中国明对虾NHE3基因克隆及其在pH胁迫下的表达
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摘要
为研究钠/氢交换体(Na+/H+-exchanger,NHE)在中国明对虾(Fenneropenaeus chinensis)响应pH胁迫过程中发挥的作用,首先采用静水毒性实验方法确定了中国明对虾酸碱半致死pH,然后利用RACE技术克隆了中国明对虾Na+/H+-exchanger isoform 3(命名为FcNHE3)基因,并通过荧光定量PCR及RNA干扰技术分析了其在pH胁迫下的表达特征及功能。结果显示,72 h酸性半致死pH和碱性半致死pH分别为5.2和9.1。克隆获得FcNHE3基因(Gen Bank:MF373587)cDNA序列全长3508 bp,开放阅读框2805 bp,编码934个氨基酸,具有信号肽和12个跨膜结构域;蛋白同源分析发现,FcNHE3与青蟹(Carcinus maenas)同源性最高,达到74%;系统进化分析显示,FcNHE3与三疣梭子蟹(Portunus trituberculatus)和青蟹亲缘关系最近。荧光定量PCR分析表明,FcNHE3基因在鳃组织中表达量显著高于其他组织(P<0.05);酸性半致死pH(pH 5.2)胁迫下,FcNHE3基因在整个胁迫过程中显著上调表达(P<0.05);碱性半致死pH(pH 9.1)胁迫下,FcNHE3基因在前48 h显著下调表达(P<0.05),12 h表达量最低,仅在72 h出现上调表达。RNA干扰后,FcNHE3基因表达受到抑制,pH 5.2胁迫下对虾存活率相比对照组显著下降。研究表明相较于高pH胁迫,FcNHE3基因在中国明对虾响应低pH胁迫过程中可能发挥更重要的调节作用。
Na~+/H~+-exchanger is a membrane-associated enzyme responsible for the active transport of Na+ and H+ions across cell membranes, and generates chemical and electrical gradients. It plays an important role in the aquatic adaptation of aquatic crustaceans. To investigate the function of the Na+/H+-exchanger in Fenneropenaeus chinensis under pH stress, Na+/H+-exchanger 3 c DNA of Fenneropenaeus chinensis, named FcNHE3(GenBank:MF373587), was cloned from the gill tissues of the animal by Reverse Transcription-Polymerase Chain Reaction(RACE). The full-length of FcNHE3 cDNA was 3508 bp(base pairs), including a 2805 bp open reading frame(ORF) encoding a 934-amino acid peptide with one signal peptide and 12 transmembrane domains. Comparison with homologous proteins showed that the deduced FcNHE3 amino acid sequence has the highest sequence identity with Carcinus maenas(74%), and along with Portunus trituberculatus, were clustered into one group by phylogenetic analysis. Results of RT-q PCR showed that FcNHE3 expression level in the gills was significantly higher than in other tissues(P<0.05). The expression level of FcNHE3 in the gills was up-regulated under low pH stress(pH 5.2) during the entire duration of exposure. The expression level of FcNHE3 in the gills was down-regulated during the first 48 h and up-regulated at 72 h under high pH stress(pH 9.1). After the knockdown of FcNHE3 expression by RNA interference(RNAi), shrimp mortality was found to be significantly higher under low pH stress when compared with the control group. The results suggest that FcNHE3 may play a more important role in regulating acid-base balance under low pH stress than under high pH stress.
Na~+/H~+-exchanger is a membrane-associated enzyme responsible for the active transport of Na+ and H+ions across cell membranes, and generates chemical and electrical gradients. It plays an important role in the aquatic adaptation of aquatic crustaceans. To investigate the function of the Na+/H+-exchanger in Fenneropenaeus chinensis under pH stress, Na+/H+-exchanger 3 c DNA of Fenneropenaeus chinensis, named FcNHE3(GenBank:MF373587), was cloned from the gill tissues of the animal by Reverse Transcription-Polymerase Chain Reaction(RACE). The full-length of FcNHE3 cDNA was 3508 bp(base pairs), including a 2805 bp open reading frame(ORF) encoding a 934-amino acid peptide with one signal peptide and 12 transmembrane domains. Comparison with homologous proteins showed that the deduced FcNHE3 amino acid sequence has the highest sequence identity with Carcinus maenas(74%), and along with Portunus trituberculatus, were clustered into one group by phylogenetic analysis. Results of RT-q PCR showed that FcNHE3 expression level in the gills was significantly higher than in other tissues(P<0.05). The expression level of FcNHE3 in the gills was up-regulated under low pH stress(pH 5.2) during the entire duration of exposure. The expression level of FcNHE3 in the gills was down-regulated during the first 48 h and up-regulated at 72 h under high pH stress(pH 9.1). After the knockdown of FcNHE3 expression by RNA interference(RNAi), shrimp mortality was found to be significantly higher under low pH stress when compared with the control group. The results suggest that FcNHE3 may play a more important role in regulating acid-base balance under low pH stress than under high pH stress.
引文
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[16]Li J,Liu P,He Y Y.Artificial selection in the new breed of Fenneropenaeus chinensis named“Yellow Sea 1”based on fast growth trait[J].Journal of Fisheries of China,2005,29(1):1-5.[李健,刘萍,何玉英.中国对虾快速生长新品种“黄海1号”的人工选育[J].水产学报,2005,29(1):1-5.]
[17]Yang F Y,Sun L M,Yang X Q.Toxicity of Carbonate alkalinity to Penaeus vannamei Juveniles[J].Fisheries Science,2004,23(9):3-6.[杨富亿,孙丽敏,杨欣乔.碳酸盐碱度对南美白对虾幼虾的毒性作用[J].水产科学,2004,23(9):3-6.]
[18]Yang F Y,Shao Q C,Li J L,et al.Toxicity of p H and carbonatealkalinity on tadpoles of frog Rana chensinensis[J].Journal of Fishery Sciences of China,2005,12(1):43-48.[杨富亿,邵庆春,李景林,等.碳酸盐碱度和p H对中国林蛙蝌蚪的毒性作用[J].中国水产科学,2005,12(1):43-48.]
[19]Pan R W,Ma L,Zhang Y C,et al.Comparison between Up-and-Down Procedure and Karber test in determining tetanus toxin LD50 in mice[J].Chinese Journal of Comparative Medicine,2012,22(12):28-30.[潘若文,马力,张勇朝,等.Up and Down法与寇氏法测定破伤风毒素LD50的比较[J].中国比较医学杂志,2012,22(12):28-30.]
[20]Elbashir S M,Harborth J,Weber K,et al.Analysis of gene function in somatic mammalian cells using small interfering RNAs[J].Methods,2002,26(2):199-213.
[21]Bu Z Y,Fu H T,Sun S M,et al.Molecular characterization and developmental expression of Ras-related nuclear protein in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference on ovarian maturation[J].Journal of Fishery Sciences of China,2017,24(3):459-469.[卜宗元,傅洪拓,孙盛明,等.青虾Ran基因的克隆、表达及其在卵巢发育中的功能[J].中国水产科学,2017,24(3):459-469.]
[22]Orlowski J,Grinstein S.Na+/H+exchangers of mammalian cells[J].The Journal of Biological Chemistry,1997,272(36):22373-22376.
[23]Numata M,Petrecca K,Lake N,et al.Identification of a mitochondrial Na+/H+exchanger[J].The Journal of Biological Chemistry,1998,273(12):6951-6959.
[24]Dibrov P,Fliegel L.Comparative molecular analysis of Na+/H+exchangers:a unified model for Na+/H+antiport?[J].FEBS Letters,1998,424(1-2):1-5.
[25]Wang Y,Li J,Zhang Z,et al.Effects of p H and ammonia-N stresses on HSP90 gene expression of Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2013,34(5):43-50.[王芸,李健,张喆,等.p H、氨氮胁迫对中国对虾HSP90基因表达的影响[J].渔业科学进展,2013,34(5):43-50.]
[26]Li Y Q.Effects of p H stress on activities of phosphatase in Exopalaemon carinicauda Holthuis[J].Journal of Southern Agriculture,2014,45(6):1098-1101.[李玉全.p H胁迫对脊尾白虾代谢酶活力的影响[J].南方农业学报,2014,45(6):1098-1101.]
[27]Zhao X Y,Li J,Chen P,et al.Effects of p H stress on survival rate and activities of iontransport enzyme,immunerelated enzymes in three species of shrimps[J].Journal of Shanghai Ocean University,2011,20(5):720-728.[赵先银,李健,陈萍,等.p H胁迫对3种对虾存活率、离子转运酶和免疫酶活力的影响[J].上海海洋大学学报,2011,20(5):720-728.]
[28]Cai Y M,Chen T,Ren C H,et al.Molecular characterization of Pacific white shrimp(Litopenaeus vannamei)sodium bicarbonate cotransporter(NBC)and its role in response to p H stress[J].Fish&Shellfish Immunology,2017,64:226-233.
[29]Dymowska A,Hwang P P,Goss G G,et al.Structure and function of ionocytes in the freshwater fish gill[J].Respiratory Physiology and Neurobiology,2012,184(3):282-292.
[30]Uchiyama M,Komiyama M,Yoshizawa H,et al.Structures and immunolocalization of Na+-K+-ATPase,Na+/H+exchanger 3 and vacuolar-type H+-ATPase in the gills of blennies(Teleostei:Blenniidae)inhabiting rocky intertidal areas[J].Journal of Fish Biology,2012,80(6):2236-2252.
[2]Pan L Q,Liu H Y.Review on the osmoregulation of crustacean[J].Journal of Fisheries of China,2005,29(1):109-114.[潘鲁青,刘泓宇.甲壳动物渗透调节生理学研究进展[J].水产学报,2005,29(1):109-114.]
[3]Wang Y,Li J,Li J T,et al.Effects of p H stress on antioxidant system enzyme activities and gene expression of Fenneropenaeus chinensis[J].Journal of Fishery Sciences of China,2011,18(3):556-564.[王芸,李健,李吉涛,等.p H胁迫对中国明对虾抗氧化系统酶活力及基因表达的影响[J].中国水产科学,2011,18(3):556-564.]
[4]Li C,Chen J.The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high p H stress[J].Fish&Shellfish Immunology,2008,25(6):701-709.
[5]Wang W,Zhou J,Wang P,et al.Oxidative stress,DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp,Litopenaeus vannamei when exposed to acute p H stress[J].Comparative Biochemistry and Physiology C:Toxicology and Pharmacology,2009,150(4):428-435.
[6]Fang W H,Wang H,Lai Q F,et al.Toxicity of carbonate-alkalinity and p H to larval Penaeus chinensis[J].Journal of Fishery Sciences of China,2000,7(4):78-81.[房文红,王慧,来琦芳,等.碳酸盐碱度、p H对中国对虾幼虾的致毒效应[J].中国水产科学,2000,7(4):78-81.]
[7]Zhang P D,Zhang X M,Li J,et al.The effects of body weight,temperature,salinity,p H,light intensity and feeding condition on lethal DO levels of whiteleg shrimp,Litopenaeus vannamei(Boone,1931)[J].Aquaculture,2006,256(1-4):579-587.
[8]Walton W E,Compton S M,Allan J D,et al.The effect of acid stress on survivorship and reproduction of Daphnia pulex(Crustacea:Cladocera)[J].Canadian Journal of Zoology,2011,60(4):573-579.
[9]Padan E,Landau M.Sodium-Proton(Na+/H+)Antiporters:Properties and Roles in Health and Disease[J].Metal Ions in Life Sciences.2016,16:391-458.
[10]Loo S Y,Chang M K,Chua C S,et al.NHE-1:A promising target for novel anti-cancer therapeutics[J].Current Pharmaceutical Design,2012,18(10):1372-1382.
[11]Hirata T,Kaneko T,Ono T,et al.Mechanism of acid adaptation of a fish living in a p H 3.5 lake[J].American Journal of Physiology Regulatory Integrative and Comparative Physiology,2003,284(5):1199-1212.
[12]Liu S T,Horng J L,Chen P Y,et al.Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka:new insights into the salt-secreting mechanism[J].Scientific Reports,2016,6:31433.
[13]Evans D H,Piermarini P M,Choe K P,et al.The multifunctional fish gill:Dominant site of gas exchange,osmoregulation,acid-base regulation,and excretion of nitrogenous waste[J].Physiological Reviews,2005,85(1):97-177.
[14]Zhou S L,Jiang N C,Lu J P,et al.Progress of the study on osmotic regulation in crustaceans I.The gill’s structure and function and its’concerned factors[J].Donghai Marine Science,2001,19(1):44-51.[周双林,姜乃澄,卢建平,等.甲壳动物渗透压调节的研究进展I.鳃的结构与功能及其影响因子[J].东海海洋,2001,19(1):44-51.]
[15]Ma J W,LüJ J,Liu P,et al.Na+/H+-exchanger in swimming crab(portunus trituberculatus):cloning,characterization and m RNA expression under salinity stress[J].Acta Hydrobiologica Sinica,2016,40(5):902-907.[马金武,吕建建,刘萍,等.三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析[J].水生生物学报,2016,40(5):902-907.]
[16]Li J,Liu P,He Y Y.Artificial selection in the new breed of Fenneropenaeus chinensis named“Yellow Sea 1”based on fast growth trait[J].Journal of Fisheries of China,2005,29(1):1-5.[李健,刘萍,何玉英.中国对虾快速生长新品种“黄海1号”的人工选育[J].水产学报,2005,29(1):1-5.]
[17]Yang F Y,Sun L M,Yang X Q.Toxicity of Carbonate alkalinity to Penaeus vannamei Juveniles[J].Fisheries Science,2004,23(9):3-6.[杨富亿,孙丽敏,杨欣乔.碳酸盐碱度对南美白对虾幼虾的毒性作用[J].水产科学,2004,23(9):3-6.]
[18]Yang F Y,Shao Q C,Li J L,et al.Toxicity of p H and carbonatealkalinity on tadpoles of frog Rana chensinensis[J].Journal of Fishery Sciences of China,2005,12(1):43-48.[杨富亿,邵庆春,李景林,等.碳酸盐碱度和p H对中国林蛙蝌蚪的毒性作用[J].中国水产科学,2005,12(1):43-48.]
[19]Pan R W,Ma L,Zhang Y C,et al.Comparison between Up-and-Down Procedure and Karber test in determining tetanus toxin LD50 in mice[J].Chinese Journal of Comparative Medicine,2012,22(12):28-30.[潘若文,马力,张勇朝,等.Up and Down法与寇氏法测定破伤风毒素LD50的比较[J].中国比较医学杂志,2012,22(12):28-30.]
[20]Elbashir S M,Harborth J,Weber K,et al.Analysis of gene function in somatic mammalian cells using small interfering RNAs[J].Methods,2002,26(2):199-213.
[21]Bu Z Y,Fu H T,Sun S M,et al.Molecular characterization and developmental expression of Ras-related nuclear protein in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference on ovarian maturation[J].Journal of Fishery Sciences of China,2017,24(3):459-469.[卜宗元,傅洪拓,孙盛明,等.青虾Ran基因的克隆、表达及其在卵巢发育中的功能[J].中国水产科学,2017,24(3):459-469.]
[22]Orlowski J,Grinstein S.Na+/H+exchangers of mammalian cells[J].The Journal of Biological Chemistry,1997,272(36):22373-22376.
[23]Numata M,Petrecca K,Lake N,et al.Identification of a mitochondrial Na+/H+exchanger[J].The Journal of Biological Chemistry,1998,273(12):6951-6959.
[24]Dibrov P,Fliegel L.Comparative molecular analysis of Na+/H+exchangers:a unified model for Na+/H+antiport?[J].FEBS Letters,1998,424(1-2):1-5.
[25]Wang Y,Li J,Zhang Z,et al.Effects of p H and ammonia-N stresses on HSP90 gene expression of Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2013,34(5):43-50.[王芸,李健,张喆,等.p H、氨氮胁迫对中国对虾HSP90基因表达的影响[J].渔业科学进展,2013,34(5):43-50.]
[26]Li Y Q.Effects of p H stress on activities of phosphatase in Exopalaemon carinicauda Holthuis[J].Journal of Southern Agriculture,2014,45(6):1098-1101.[李玉全.p H胁迫对脊尾白虾代谢酶活力的影响[J].南方农业学报,2014,45(6):1098-1101.]
[27]Zhao X Y,Li J,Chen P,et al.Effects of p H stress on survival rate and activities of iontransport enzyme,immunerelated enzymes in three species of shrimps[J].Journal of Shanghai Ocean University,2011,20(5):720-728.[赵先银,李健,陈萍,等.p H胁迫对3种对虾存活率、离子转运酶和免疫酶活力的影响[J].上海海洋大学学报,2011,20(5):720-728.]
[28]Cai Y M,Chen T,Ren C H,et al.Molecular characterization of Pacific white shrimp(Litopenaeus vannamei)sodium bicarbonate cotransporter(NBC)and its role in response to p H stress[J].Fish&Shellfish Immunology,2017,64:226-233.
[29]Dymowska A,Hwang P P,Goss G G,et al.Structure and function of ionocytes in the freshwater fish gill[J].Respiratory Physiology and Neurobiology,2012,184(3):282-292.
[30]Uchiyama M,Komiyama M,Yoshizawa H,et al.Structures and immunolocalization of Na+-K+-ATPase,Na+/H+exchanger 3 and vacuolar-type H+-ATPase in the gills of blennies(Teleostei:Blenniidae)inhabiting rocky intertidal areas[J].Journal of Fish Biology,2012,80(6):2236-2252.