]ANDERSEN C L,JENSEN J L,ORNTOFT T F.Normalization of real-time quantitative reverse transcription-PCR data:A model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Research,2004,64(15):5245-5250.
[5]XIE F L,SUN G L,STILLER J W,et al.Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database[J].PLoS ONE,2011,6(11):e26980.
[6]BODIN N,BURGEOT T,STANISIERE J Y,et al.Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea[J].Comparative Biochemistry and Physiology C-toxicology&Pharmacology,2004,138(4):411-427.
[7]蔡英亚,黄翔鹄,吴洞科.红树蚬的生态观察[J].热带海洋,1995,4(1):94-98.CAI Y Y,HUANG X H,WU D K.Studies on the ecology of Polymesoda Erosa(solander)[J].Tropic O-ceanology,1995,4(1):94-98.
[8]赖廷和,何斌源.广西红树林区大型底栖动物种类多样性研究[J].广西科学,1998,5(3):166-172.LAI T H,HE B Y.Studies on the macrobenthos species diversity for Guangxi mangrove areas[J].Guangxi Sciences,1998,5(3):166-172.
[9]周浩郎,张俊杰,邢永泽,等.广西红树蚬的分布特征及影响因素分析[J].广西科学,2014,21(2):147-152.ZHOU H L,ZHANG J J,XING Y Z,et al.Characteristics of distribution and the influential factors of mangrove clam,Polymesoda erosa(Solander 1768),in Guangxi[J].Guangxi Sciences,2014,21(2):147-152.
[10]DSIKOWITZKY L,NORDHAUS I,JENNERJAHNT C,et al.Anthropogenic organic contaminants in water,sediments and benthic organisms of the mangrove-fringed Segara Anakan Lagoon,Java,Indonesia[J].Marine Pollution Bulletin,2011,62(4):851-862.
[11]赖廷和,何斌源,范航清,等.重金属Cd胁迫对红树蚬的抗氧化酶、消化酶活性和MDA含量的影响[J].生态学报,2011,31(11):3044-3053.LAI T H,HE B Y,FAN H Q,et al.Effects of cadmium stress on the activities of antioxidant enzymes,digestive enzymes and the membrane lipid peroxidation of the mangrove mud clamGeloina coaxans(Gmelin)[J].Acta Ecologica Sinica,2011,31(11):3044-3053.
[12]武文丽,杨明柳,吴斌,等.露空对红树蚬生物标志物的影响[J].广西科学,2014,21(2):153-157,163.WU W L,YANG M L,WU B,et al.The effect of air exposure on biomarkers of mangrove mud clam(Polymesoda erosa)[J].Guangxi Sciences,2014,21(2):153-157,163.
[13]GIBSON R,BARKER P.The decapod hepatopancreas[J].Oceanogr Mar Biol Ann Rev,1979,17(17):285-346.
[14]MCLEESE D W,METCALFE C D.Toxicity of creosote to larval and adult lobsters and Crangon and its accumulation in lobster hepatopancreas[J].Bulletin of Environmental Contamination and Toxicology,1979,22(6):796-799.
[15]卢彤岩,杨雨辉,徐连伟,等.达氟沙星对施氏鲟的急性毒性及组织残留检测[J].中国水产科学,2004,11(6):542-547.LU T Y,YANG Y H,XU L W,et al.Acute toxicity of danofloxacin in Amur sturgeon and the body residue[J].Journal of Fishery Sciences of China,2004,11(6):542-547.
[16]吕晓华,古燕,宋艳.壬基酚对红鲫、草鱼和鲢鱼的毒性及组织蓄积研究[J].卫生研究,2012,41(5):785-789.LV X H,GU Y,SONG Y.Toxicity and tissue accumulation of nonylphenol in Carassius auratus red variety,Grass Carp and Sliver Carp[J].Journal of Hygiene Research,2012,41(5):785-789.
[17]许星鸿,阎斌伦,徐加涛,等.重金属镉胁迫对日本蟳抗氧化酶活力、丙二醛含量及组织蓄积的影响[J].水产科学,2014,33(9):551-555.XU X H,YAN B L,XU J T,et al.Effects of cadmium stress on activities of antioxidant enzymes,malonaldehyde content and cadmium accumulation in asian swimming crab Charybdis japonica[J].Fisheries Science,2014,33(9):551-555.
[18]ZHANG H,PAN L Q,TAO Y X,et al.Identification and expression of differentially expressed genes in clamVenerupis philippinarumin response to environmental pollutant hexabromocyclododecane(HB-CD)[J].Journal of Experimental Marine Biology and Ecology,2013,445(3):166-173.
[19]ZHANG H,PAN L Q,TAO Y X.Antioxidant responses in clamVenerupis philippinarumexposed to environmental pollutant hexabromocyclododecane[J].Environmental Science and Pollution Research,2014,21(13):8206-8215.
[20]ETSCHMANN B,WILCKEN B,STOEVESAND K,et al.Selection of reference genes for quantitative realtime PCR analysis in canine mammary tumors using the geNorm algorithm[J].Veterinary Pathology,2006,43(6):934-942.
[21]张艳君,朱志峰,陆融,等.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展,2007,3(5):546-550.ZHANG Y J,ZHU Z F,LU R,et al.Selection of control genes in transcription analysis of gene expression[J].Progress in Biochemistry and Biophysics,2007,3(5):546-550.
[22]马飞.极大节旋藻实时荧光定量PCR内参基因的选择[D].青岛:中国海洋大学,2012.MA F.Reference genes for Arthrospira maxima when using quantitative real-time PCR[D].Qingdao:Ocean University of China,2012.
[23]BUSTIN S A,BENES V,GARSON J A,et al.The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments[J].Clinical Chemistry,2009,55(4):611-622.
[24]MALLONA I,LISCHEWSKI S,WEISS J,et al.Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J].BMC Plant Biology,2010,10:4.
[25]车志群,莫祖英,孙仁杰,等.荧光定量PCR方法及分子标志物在海洋污染监测中的应用进展[J].生态科学,2015,34(5):233-240.CHE Z Q,MO Z Y,SUN R J,et al.Review on application of methods of realtime fluorescent quantitative PCR and molecular marker in marine pollution monitoring[J].Ecological Science,2015,34(5):233-240.
[26]BAI Z Y,LIN J Y,MA K Y,et al.Identification of housekeeping genes suitable for gene expression analysis in the pearl mussel,Hyriopsis cumingii,during biomineralization[J].Molecular Genetics and Genomics,2014,289(4):717-725.
[27]FENG L Y,YU Q,LI X,et al.Identification of reference genes for qRT-PCR analysis in Yesso Scallop patinopecten yessoensis[J].PLoS ONE,2013,8(9):e75609.
[28]CUBERO-LEON E,CIOCAN C M,MINIER C,et al.Reference gene selection for qPCR in mussel,Mytilus edulis,during gametoge nesis and exogenous estrogen exposure[J].Environmental Science and Pollution Research,2012,19(7):2728-2733.
[29]MORGA B,ARZUL I,FAURY N,et al.Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR[J].Fish&Shellfish Immunology,2010,29(6):937-945.
[30]MAURIZ O,MANEIRO V,PEREZ-PARALLE M,et al.Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.[J].Aquaculture,2012,370-371(2):158-165.
[31]LLERA-HERRERA R,GARCIA-GASCA A,HU-VET A,et al.Identification of a tubulin?αgene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus[J].Marine Genomics,2012,6:33-44.
[32]DU Y S,ZHANG L L,XU F,et al.Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster(Crassostrea gigas)development by quantitative real-time PCR[J].Fish&Shellfish Immunology,2013,34(3):939-945.
[33]王琦,何毛贤.合浦珠母贝实时定量PCR内参基因的稳定性比较[J].南方水产科学,2013,9(6):33-40.WANG Q,HE M X.Stability comparison of reference genes of Pinctada fucata by real-time qPCR[J].South China Fisheries Science,2013,9(6):33-40.
[34]刘颖,王双耀,安立会,等.栉孔扇贝内参基因稳定性研究[J].生态毒理学报,2013,8(4):616-622.LIU Y,WANG S Y,AN L H,et al.Study on the expression stability of reference genes in Chlamys farreri[J].Asian Journal of Ecotoxicology,2013,8(4):616-622.
[35]鲍相渤,刘卫东,姜冰,等.内参基因在虾夷扇贝定量PCR中表达稳定性的比较[J].水产科学,2011,30(10):603-608.BAO X B,LIU W D,JIANG B,et al.Expression stability of reference genes for quantitative PCR in Japanese Scallop Patinopecten yessoensis[J].Fisheries Science,2011,30(10):603-608.
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