岩牡蛎正常和低盐胁迫下定量PCR内参基因的筛选与验证
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摘要
实时荧光定量PCR已广泛用于基因表达的分析,合适的内参基因选择是获得准确分析结果的关键。本研究选用正常生理状态下岩牡蛎(Crassostreanippona)的鳃、外套膜、闭壳肌和内脏团4种组织以及盐度10、20和30暂养1周后的鳃组织为材料,对已报道的常见内参基因(EF1A、GAPDH、RO21、TUB和TUA)的稳定性通过3种方法(geNorm、NormFinder和BestKeeper)进行分析和筛选,发现针对鳃单一组织在不同盐度胁迫下,GAPDH和RO21可以作为合适内参基因,而在不同组织中,需要更多的内参基因联合分析才能获得准确的定量表达结果。本研究是首次利用q-PCR对岩牡蛎进行内参基因的筛选和验证,为今后该物种低盐胁迫相关的基因表达和功能基因的研究提供重要依据。
Crassostrea nippona is a commercially important oyster species in East Asia as it is edible during the summer when the other oyster species are unavailable. Salinity is one of the important limiting factors to the survival and distribution of this stenohaline species. The molecular mechanism behind the response of this species to hypo-salinity stress remains unclear. Quantitative Real-Time PCR(qRT-PCR) has been widely used for the analysis of gene expression. The optimal reference gene is constantly transcribed in different types of cells, tissues, and species and under various experimental conditions. However, reference genes that meet all of these conditions are almost non-existent. The selection of a proper reference gene is a precondition for accurate analysis of the expression level of a target gene in quantitative real-time PCR. A total of five candidate reference genes, elongation factor 1a(EF1A), glyceraldehyde 3-phosphate dehydrogenase(GAPDH), heterogeneous nuclear ribonucleoprotein A2/B1(RO21), β-tubulin(TUB), and α-tubulin(TUA), were analyzed by qRT-PCR in four tissues(gill, mantle,visceral mass, and adductor muscle) under three salinity conditions of 10, 20, and 30 psu for one week. Three algorithms, geNorm, NormFinder, and BestKeeper, were used to evaluate the expression stability of the candidate reference genes. The results showed that EF1A was most stable in the different tissues under normal conditions.Under salinity stress, GAPDH was the most stable gene according to overall ranking. In contrast, TUB and TUA were the least stable genes and were not suitable as reference genes. This study showed that different algorithms may generate inconsistent results. Therefore, a combination of several reference genes should be selected to accurately calibrate system errors, especially for studies of different tissues in which candidate reference genes have more unstable expression. The present study was the first to select C. nippona reference genes by qRT-PCR and to provide a useful basis for selecting appropriate C. nippona reference genes. The present study also has important implications for gene expression and functional genomics research related to salinity stress in this species or other bivalve species.
Crassostrea nippona is a commercially important oyster species in East Asia as it is edible during the summer when the other oyster species are unavailable. Salinity is one of the important limiting factors to the survival and distribution of this stenohaline species. The molecular mechanism behind the response of this species to hypo-salinity stress remains unclear. Quantitative Real-Time PCR(qRT-PCR) has been widely used for the analysis of gene expression. The optimal reference gene is constantly transcribed in different types of cells, tissues, and species and under various experimental conditions. However, reference genes that meet all of these conditions are almost non-existent. The selection of a proper reference gene is a precondition for accurate analysis of the expression level of a target gene in quantitative real-time PCR. A total of five candidate reference genes, elongation factor 1a(EF1A), glyceraldehyde 3-phosphate dehydrogenase(GAPDH), heterogeneous nuclear ribonucleoprotein A2/B1(RO21), β-tubulin(TUB), and α-tubulin(TUA), were analyzed by qRT-PCR in four tissues(gill, mantle,visceral mass, and adductor muscle) under three salinity conditions of 10, 20, and 30 psu for one week. Three algorithms, geNorm, NormFinder, and BestKeeper, were used to evaluate the expression stability of the candidate reference genes. The results showed that EF1A was most stable in the different tissues under normal conditions.Under salinity stress, GAPDH was the most stable gene according to overall ranking. In contrast, TUB and TUA were the least stable genes and were not suitable as reference genes. This study showed that different algorithms may generate inconsistent results. Therefore, a combination of several reference genes should be selected to accurately calibrate system errors, especially for studies of different tissues in which candidate reference genes have more unstable expression. The present study was the first to select C. nippona reference genes by qRT-PCR and to provide a useful basis for selecting appropriate C. nippona reference genes. The present study also has important implications for gene expression and functional genomics research related to salinity stress in this species or other bivalve species.
引文
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[14]Zhao X L,Yu H,Kong L F,et al.Transcriptomic responses to salinity stress in the Pacific oyster Crassostrea gigas[J].PLoS ONE,2012,7(9):e46244.
[15]Meng J,Zhu Q H,Zhang L L,et al.Genome and transcriptome analyses provide insight into the euryhaline adaptation mechanism of Crassostrea gigas[J].PLoS ONE,2013,8(3):e58563.
[16]Dheda K,Huggett J F,Chang J S,et al.The implications of using an appropriate reference gene for real-time reverse transcription PCR data normalization[J].Analytical Biochemistry,2005,344(1):141-143.
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[18]Cubero-Leon E,Ciocan C M,Minier C,et al.Reference gene selection for qPCR in mussel,Mytilus edulis,during gametogenesis and exogenous estrogen exposure[J].Environmental Science and Pollution Research,2012,19(7):2728-2733.
[19]Sirakov M,Zarrella I,Borra M,et al.Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris[J].BMC Molecular Biology,2009,10:70.
[20]Wang H L,Wen H S,Li Y,et al.Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass(Lateolabrax maculatus)under normal and salinity stress conditions[J].PeerJ,2018,6:e5631.
[21]Zhang Y F,Zhao L J,Zeng Y L.Selection of reference genes in transcription analysis of gene expression[J].Plant Physiology Journal,2014,50(8):1119-1125.[张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125.]
[22]Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):Trends and problems[J].Journal of Molecular Endocrinology,2002,29(1):23-39.
[23]Dheilly N M,Lelong C,Huvet A,et al.Development of a Pacific oyster(Crassostrea gigas)31,918-feature microarray:identification of reference genes and tissue-enriched expression patterns[J].BMC Genomics,2011,12:468.
[24]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.
[25]Guo R,Lee M A,Ki J S.Normalization genes for mRNAexpression in the marine diatom Ditylum brightwellii following exposure to thermal and toxic chemical stresses[J].Journal of Applied Phycology,2010,25(4):1101-1109.
[26]Fernandes J M O,Mommens M,Hagen O,et al.Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,150(1):23-32.
[27]Fernández-Aparicio M,Huang K,Wafula E K,et al.Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development[J].Molecular Biology Reports,2013,40(4):3395-3407.
[28]Temel A,Gozukirmizi N.Physiological and molecular changes in barley and wheat under salinity[J].Applied Biochemistry and Biotechnology,2015,17(6):2950-2960.
[29]Hiller G,Weber K.Radioimmunoassay for tubulin:a quantitative comparison of the tubulin content of different established tissue culture cells and tissues[J].Cell,1978,14(4):795-804.
[30]Browne G J,Proud C G.Regulation of peptide-chain elongation in mammalian cells[J].European Journal of Biochemistry,2002,269:5360-5368.
[31]Siomi H,Dreyfuss G.RNA-binding proteins as regulators of gene expression[J].Current Opinion in Genetics and Development,1997,7(3):345-353.
[32]Xiong X H,Feng Q L,Xie L P,et al.Cloning and characterization of a heterogeneous nuclear ribonucleoprotein homolog from pearl oyster,Pinctada fucata[J].Acta Biochimica et Biophysica Sinica,2007,39:955-963.
[2]Drouin G,Himmelman J H,Pierre B.Impact of tidal salinity fluctuations on echinoderm and mollusc populations[J].Canadian Journal of Zoology,1985,63(6):1377-1387.
[3]Philippart C J M,Anadón R,Danovaro R,et al.Impacts of climate change on European marine ecosystems:Observations,expectations and indicators[J].Journal of Experimental Marine Biology and Ecology,2011,400(1):52-69.
[4]Bourque C W.Central mechanisms of osmosensation and systemic osmoregulation[J].Nature Reviews Neuroscience,2008,9(7):519-531.
[5]Bruckner P,van der Rest M.Structure and function of cartilage collagens[J].Microscopy Research&Technique,2010,28(5):378-384.
[6]Mcallen R,Walker D,Taylor A.The environmental effects of salinity and temperature on the oxygen consumption and total body osmolality of the marine flatworm Procerodes littoralis[J].Journal of Experimental Marine Biology and Ecology,2002,268(1):103-113.
[7]Ma H M,Liu X W,Mai K S,et al.Effects of abrupt salinity decline on anti-disease ability of scallop Chlamys farrer[J].Chinese High Technology Letters,2006,16(7):746-751.[马洪明,刘晓伟,麦康森,等.盐度突降对栉孔扇贝(Chlamys farreri)抗病力指标的影响[J].高技术通讯,2006,16(7):746-751.]
[8]Butt D,Shaddick K,Raftos D,et al.The effect of low salinity on phenoloxidase activity in the Sydney rock oyster,Saccostrea glomerata[J].Aquaculture,2006,251(2-4):159-166.
[9]Navarro J M.The effects of salinity on the physiological ecology of Choromytilus chorus(Molina,1782)(Bivalvia:Mytilidae)[J].Journal of Experimental Marine Biology and Ecology,1988,122(1):19-33.
[10]Meng X L,Dong Y W,Dong S L,et al.Mortality of the sea cucumber,Apostichopus japonicus Selenka,exposed to acute salinity decrease and related physiological responses:Osmoregulation and heat shock protein expression[J].Aquaculture,2011,316(1-4):88-92.
[11]Li W J.Biology and cultivation of oyster Crassostrea nippona[J].Fisheries Science,2007,26(12):689-690.[李文姬.岩牡蛎的生物学及其养殖[J].水产科学,2007,26(12):689-690.]
[12]Yang C,Su X R,Li T W.The cultural technique of Pacific oyster,Crassostrea gigas[J].Fisheries Science,2003,22(5):31-33.[杨春,苏秀榕,李太武.牡蛎养殖技术[J].水产科学,2003,22(5):31-33.]
[13]Berger V J,Kharazova A D.Mechanisms of salinity adaptations in marine molluscs[J].Hydrobiologia,1997,355(1-3):115-126.
[14]Zhao X L,Yu H,Kong L F,et al.Transcriptomic responses to salinity stress in the Pacific oyster Crassostrea gigas[J].PLoS ONE,2012,7(9):e46244.
[15]Meng J,Zhu Q H,Zhang L L,et al.Genome and transcriptome analyses provide insight into the euryhaline adaptation mechanism of Crassostrea gigas[J].PLoS ONE,2013,8(3):e58563.
[16]Dheda K,Huggett J F,Chang J S,et al.The implications of using an appropriate reference gene for real-time reverse transcription PCR data normalization[J].Analytical Biochemistry,2005,344(1):141-143.
[17]Du Y S,Zhang 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.
[18]Cubero-Leon E,Ciocan C M,Minier C,et al.Reference gene selection for qPCR in mussel,Mytilus edulis,during gametogenesis and exogenous estrogen exposure[J].Environmental Science and Pollution Research,2012,19(7):2728-2733.
[19]Sirakov M,Zarrella I,Borra M,et al.Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris[J].BMC Molecular Biology,2009,10:70.
[20]Wang H L,Wen H S,Li Y,et al.Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass(Lateolabrax maculatus)under normal and salinity stress conditions[J].PeerJ,2018,6:e5631.
[21]Zhang Y F,Zhao L J,Zeng Y L.Selection of reference genes in transcription analysis of gene expression[J].Plant Physiology Journal,2014,50(8):1119-1125.[张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125.]
[22]Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):Trends and problems[J].Journal of Molecular Endocrinology,2002,29(1):23-39.
[23]Dheilly N M,Lelong C,Huvet A,et al.Development of a Pacific oyster(Crassostrea gigas)31,918-feature microarray:identification of reference genes and tissue-enriched expression patterns[J].BMC Genomics,2011,12:468.
[24]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.
[25]Guo R,Lee M A,Ki J S.Normalization genes for mRNAexpression in the marine diatom Ditylum brightwellii following exposure to thermal and toxic chemical stresses[J].Journal of Applied Phycology,2010,25(4):1101-1109.
[26]Fernandes J M O,Mommens M,Hagen O,et al.Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,150(1):23-32.
[27]Fernández-Aparicio M,Huang K,Wafula E K,et al.Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development[J].Molecular Biology Reports,2013,40(4):3395-3407.
[28]Temel A,Gozukirmizi N.Physiological and molecular changes in barley and wheat under salinity[J].Applied Biochemistry and Biotechnology,2015,17(6):2950-2960.
[29]Hiller G,Weber K.Radioimmunoassay for tubulin:a quantitative comparison of the tubulin content of different established tissue culture cells and tissues[J].Cell,1978,14(4):795-804.
[30]Browne G J,Proud C G.Regulation of peptide-chain elongation in mammalian cells[J].European Journal of Biochemistry,2002,269:5360-5368.
[31]Siomi H,Dreyfuss G.RNA-binding proteins as regulators of gene expression[J].Current Opinion in Genetics and Development,1997,7(3):345-353.
[32]Xiong X H,Feng Q L,Xie L P,et al.Cloning and characterization of a heterogeneous nuclear ribonucleoprotein homolog from pearl oyster,Pinctada fucata[J].Acta Biochimica et Biophysica Sinica,2007,39:955-963.