魁蚶HIF-1α基因结构特征及对低氧胁迫的响应
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摘要
低氧诱导因子(hypoxia-induciblefactor,HIF)是低氧信号传导途径中的关键因子,在动物低氧应答反应中发挥重要作用。为探讨魁蚶(Scapharca broughtonii) HIF-1α基因结构特征及在低氧胁迫下的应答规律,本研究以魁蚶转录组数据库中的部分序列为基础,通过cDNA末端快速扩增(rapid amplication of cDNA ends, RACE)技术克隆获得了魁蚶HIF-1α基因cDNA全长序列(命名为SbHIF-1α),并检测了其mRNA的组织分布和低氧胁迫下的表达规律。序列和结构分析显示, SbHIF-1α基因cDNA全长为2741 bp,其中包括2136 bp的ORF,编码711个氨基酸,含有HIF保守的HLH、PAS-A、PAS-B和PAC结构域,预测蛋白分子量为80.8kDa,理论等电点为5.57;SbHIF-1α与所选其他物种HIF-1α的序列相似度为56%～95%;系统进化分析显示SbHIF-1α与软体动物的遗传距离最近。qRT-PCR结果显示,在魁蚶的血淋巴、鳃、外套膜、斧足、闭壳肌和肝胰腺6个组织内均能检测到SbHIF-1α基因,在血淋巴中表达量最高,鳃次之,与其他4个组织都存在显著差异(P<0.05)。海水溶解氧(DO)为0.5 mg/L、2.5 mg/L、4.5mg/L的低氧胁迫下,每个组织中SbHIF-1α都积极响应,其中血淋巴和鳃比其他四个组织的响应程度大;血淋巴中, DO为0.5 mg/L胁迫4 h后, SbHIF-1α的表达量较对照组即达到极显著差异(P<0.01),胁迫64 h后,表达量达到最高,是对照组的519.43倍;每个组织对不同浓度DO处理响应结果表明,3个处理浓度中0.5mg/L处理对SbHIF-1α激活程度相对较大。本研究明确了SbHIF-1α的基因结构特征、时空表达特征及对低氧胁迫的响应规律,丰富了海洋贝类HIF基因的研究资料。
Hypoxia-inducible factor is a key factor in HIF signaling pathway and plays an significant role in Hypoxia response in animals. To determine the response of hypoxia-inducible factor(HIF-1) gene of Scapharca broughtonii under hypoxia stress and to reveal the unique hypoxia-adapting mechanism of Scapharca broughtonii.The cDNA sequence of HIF-1α(named SbHIF-1α) by EST and RACE methods were obtained. The mRNA expression in the gene under hypoxia stress was also studied.Sequence analysis revealed that the SbHIF-1α cDNA was 2741 bp in length, including an open reading frame(ORF) of 2136 bp encoding a polypeptide of 711 amino acid residues with conserved HLH, PAS-A, PAS-B and PAC motif. The predicted molecular weight is 80.8 kDa and isoelectric point(pI) is 5.57. The analysis of structure and putative functional sites showed SBHIF-1α shared56%-95% with other species, the highest with Chlamys farreri HIF-1α for 95%. Phylogenetic analysis showed that SbHIF-1α and the corresponding homologous molecule in mollusks clustered into one branch. The mRNA expression analysis of SbHIF-1α in tested tissues by quantitative real-time PCR(qRT-PCR) revealed that the mRNA of the the gene could be all detected in foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas. The expression level of SbHIF-1α in haemocytes were more than that in other tissues. Under the hypoxic stress of0.5 mg/L, 2.5 mg/L and 4.5 mg/L dissolved oxygen(DO) in seawater, SbHIF-1α in each tissue responded positively, and hemolymph and gill were more responsive than the other four tissues. n haemolymph, the expression of SbHIF-1α was significantly different from that of control group after 4 hours of DO stress(P<0.01). After 64 hours of stress, the expression of SbHIF-1α reached the highest level, which was 519.43 times of that of control group. The response of each tissue to at different DO concentrations showed that the activation of SbHIF-1α was relatively high in 0.5 mg/L of the three treatment concentrations. This study clarified the structural characteristics,temporal and spatial expression characteristics of SbHIF-1α gene and its response to hypoxia stress, which enriched the research data of marine shellfish HIF gene.
Hypoxia-inducible factor is a key factor in HIF signaling pathway and plays an significant role in Hypoxia response in animals. To determine the response of hypoxia-inducible factor(HIF-1) gene of Scapharca broughtonii under hypoxia stress and to reveal the unique hypoxia-adapting mechanism of Scapharca broughtonii.The cDNA sequence of HIF-1α(named SbHIF-1α) by EST and RACE methods were obtained. The mRNA expression in the gene under hypoxia stress was also studied.Sequence analysis revealed that the SbHIF-1α cDNA was 2741 bp in length, including an open reading frame(ORF) of 2136 bp encoding a polypeptide of 711 amino acid residues with conserved HLH, PAS-A, PAS-B and PAC motif. The predicted molecular weight is 80.8 kDa and isoelectric point(pI) is 5.57. The analysis of structure and putative functional sites showed SBHIF-1α shared56%-95% with other species, the highest with Chlamys farreri HIF-1α for 95%. Phylogenetic analysis showed that SbHIF-1α and the corresponding homologous molecule in mollusks clustered into one branch. The mRNA expression analysis of SbHIF-1α in tested tissues by quantitative real-time PCR(qRT-PCR) revealed that the mRNA of the the gene could be all detected in foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas. The expression level of SbHIF-1α in haemocytes were more than that in other tissues. Under the hypoxic stress of0.5 mg/L, 2.5 mg/L and 4.5 mg/L dissolved oxygen(DO) in seawater, SbHIF-1α in each tissue responded positively, and hemolymph and gill were more responsive than the other four tissues. n haemolymph, the expression of SbHIF-1α was significantly different from that of control group after 4 hours of DO stress(P<0.01). After 64 hours of stress, the expression of SbHIF-1α reached the highest level, which was 519.43 times of that of control group. The response of each tissue to at different DO concentrations showed that the activation of SbHIF-1α was relatively high in 0.5 mg/L of the three treatment concentrations. This study clarified the structural characteristics,temporal and spatial expression characteristics of SbHIF-1α gene and its response to hypoxia stress, which enriched the research data of marine shellfish HIF gene.
引文
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[19]Zhao Q.Molecular cloning and functional analysis of hemoglobin genes from Ark Shell Scapharca Broughtonii[D].Shanghai:Shanghai Ocean University,2018.[赵庆.血红蛋白基因的克隆及功能分析[D].上海:上海海洋大学,2018.]
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[22]Cheng W,Liu C H,Cheng S Y,et al.Effect of dissolved oxygen on the acid-base balance and ion concentration of Taiwan abalone Haliotis diversicolor supertexta[J].Aquaculture,2004,231(1-4):573-586.
[23]Yuan Y,Zhong H.Progress of structure and function of hypoxia-inducible factor-1[J].Progress in Modern Biomedicine,2010,10(5):961-963.[袁源,钟竑.缺氧诱导因子-1结构及功能的研究进展[J].现代生物医学进展,2010,10(5):961-963.]
[24]Rolfs A,Kvietikova I,Gassmann M,et al.Oxygenregulated transferrin expression is mediated by hypoxiainducible factor-1[J].Journal of Biological Chemistry,1997,272(32):20055-20062.
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[26]Kawabe S,Yokoyama Y.Role of hypoxia-inducible factorαin response to hypoxia and heat shock in the Pacific oyster Crassostrea gigas[J].Marine Biotechnology,2011,14(1):106-119.]
[27]Li T,Brouwer M.Hypoxia-inducible factor,gs HIF,of the grass shrimp Palaemonetes pugio:Molecular characterization and response to hypoxia[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2007,147(1):11-19.
[28]Semenza G L,Jiang B H,Leung S W,et al.Hypoxia response elements in the aldolase A,enolase 1,and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1[J].Journal of Biological Chemistry,1996,271(51):32529-32537.
[29]Xiao W H.Hypoxic signal transduction pathway and hypoxic adaptation in fish[J].Scientia Sinica Vitae,2012,44(12):1227-1235.[肖武汉.低氧信号传导途径与鱼类低氧适应[J].中国科学:生命科学,2012,44(12):1227-1235.]
[30]Han Q Q,Wang H,Li X,et al.Research progress of HIF-1in Cyprinidae[J].Journal of Shanxi Agricultural Sciences,2018,46(9):1587-1590.[韩青青,王华,李鑫,等.鲤科鱼类HIF-1的研究进展[J].山西农业科学,2018,46(9):1587-1590.]
[31]Piontkivska H,Chung J S,Ivanina A V,et al.Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica(Gmelin):Hypoxia-inducible factorα(HIF-α)and HIF-prolyl hydroxylase(PHD)[J].Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,2011,6(2):103-114.
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[33]Cai X H,Huang Y T,Zhang X,et al.Cloning,characterization,hypoxia and heat shock response of hypoxia inducible factor-1(HIF-1)from the small abalone Haliotis diversicolor[J].Gene,2014,534(2):256-264.
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[2]Cai X H,Huang Y T,Zhang Z P,et al.Hypoxia inducible factor-1(HIF-1)and its research advance in aquatic[J].Journal of Agricultural Biotechnology,2014,22(1):119-132.[蔡秀红,黄贻涛,张子平,等.缺氧诱导因子-1(HIF-1)及其在水生动物中的研究进展[J].农业生物技术学报,2014,22(1):119-132.]
[3]Manolescu B,Oprea E,Busu C,et al.Natural compounds and the hypoxia-inducible factor(HIF)signalling pathway[J].Biochimie,2009,91(11-12):1347-1358.
[4]Li Q F.Regulation of gene expression by HIF-1αand HIF-2α[J].Journal of International Pathology and Clinical Medicine,2005,25(5):447-449.[李启芳.HIF-1α和HIF-2α对基因表达的调控[J].国际病理科学与临床杂志,2005,25(5):447-449.]
[5]Jewell U R,Kvietikova I,Scheid A,et al.Induction of HIF-1αin response to hypoxia is instantaneous[J].The FASEB Journal,2001,15(7):1312-1314.
[6]Li C Y,Wang L,Wu K N,et al.Cloning and bioinformatics analysis of HIF-3A in Jinchuan yak[J].Biotechnology,2017,5(12):472-477.[李晨阳,王利,吴开年,等.金川牦牛HIF-3A克隆及生物信息学分析[J].生物技术,2017,5(12):472-477.]
[7]LüY Q,Li X,Cao Y B,et al.Site-directed mutagenesis and eukaryotic expression of HIF-2αin Myospalax baileyi[J].Journal of Shanxi Agricultural Sciences,2017,45(8):1228-1231.[吕雅琪,李鑫,曹诣斌,等.高原鼢鼠HIF-2α的定点突变与真核表达[J].山西农业科学,2017,45(8):1228-1231.]
[8]Xu G,Zhao X J,Xiang X Y,et al.Pulmonary vascular remodeling in a rat model and the role of HIF-1αin the pathogenesis[J].Journal of Medical Research,2016,2(4):127-130.[许果,赵兴吉,向小勇,等.大鼠肺血管重塑动物模型的建立及HIF-1α在其中的作用[J].医学研究杂志,2016,2(4):127-130.]
[9]Shen R J,Jiang X Y,Pu J W,et al.HIF-1αand-2αgenes in a hypoxia-sensitive teleost species Megalobrama amblycephala:cDNA cloning,expression and different responses to hypoxia[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2010,157(3):273-280.
[10]Chen N,Chen L P,Zhang J,et al.Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits,HIF-1α/2α/3αof the hypoxia-sensitive freshwater species,Chinese sucker[J].Gene,2012,498(1):81-90.
[11]Rimoldi S,Terova G,Ceccuzzi P,et al.HIF-1αmRNA levels in Eurasian perch(Perca fluviatilis)exposed to acute and chronic hypoxia[J].Molecular Biology Reports,2012,39(4):4009-4015.
[12]Wang Z S,Qi Z T,Qiu M,et al.Changes of globins expression in the half-smooth tongue sole(Cynoglossus semilaevis)in response to short-term hypoxia[J].Acta Ichthyologica et Piscatoria,2011,41(3):179-184.
[13]Zhang H.Cloning of the hypoxia inducible factor-1αand gene expression analysis of Pelteonagrus fulvidsco[D].Baoding:Hebei University,2010.[张慧.黄颡鱼HIF-1α基因克隆与表达特征分析[D].保定:河北大学,2010.]
[14]Kawabe S,Yokoyama Y.Role of hypoxia-inducible factorαin response to hypoxia and heat shock in the Pacific oyster Crassostrea gigas[J].Marine Biotechnology,2011,14(1):106-119.
[15]Cai X H,Huang Y T,Zhang X,et al.Cloning,characterization,hypoxia and heat shock response of hypoxia inducible factor-1(HIF-1)from the small abalone Haliotis diversicolor[J].Gene,2014,534(2):256-264.
[16]Wang Z S.Cloning of Hif-1,Hb-1 and Tf genes in half-smooth tongue sole(Cynoglossus semilaevis)and expression analysis under hypoxia[D].Zhenjiang:Jiangsu University of Science and Technology,2012.[王资生.半滑舌鳎HIF-1α、Hb-α1和Tf基因克隆及在低氧胁迫下的表达分析[D].镇江:江苏科技大学,2012.]
[17]David E,Tanguy A,Pichavant K,et al.Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions[J].FEBS Journal,2005,272(21):5635-565.
[18]Wang S F,Bao Y B,Shi M J,et al.Purification and antibacterial activity of hemoglobin from Tegillarca granosa[J].Acta Oceanologica Sinica,2014,36(12):67-73.[王素芳,包永波,施淼江,等.泥蚶血红蛋白的制备及其抗菌活性研究[J].海洋学报,2014,36(12):67-73.]
[19]Zhao Q.Molecular cloning and functional analysis of hemoglobin genes from Ark Shell Scapharca Broughtonii[D].Shanghai:Shanghai Ocean University,2018.[赵庆.血红蛋白基因的克隆及功能分析[D].上海:上海海洋大学,2018.]
[20]Sun J,Wang B J,Sun S J,et al.cDNA cloning and sequence analysis of hemocyanin in Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2010,31(1):82-90.[孙杰,王宝杰,孙姝娟,等.中国对虾血蓝蛋白基因c DNA的克隆与序列分析[J].渔业科学进展,2010,31(1):82-90.]
[21]Semenza G L,Agani F,Feldser D,et al.Hypoxia,HIF-1,and the pathophysiologi of common human diseases[M]//Oxygen Sensing.Boston,Springer,2002,475:123-130.
[22]Cheng W,Liu C H,Cheng S Y,et al.Effect of dissolved oxygen on the acid-base balance and ion concentration of Taiwan abalone Haliotis diversicolor supertexta[J].Aquaculture,2004,231(1-4):573-586.
[23]Yuan Y,Zhong H.Progress of structure and function of hypoxia-inducible factor-1[J].Progress in Modern Biomedicine,2010,10(5):961-963.[袁源,钟竑.缺氧诱导因子-1结构及功能的研究进展[J].现代生物医学进展,2010,10(5):961-963.]
[24]Rolfs A,Kvietikova I,Gassmann M,et al.Oxygenregulated transferrin expression is mediated by hypoxiainducible factor-1[J].Journal of Biological Chemistry,1997,272(32):20055-20062.
[25]Shen R J.Structural and functional studies of hypoxia inducible factors in Megalobrama amblycephala[D].Shanghai:Shanghai Ocean University,2011.[沈睿杰.团头鲂缺氧诱导因子的结构和功能研究[D].上海:上海海洋大学,2011.]
[26]Kawabe S,Yokoyama Y.Role of hypoxia-inducible factorαin response to hypoxia and heat shock in the Pacific oyster Crassostrea gigas[J].Marine Biotechnology,2011,14(1):106-119.]
[27]Li T,Brouwer M.Hypoxia-inducible factor,gs HIF,of the grass shrimp Palaemonetes pugio:Molecular characterization and response to hypoxia[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2007,147(1):11-19.
[28]Semenza G L,Jiang B H,Leung S W,et al.Hypoxia response elements in the aldolase A,enolase 1,and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1[J].Journal of Biological Chemistry,1996,271(51):32529-32537.
[29]Xiao W H.Hypoxic signal transduction pathway and hypoxic adaptation in fish[J].Scientia Sinica Vitae,2012,44(12):1227-1235.[肖武汉.低氧信号传导途径与鱼类低氧适应[J].中国科学:生命科学,2012,44(12):1227-1235.]
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