中间球海胆乳酸脱氢酶基因克隆及其对海水酸化的响应
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摘要
为明确中间球海胆乳酸脱氢酶(LDH)基因序列及表达特征,研究其对海水酸化胁迫的响应,实验利用cDNA末端快速扩增(RACE)技术获得中间球海胆LDH基因(命名为SiLDH)的全长cDNA,并且比较了海水酸化胁迫下,中间球海胆不同组织SiLDH基因表达及总LDH活性的变化情况。结果显示:①SiLDH基因的cDNA全长为1 499 bp,包含133 bp的5′非编码区,349 bp的3′非编码区和1 017 bp编码338个氨基酸的开放阅读框(ORF)。SiLDH编码蛋白的相对分子量为36.40 ku,理论等电点为6.55,属于无信号肽的非跨膜、温和疏水蛋白。②生物信息学分析显示,SiLDH蛋白序列与紫球海胆LDH X4蛋白序列一致性最高(90.88%)。③实时荧光定量PCR(qRT-PCR)发现,SiLDH基因在检测的4种组织中均有表达,相对表达量从高到低为性腺>管足>肠>体腔液;总LDH活性检测显示,中间球海胆总LDH活性从高到低为性腺>管足>体腔液>肠;④海水酸化处理60 d后发现,与自然海水组(pH 8.06±0.01)相比,SiLDH基因在中间球海胆性腺、管足和肠道中呈现总体降低趋势,在体腔液中则呈现随海水pH降低先降低后显著升高的趋势;总LDH活性在性腺、管足和体腔液中呈现随海水pH降低而降低的趋势,在肠道中则呈现随海水pH降低先降低后显著升高的趋势。研究表明,中间球海胆面对海水酸化时可能通过调节SiLDH基因的表达和LDH活性来缓解海水酸化带来的负面影响。
In order to identify and characterize the lactate dehydrogenase(LDH) gene and investigate its expression and activity alterations in response to seawater acidification stress in sea urchins, we used database data mining and rapid amplification of cDNA ends(RACE) to identify a novel LDH gene in the sea urchin Strongylocentrotus intermedius(here designated SiLDH). The full-length cDNA of SiLDH was 1 499 bp, including a 1 017 bp open reading frame encoding 338 amino acid residues. Sequence analysis indicated that the predicted SiLDH protein was similar to the LDH homolog in the sea urchin S. purpuratus. Quantitative real-time PCR analysis showed that SiLDH was ubiquitously expressed in coelomic fluid, gonad, tube foot and intestine of healthy adult S. intermedius, with the highest level of expression identified in the gonad. Total LDH enzyme activity in different tissues of S. intermedius from high to low was as gonad > tubefoot > coelomic fluid >intestine. Both SiLDH mRNA expression profiles and total LDH enzyme activity were altered in the coelomic fluid, gonad, tube foot and intestine after 60-day seawater acidification treatments(Control: natural seawater; OA1:pH=-0.3; OA2: pH=-0.4; OA3: pH=-0.5). Our results provide more information about the characteristics and biological functions of the LDH homolog in sea urchins.
In order to identify and characterize the lactate dehydrogenase(LDH) gene and investigate its expression and activity alterations in response to seawater acidification stress in sea urchins, we used database data mining and rapid amplification of cDNA ends(RACE) to identify a novel LDH gene in the sea urchin Strongylocentrotus intermedius(here designated SiLDH). The full-length cDNA of SiLDH was 1 499 bp, including a 1 017 bp open reading frame encoding 338 amino acid residues. Sequence analysis indicated that the predicted SiLDH protein was similar to the LDH homolog in the sea urchin S. purpuratus. Quantitative real-time PCR analysis showed that SiLDH was ubiquitously expressed in coelomic fluid, gonad, tube foot and intestine of healthy adult S. intermedius, with the highest level of expression identified in the gonad. Total LDH enzyme activity in different tissues of S. intermedius from high to low was as gonad > tubefoot > coelomic fluid >intestine. Both SiLDH mRNA expression profiles and total LDH enzyme activity were altered in the coelomic fluid, gonad, tube foot and intestine after 60-day seawater acidification treatments(Control: natural seawater; OA1:pH=-0.3; OA2: pH=-0.4; OA3: pH=-0.5). Our results provide more information about the characteristics and biological functions of the LDH homolog in sea urchins.
引文
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[22]王群,赵云龙,陈立侨.中华绒螯蟹雄性生殖系统生化组成及精子代谢[J].水产学报,2002,26(5):411-416.Wang Q,Zhao Y L,Chen L Q.Biochemical composition and sperm metabolism in the reproductive system of the male,Eriocheir sinensis[J].Journal of Fisheries of China,2002,26(5):411-416(in Chinese).
[23]孙华宾,刘雅峰,刘燕,等.男性不育者精浆及精子乳酸脱氢酶活性的研究[J].中华全科医学,2010,8(8):974,1011.Sun H B,Liu Y F,Liu Y,et al.Activity of lactate dehydrogenase isoenzyme in seminal plasma or spermatozoa of male infertility patients[J].Chinese Journal of General Practice,2010,8(8):974,1011(in Chinese).
[24]Uthicke S,Liddy M,Nguyen H D,et al.Interactive effects of near-future temperature increase and ocean acidification on physiology and gonad development in adult Pacific sea urchin,Echinometra sp.[J].Coral Reefs,2014,33(3):831-845.
[25]张颖.虾夷马粪海胆体腔液免疫因子及吞噬细胞活性的研究[D].大连:大连海洋大学,2014.Zhang Y.Studies on immune factor and activity ofphagocytes in coelomic fluid of Strongylocentrotus intermedius[D].Dalian:Dalian Ocean University,2014(in Chinese).
[26]An Z F,Wei D B,Wei L,et al.The expression of Ldh-c in the skeletal muscle of plateau pika(Ochotona curzoniae)enhances adaptation to a hypoxic environment[J].Biology Open,2017,6(9):1336-1341.
[27]Collard M,Laitat K,Moulin L,et al.Buffer capacity of the coelomic fluid in echinoderms[J].Comparative Biochemistry and Physiology-Part A:Molecular&Integrative Physiology,2013,166(1):199-206.
[28]Wang G N,Yagi M,Yin R,et al.Effects of elevated seawater CO2 on feed intake,oxygen consumption and morphology of Aristotle’s lantern in the sea urchin Anthocidaris crassispina[J].Journal of Marine Science and Technology,2013,21(Suppl.1):192-200.
[29]Heuer R M,Esbaugh A J,Grosell M.Ocean acidification leads to counterproductive intestinal base loss in the gulf toadfish(Opsanus beta)[J].Physiological and Biochemical Zoology:Ecological and Evolutionary Approaches,2012,85(5):450-459.
[2]贾旭颖,国先涛,王芳,等.非离子氨胁迫对淡水和海水养殖凡纳滨对虾呼吸代谢酶活力影响的比较[J].水产学报,2014,38(11):1837-1846.Jia X Y,Guo X T,Wang F,et al.Comparison of the effect of nonionic ammonia stress on respiratory metabolic enzyme of Litopenaeus vannamei in seawater and freshwater[J].Journal of Fisheries of China,2014,38(11):1837-1846(in Chinese).
[3]李泽健.低氧胁迫对中华绒螯蟹能量代谢、呼吸代谢及抗氧化代谢的影响[D].保定:河北大学,2012.Li Z J.Effects of hypoxia stress on energy metabolism,respiratory metabolism and antioxidant metabolism of Eriocheir sinensis[D].Baoding:Hebei University,2012(in Chinese).
[4]陶易凡,强俊,王辉,等.高pH胁迫对克氏原螯虾的急性毒性和鳃、肝胰腺中酶活性及组织结构的影响[J].水产学报,2016,40(11):1694-1704.Tao Y F,Qiang J,Wang H,et al.Acute toxicity of high pH stress and its effect on enzymes activity and histological structure of gill and hepatopancreas in Procambarus clarkii[J].Journal of Fisheries of China,2016,40(11):1694-1704(in Chinese).
[5]Caldeira K,Wickett M E.Oceanography:anthropogenic carbon and ocean pH[J].Nature,2003,425(6956):365.
[6]丁兆坤,王福平,许友卿.海水酸化对海洋生物代谢的影响及机理[J].水产科学,2015,34(5):331-334.Ding Z K,Wang F P,Xu Y Q.Effect of ocean acidification on metabolism of marine organisms[J].Fisheries Science,2015,34(5):331-334(in Chinese).
[7]Gaitán-Espitia J D,Marshall D,Dupont S,et al.Geographical gradients in selection can reveal genetic constraints for evolutionary responses to ocean acidification[J].Biology Letters,2017,13(2):10.1098/rsbl.2016.0784.
[8]湛垚垚,黄显雅,段立柱,等.海洋酸化对近岸海洋生物的影响[J].大连大学学报,2013,34(3):79-84.Zhan Y Y,Huang X Y,Duan L Z,et al.Review on the impacts of ocean acidification on nearshore marine life[J].Journal of Dalian University,2013,34(3):79-84(in Chinese).
[9]Comeau S,Jeffree R,TeyssiéJ L,et al.Response of the arctic pteropod Limacina helicina to projected future environmental conditions[J].PLoS One,2010,5(6):e11362.
[10]Wootton J T,Pfister C A,Forester J D.Dynamic patterns and ecological impacts of declining ocean pH in a highresolution multi-year dataset[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(48):18848-18853.
[11]Anthony K R N,Kline D I,Diaz-Pulido G,et al.Ocean acidification causes bleaching and productivity loss in coral reef builders[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(45):17442-17446.
[12]Albright R,Caldeira L,Hosfelt J,et al.Reversal of ocean acidification enhances net coral reef calcification[J].Nature,2016,531(7594):362-365.
[13]Beniash E,Ivanina A,Lieb N S,et al.Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica[J].Marine Ecology Progress Series,2010,419:95-108.
[14]Zhan Y Y,Hu W B,Zhang W J,et al.The impact of CO2-driven ocean acidification on early development and calcification in the sea urchin Strongylocentrotus intermedius[J].Marine Pollution Bulletin,2016,112(1/2):291-302.
[15]Fang J K H,Sch?Nberg C H L,Mello-Athayde M A,et al.Effects of ocean warming and acidification on the energy budget of an excavating sponge[J].Global Change Biology,2014,20(4):1043-1054.
[16]Hu M Y,Casties I,Stumpp M,et al.Energy metabolism and regeneration are impaired by seawater acidification in the infaunal brittlestar Amphiura filiformis[J].Journal of Experimental Biology,2014,217:2411-2421.
[17]常亚青,丁君,宋坚,等.海参、海胆生物学研究与养殖[M].北京:海洋出版社,2004.Chang Y Q,Ding J,Song J,et al.Biology research and breeding of sea cucumber and sea urchin[M].Beijing:China Ocean Press,2004(in Chinese).
[18]湛垚垚,黄显雅,段立柱,等.实验室模拟海水酸化系统:201320267332.7[P].2013-12-11.Zhan Y Y,Huang X Y,Duan L Z,et al.Laboratory simulation of ocean acidification system:201320267332.7[P].2013-12-11.
[19]Stocker T F,Qin D,Plattner G K,et al.Climate change2013:the physical science basis.Contribution of working group I to the fifth assessment report of the intergovernmental panel on climate change[R].Cambridge,United Kingdom and New York,NY,USA:Cambridge University Press,2014.
[20]Mehrbach C,Culberson C H,Hawley J E,et al.Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure[J].Limnology and Oceanography,1973,18(6):897-907.
[21]姬南京,杨芸菲,丁君,等.虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析[J].中国水产科学,2013,20(5):950-957.Ji N J,Yang Y F,Ding J,et al.Cloning and expression analysis of a sea urchin(Strongylocentrotus intermedius)lysozyme gene[J].Journal of Fishery Sciences of China,2013,20(5):950-957(in Chinese).
[22]王群,赵云龙,陈立侨.中华绒螯蟹雄性生殖系统生化组成及精子代谢[J].水产学报,2002,26(5):411-416.Wang Q,Zhao Y L,Chen L Q.Biochemical composition and sperm metabolism in the reproductive system of the male,Eriocheir sinensis[J].Journal of Fisheries of China,2002,26(5):411-416(in Chinese).
[23]孙华宾,刘雅峰,刘燕,等.男性不育者精浆及精子乳酸脱氢酶活性的研究[J].中华全科医学,2010,8(8):974,1011.Sun H B,Liu Y F,Liu Y,et al.Activity of lactate dehydrogenase isoenzyme in seminal plasma or spermatozoa of male infertility patients[J].Chinese Journal of General Practice,2010,8(8):974,1011(in Chinese).
[24]Uthicke S,Liddy M,Nguyen H D,et al.Interactive effects of near-future temperature increase and ocean acidification on physiology and gonad development in adult Pacific sea urchin,Echinometra sp.[J].Coral Reefs,2014,33(3):831-845.
[25]张颖.虾夷马粪海胆体腔液免疫因子及吞噬细胞活性的研究[D].大连:大连海洋大学,2014.Zhang Y.Studies on immune factor and activity ofphagocytes in coelomic fluid of Strongylocentrotus intermedius[D].Dalian:Dalian Ocean University,2014(in Chinese).
[26]An Z F,Wei D B,Wei L,et al.The expression of Ldh-c in the skeletal muscle of plateau pika(Ochotona curzoniae)enhances adaptation to a hypoxic environment[J].Biology Open,2017,6(9):1336-1341.
[27]Collard M,Laitat K,Moulin L,et al.Buffer capacity of the coelomic fluid in echinoderms[J].Comparative Biochemistry and Physiology-Part A:Molecular&Integrative Physiology,2013,166(1):199-206.
[28]Wang G N,Yagi M,Yin R,et al.Effects of elevated seawater CO2 on feed intake,oxygen consumption and morphology of Aristotle’s lantern in the sea urchin Anthocidaris crassispina[J].Journal of Marine Science and Technology,2013,21(Suppl.1):192-200.
[29]Heuer R M,Esbaugh A J,Grosell M.Ocean acidification leads to counterproductive intestinal base loss in the gulf toadfish(Opsanus beta)[J].Physiological and Biochemical Zoology:Ecological and Evolutionary Approaches,2012,85(5):450-459.
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