一种三角帆蚌肌浆网Ca~(2+)-ATP酶基因cDNA的全长克隆与表达分析
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摘要
采用RACE技术,从三角帆蚌(Hyriopsis cumingii)鳃组织中成功克隆得到一种肌浆网Ca~(2+)-ATP酶(sarco/endoplasmic reticulum calcium ATPase,SERCA)基因的全长cDNA序列,共3 326 bp,包含201-bp 5’-UTR区域、3 060-bp编码框(ORF)和65-bp 3’-UTR。ORF共编码1 019个氨基酸,预测无信号肽。该基因氨基酸序列呈现出典型的Ca~(2+)-ATP酶特征,由Cation_ATPase_N、E1-E2_ATPase、Hydrolase、Cation_ATPase_C四种类型结构域组成,其内含SERCAs的常见结构组成包括磷酸化区域、异硫氰酸荧光素位点、FSBA结合位点、受磷蛋白结合区以及毒胡萝卜素位点。分析显示,该基因序列高度保守且与海洋软体动物具有最高同源性。荧光定量PCR检测,该基因在三角帆蚌外套膜、斧足、鳃、肝胰腺、性腺等5个组织中均有表达,且在鳃、外套膜、肝胰腺组织中表达较高。不同Ca~(2+)浓度处理试验的结果表明,随水体中Ca~(2+)浓度逐渐升高,该基因在外套膜中的表达水平呈先下降后上升趋势,并在Ca~(2+)浓度为60 mg·L~(-1)时达到最低值,80 mg·L~(-1)时达到最高值。同时在60 mg·L~(-1) Ca~(2+)浓度条件下,外套膜中SERCA基因的表达量随时间推移先上升,并于48 h时达到最高,而后逐渐下降。上述结果为进一步深入研究SERCA基因的功能及其调控机理奠定了基础。
The full-length cDNA sequence of sarco/endoplasmic reticulum calcium ATPase isoform gene was isolated from the gill of Hyriopsis cumingii by using SMART RACE technique. The entire SERCA cDNA was 3 326 bp, including a 201-bp 5′-UTR, a 3 060-bp ORF and a 65-bp 3′-UTR, encoding a 1 019-amino acid protein, and no putative signal peptide was predicted. Four types of domains were found and the general structures of SERCA gene, containing phosphorylation region, fluorescein isothiocyanate site, 5′-(p-fluorosulfonyl) benzoyladenosinebinding site, phospholamban-binding motif and thapsigargin sites were also identified. Compared with SERCA homologs from seawater mollusks and other animals, the SERCA of H. cumingii had high similarity with them in both se￣quence and structure. Tissue-specific expression analysis revealed that SERCA mRNA was detected in all the sampled tissues, but was prominently expressed in the gill, mantle and hepatopancreas. When exposed to a series of increasing Ca~(2+) that lasted 7 days, the mRNA expression of SERCA gene in mantle was shown to be cove-shaped, ascending when Ca~(2+) concentration was more than 60 mg·L~(-1) and peaking at 80 mg·L~(-1). Moreover, the temporal expression of SERCA transcripts in mantle after 60 mg·L~(-1) Ca~(2+) exposure was slightly down-regulated at 24 h, but up-regulated from 24 h to 48 h post-treatment, peaking at 48 h. The results of present study will provide useful information for further studies on function and regulation mechanism of SERCA gene.
The full-length cDNA sequence of sarco/endoplasmic reticulum calcium ATPase isoform gene was isolated from the gill of Hyriopsis cumingii by using SMART RACE technique. The entire SERCA cDNA was 3 326 bp, including a 201-bp 5′-UTR, a 3 060-bp ORF and a 65-bp 3′-UTR, encoding a 1 019-amino acid protein, and no putative signal peptide was predicted. Four types of domains were found and the general structures of SERCA gene, containing phosphorylation region, fluorescein isothiocyanate site, 5′-(p-fluorosulfonyl) benzoyladenosinebinding site, phospholamban-binding motif and thapsigargin sites were also identified. Compared with SERCA homologs from seawater mollusks and other animals, the SERCA of H. cumingii had high similarity with them in both se￣quence and structure. Tissue-specific expression analysis revealed that SERCA mRNA was detected in all the sampled tissues, but was prominently expressed in the gill, mantle and hepatopancreas. When exposed to a series of increasing Ca~(2+) that lasted 7 days, the mRNA expression of SERCA gene in mantle was shown to be cove-shaped, ascending when Ca~(2+) concentration was more than 60 mg·L~(-1) and peaking at 80 mg·L~(-1). Moreover, the temporal expression of SERCA transcripts in mantle after 60 mg·L~(-1) Ca~(2+) exposure was slightly down-regulated at 24 h, but up-regulated from 24 h to 48 h post-treatment, peaking at 48 h. The results of present study will provide useful information for further studies on function and regulation mechanism of SERCA gene.
引文
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[3]PETERSON J A,OBLAD R V,MECHAM J C,et al.Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium[J].Biochemistry and Biophysics Reports,2016,7:253-258.
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[17]GUNTESKI-HAMBLIN A M,GREEB J,SHULL G E.A novel Ca2+pump expressed in brain,kidney,and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene.Identification of c D-NAs encoding Ca2+and other cation-transporting ATPases using an oligonucleotide probe derived from the ATP-binding site[J].The Journal of Biological Chemistry,1988,263(29):15032-15040.
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[20]ZHANG Z,CHEN D,WHEATLY M G.Cloning and characterization of sarco/endoplasmic reticulum Ca2+-ATPase(SERCA)from crayfish axial muscle[J].Journal of Experimental Biology,2000,203(22):3411-3423.
[21]ESCALANTE R,SASTRE L.Similar alternative splicing events generate two sarcoplasmic or endoplasmic reticulum CaATPase isoforms in the crustacean Artemia franciscana and in vertebrates[J].Journal of Biological Chemistry,1993,268(19):14090-14095.
[22]DODE L,DE GREEF C,MOUNTIAN I,et al.Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene[J].Journal of Biological Chemistry,1998,273(22):13982-13994.
[23]DAVIS W L,JONES R G,HAGLER H K.Calcium containing lysosomes in the normal chick duodenum:a histochemical and analytical electron microscopic study[J].Tissue and Cell,1979,11(1):127-138.
[24]郝莹莹.三种环境因子对三解帆蚌外套膜Ca2+代谢和ALP基因表达的影响[D].上海:上海海洋大学,2011.HAO Y Y.Effect of three different factors on the calcium ion metabolism and expression of alkaline phosphatase gene in Hyriopsis cumingii[D].Shanghai:Shanghai Ocean University,2011.(in Chinese with English abstract)
[25]CHO J H,BANDYOPADHYAY J,LEE J,et al.Two isoforms of sarco/endoplasmic reticulum calcium ATPase(SER-CA)are essential in Caenorhabditis elegans[J].Gene,2000,261(2):211-219.
[26]BEIRAO P.Sodium-and calcium-dependent mechanisms in the action of the secretary epithelium of a clam mantle[J].Journal of Experimental Biology,1989,145(1):395-402.
[27]KIRSCHNER L B,SORENSON A L,KRIEBEL M.Calcium and electric potential across the clam mantle[J].Science,1960,131(3402):735.
[28]COIMBRA J,MACHADO J,FERNANDES P L,et al.Electrophysiology of the mantle of Anodonta cygnea[J].Journal of Experimental Biololgy,1988,140:65-88.
[29]唐敏,石安静.环境钙浓度对淡水育珠蚌外套膜及珍珠囊钙代谢的影响[J].四川大学学报(自然科学版),2000,37(5):741-747.TANG M,SHI A J.Studies of environmental calcium concentration effect on the calcium metabolism of the mantle and pearl sac of the freshwater pearl mussel[J].Journal of Sichuan University(Natural Science Edition),2000,37(5):741-747.(in Chinese with English abstract)
[30]李静,石安静,莫尚武,等.45Ca在圆背角无齿蚌体内的代谢与分布[J].水生生物学报,2002,26(2):188-191.LI J,SHI A J,MO S W,et al.Metabolism and distribution of calcium in Anodonta woodiano pacifica(Heuda)traced by45Ca[J].Acta Hydrobiologica Sinica,2002,26(2):188-191.(in Chinese with English abstract)
[31]NAGATA Y,YAMAMOTO T,EMA M,et al.c DNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca2+-ATPase[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1998,119(4):777-785.
[32]INESI G,SUMBILLA C,KIRTLEY M E.Relationships of molecular structure and function in Ca2(+)-transport ATPase[J].Physiological Reviews,1990,70(3):749-760.
[33]李文娟,施志仪,郝莹莹,等.运用微电极技术测定三角帆蚌外套膜Ca2+流量的研究[J].水生生物学报,2011,35(3):545-549.LI W J,SHI Z Y,HAO Y Y,et al.Research of calcium flux by ion-selective microelectrodes in mantle tissue of Hyriopsis cumingii[J].Acta Hydrobiologica Sinica,2011,35(3):545-549.(in Chinese with English abstract)
[34]舒妙安,胡杭娇,陆晶莹,等.三角帆蚌钙网蛋白基因c D-NA的全长克隆与表达分析[J].水生生物学报,2013,37(6):999-1006.SHU M A,HU H J,LU J Y,et al.Full-length c DNA cloning and expression analysis of calreticulin gene from Hyriopsis cumingii[J].Acta Hydrobiologica Sinica,2013,37(6):999-1006.(in Chinese with English abstract)
[2]SIMKISS K,WILBUR K M.Biomineralization:cell biology and mineral deposition[J].Quarterly Review of Biology,1989,1(3):257-264.
[3]PETERSON J A,OBLAD R V,MECHAM J C,et al.Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium[J].Biochemistry and Biophysics Reports,2016,7:253-258.
[4]小林新二朗,渡部哲光.珍珠的研究[M].熊大仁译.北京:农业出版社,1966.
[5]MANDAL A,ARUNACHALAM S C,MELESHKEVITCH EA,et al.Cloning of sarco-endoplasmic reticulum Ca2+-AT-Pase(SERCA)from Caribbean spiny lobster Panulirus argus[J].Journal of Comparative Physiology B,2009,179(2):205-214.
[6]CARAFOLI E,STAUFFER T.The plasma membrane calcium pump:functional domains,regulation of the activity,and tissue specificity of isoform expression[J].Journal of Neurobiology,1994,25(3):312-324.
[7]HUSSAIN A.Involvement of sarco/endoplasmic reticulum Ca2+ATPases in cell function and the cellular consequences of their inhibition[J].Journal of Membrane Biology,1999,172(2):91-99.
[8]FAN W M,LI C Z,LI S,et al.Cloning,characterization,and expression patterns of three Sarco/Endoplasmic reticulum Ca2+-ATPase isoforms from pearl oyster(Pinctada fucata)[J].Acta Biochimica et Biophysica Sinica,2007,39(9):722-730.
[9]SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology&Evolution,1987,4(4):406-425.
[10]LI X C,ZHU L,LI L G,et al.A novel myeloid differentiation factor 88 homolog,Sp My D88,exhibiting Sp Toll-binding activity in the mud crab Scylla paramamosain[J].Developmental&Comparative Immunology,2013,39(4):313-322.
[11]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[12]MФLLER J V,NISSEN P,SФRENSEN T L,et al.Transport mechanism of the sarcoplasmic reticulum Ca2+-ATPase pump[J].Current Opinion in Structural Biology,2005,15(4):387-393.
[13]DETTBARN C,PALADE P.Effects of alkaline p H on sarcoplasmic reticulum Ca2+release and Ca2+uptake[J].Journal of Biological Chemistry,1991,266(14):8993-9001.
[14]LEE A G,EAST J M.What the structure of a calcium pump tells us about its mechanism[J].Biochemical Journal,2001,356(Pt 3):665-683.
[15]BRANDL C J,GREEN N M,KORCZAK B,et al.Two Ca2+ATPase genes:homologies and mechanistic implications of deduced amino acid sequences[J].Cell,1986,44(4):597-607.
[16]CHABANE C,CORVAZIER E,BREDOUX R,et al.Sarco/endoplasmic reticulum Ca2+ATPase type 3 isoforms(SERCA3b and SERCA3f):Distinct roles in cell adhesion and ER stress[J].Biochemical and Biophysical Research Communications,2006,345(4):1377-1385.
[17]GUNTESKI-HAMBLIN A M,GREEB J,SHULL G E.A novel Ca2+pump expressed in brain,kidney,and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene.Identification of c D-NAs encoding Ca2+and other cation-transporting ATPases using an oligonucleotide probe derived from the ATP-binding site[J].The Journal of Biological Chemistry,1988,263(29):15032-15040.
[18]BOBE R,BREDOUX R,CORVAZIER E,et al.How many Ca2+ATPase isoforms are expressed in a cell type?A growing family of membrane proteins illustrated by studies in platelets[J].Platelets,2005,16(3/4):133-150.
[19]CHEN D D,ZHANG Z P,WHEATLY M G,et al.Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ATPase(SERCA)from crayfish[J].The Journal of Experimental Biology,2002,205(Pt17):2677-2686.
[20]ZHANG Z,CHEN D,WHEATLY M G.Cloning and characterization of sarco/endoplasmic reticulum Ca2+-ATPase(SERCA)from crayfish axial muscle[J].Journal of Experimental Biology,2000,203(22):3411-3423.
[21]ESCALANTE R,SASTRE L.Similar alternative splicing events generate two sarcoplasmic or endoplasmic reticulum CaATPase isoforms in the crustacean Artemia franciscana and in vertebrates[J].Journal of Biological Chemistry,1993,268(19):14090-14095.
[22]DODE L,DE GREEF C,MOUNTIAN I,et al.Structure of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene[J].Journal of Biological Chemistry,1998,273(22):13982-13994.
[23]DAVIS W L,JONES R G,HAGLER H K.Calcium containing lysosomes in the normal chick duodenum:a histochemical and analytical electron microscopic study[J].Tissue and Cell,1979,11(1):127-138.
[24]郝莹莹.三种环境因子对三解帆蚌外套膜Ca2+代谢和ALP基因表达的影响[D].上海:上海海洋大学,2011.HAO Y Y.Effect of three different factors on the calcium ion metabolism and expression of alkaline phosphatase gene in Hyriopsis cumingii[D].Shanghai:Shanghai Ocean University,2011.(in Chinese with English abstract)
[25]CHO J H,BANDYOPADHYAY J,LEE J,et al.Two isoforms of sarco/endoplasmic reticulum calcium ATPase(SER-CA)are essential in Caenorhabditis elegans[J].Gene,2000,261(2):211-219.
[26]BEIRAO P.Sodium-and calcium-dependent mechanisms in the action of the secretary epithelium of a clam mantle[J].Journal of Experimental Biology,1989,145(1):395-402.
[27]KIRSCHNER L B,SORENSON A L,KRIEBEL M.Calcium and electric potential across the clam mantle[J].Science,1960,131(3402):735.
[28]COIMBRA J,MACHADO J,FERNANDES P L,et al.Electrophysiology of the mantle of Anodonta cygnea[J].Journal of Experimental Biololgy,1988,140:65-88.
[29]唐敏,石安静.环境钙浓度对淡水育珠蚌外套膜及珍珠囊钙代谢的影响[J].四川大学学报(自然科学版),2000,37(5):741-747.TANG M,SHI A J.Studies of environmental calcium concentration effect on the calcium metabolism of the mantle and pearl sac of the freshwater pearl mussel[J].Journal of Sichuan University(Natural Science Edition),2000,37(5):741-747.(in Chinese with English abstract)
[30]李静,石安静,莫尚武,等.45Ca在圆背角无齿蚌体内的代谢与分布[J].水生生物学报,2002,26(2):188-191.LI J,SHI A J,MO S W,et al.Metabolism and distribution of calcium in Anodonta woodiano pacifica(Heuda)traced by45Ca[J].Acta Hydrobiologica Sinica,2002,26(2):188-191.(in Chinese with English abstract)
[31]NAGATA Y,YAMAMOTO T,EMA M,et al.c DNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca2+-ATPase[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1998,119(4):777-785.
[32]INESI G,SUMBILLA C,KIRTLEY M E.Relationships of molecular structure and function in Ca2(+)-transport ATPase[J].Physiological Reviews,1990,70(3):749-760.
[33]李文娟,施志仪,郝莹莹,等.运用微电极技术测定三角帆蚌外套膜Ca2+流量的研究[J].水生生物学报,2011,35(3):545-549.LI W J,SHI Z Y,HAO Y Y,et al.Research of calcium flux by ion-selective microelectrodes in mantle tissue of Hyriopsis cumingii[J].Acta Hydrobiologica Sinica,2011,35(3):545-549.(in Chinese with English abstract)
[34]舒妙安,胡杭娇,陆晶莹,等.三角帆蚌钙网蛋白基因c D-NA的全长克隆与表达分析[J].水生生物学报,2013,37(6):999-1006.SHU M A,HU H J,LU J Y,et al.Full-length c DNA cloning and expression analysis of calreticulin gene from Hyriopsis cumingii[J].Acta Hydrobiologica Sinica,2013,37(6):999-1006.(in Chinese with English abstract)