中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析
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摘要
为探讨中国绿水螅(Hydra sinensis)抗坏血酸过氧化物酶(Ascorbate peroxidase,APX)基因的起源及功能,研究采用RACE方法克隆了中国绿水螅APX基因的全长cDNA序列。该cDNA序列总长1357 bp,包括5′非编码区107 bp,3′非编码区146 bp及开放阅读框(Open reading frame,ORF) 1104 bp,共编码367个氨基酸,预测蛋白质分子量为40.79 kD。BLAST结果表明中国绿水螅APX蛋白同源序列绝大部分来自植物界;通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)进行的系统发生分析显示植物界及动物界物种的APX序列各自形成单系群。把APX基因ORF全长序列克隆到原核表达质粒pET-GST中,重组质粒转化E.coliBL21 (DE3)菌株,IPTG诱导后成功表达重组融合蛋白GST-APX,再使用纯化的重组蛋白免疫新西兰兔制备多克隆抗体用于APX蛋白的免疫印迹分析(Western blotting assay,WB)。在不同光照时长梯度(光强度2000 lx,每天分别光照0、4h、8h、12h、16h、20h及24h)下培养中国绿水螅30d,实时定量PCR (Quantitative real-timePCR,qPCR)及WB检测结果均表明光照时间较长时(每天光照12h以上)绿水螅APX表达呈现一定程度的上调。在长时间光辐射下水螅体内共生绿藻连续进行光合作用所累积的大量活性氧能够扩散到水螅细胞内,此时水螅体内表达上调的APX可能参与清除其细胞内的活性氧。
In order to explore the origin and physiological function of ascorbate peroxidase(APX) gene of Hydra sinensis,a full-length cDNA of APX gene of H.sinensis was isolated by rapid amplification of cDNA ends(RACE) technique.The full-length cDNA of APX gene was 1357 bp,containing 107 bp 5′UTR(Untranslated region),146 bp 3′UTR and a 1104 bp open reading frame(ORF) which encodes a polypeptide of 367 amino-acid residues with a molecular weight of 40.79 kD.BLAST(Basic Local Alignmen Search Tool) showed that most of homologous protein sequences of APX in H.sinensis belonged to the plant kingdom.Phylogenetic results by MaximumLikelihood(ML) method and Bayesian analyses revealed that homologous sequences of APX from plant and animal kingdom each formed a single cluster.The ORF of APX gene was subcloned into plasmid pET-GST and transferred into Escherichia coli BL21(DE3).The recombinant GST-APX fusion protein mainly expressed in the form of soluble were immunized New Zealand rabbits get the polyclonal antibody against APX for Western blotting assay(WB).H.sinensis was cultured(0,4,8,12,16,20 and 24 hours per day with illumination intensity 2000 lx) for 30 days,which up-regulated APX expression under longer illumination time(more than 16 hours per day).Due to continuous photosynthetic activity,a large number of reactive oxygen species(ROS) accumulating in symbiotic green algae could spread to the host cell(hydra cell),and the upregulation of APX expression in H.sinensis may mediate the removal of intracellular ROS.
In order to explore the origin and physiological function of ascorbate peroxidase(APX) gene of Hydra sinensis,a full-length cDNA of APX gene of H.sinensis was isolated by rapid amplification of cDNA ends(RACE) technique.The full-length cDNA of APX gene was 1357 bp,containing 107 bp 5′UTR(Untranslated region),146 bp 3′UTR and a 1104 bp open reading frame(ORF) which encodes a polypeptide of 367 amino-acid residues with a molecular weight of 40.79 kD.BLAST(Basic Local Alignmen Search Tool) showed that most of homologous protein sequences of APX in H.sinensis belonged to the plant kingdom.Phylogenetic results by MaximumLikelihood(ML) method and Bayesian analyses revealed that homologous sequences of APX from plant and animal kingdom each formed a single cluster.The ORF of APX gene was subcloned into plasmid pET-GST and transferred into Escherichia coli BL21(DE3).The recombinant GST-APX fusion protein mainly expressed in the form of soluble were immunized New Zealand rabbits get the polyclonal antibody against APX for Western blotting assay(WB).H.sinensis was cultured(0,4,8,12,16,20 and 24 hours per day with illumination intensity 2000 lx) for 30 days,which up-regulated APX expression under longer illumination time(more than 16 hours per day).Due to continuous photosynthetic activity,a large number of reactive oxygen species(ROS) accumulating in symbiotic green algae could spread to the host cell(hydra cell),and the upregulation of APX expression in H.sinensis may mediate the removal of intracellular ROS.
引文
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[18]Posada D,Buckley T R.Model selection and model averaging in phylogenetics:advantages of akaike information criterion and bayesian approaches over likelihood ratio tests[J].Systematic Biology,2004,53(5):793-808
[19]Jeon H K,Park H,Lee D,et al.Molecular detection of Spirometra decipiens in the United States[J].The Korean Journal of Parasitology,2016,54(4):503-507
[20]Ronquist F,Teslenko M,van der Mark P,et al.MrBayes3.2:efficient Bayesian phylogenetic inference and model choice across a large model space[J].Systematic Biology,2012,61(3):539-542
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[22]Pan H C,Song D X,Zhou K Y,et al.Cloning and prokaryotic expression of major ampullate spidroin gene of spider[J].Chinese Journal of Biotechnology,2007,23(3):446-451[潘红春,宋大祥,周开亚,等.蜘蛛大壶状丝蛋白基因的克隆与原核表达.生物工程学报,2007,23(3):446-451]
[23]Koolivand D,Bashir N S,Behjatnia S A,et al.Production of polyclonal antibody against Grapevine fanleaf virus movement protein expressed in Escherichia coli[J].Journal of Plant Pathology,2016,32(5):452-459
[24]Ali-Benali M A,Alary R,Joudrier P,et al.Comparative expression of five Lea genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR[J].Biochimica et Biophysica Acta,2005,1730(1):56-65
[25]Fraune J,Alsheimer M,Volff J N,et al.Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(41):16588-16593
[26]Passardi F,Bakalovic N,Teixeira F K,et al.Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes[J].Genomics,2007,89(5):567-579
[27]Shostak S A.symbiogenetic theory for the origins of cnidocysts in Cnidaria[J].Biosystems,1993,29(1):49-58
[28]Venn A A,Loram J E,Douglas A E.Photosynthetic symbioses in animals[J].Journal of Experimental Botany,2008,59(5):1069-1080
[29]Duroux L,Welinder K G.The peroxidase gene family in plants:a phylogenetic overview[J].Journal of Molecular Evolution,2003,57(4):397-407
[30]Pan H C,Qian X C,Li P,et al.The complete mitochondrial genome of Chinese green hydra,Hydra sinensis(Hydroida:Hydridae)[J].Mitochondrial DNA,2014,25(1):44-45
[31]Huss V A R,Holweg C,Kessler E,et al.There is an ecological basis for host/symbiont specificity in Chlorella/Hydra symbioses[J].Endocytobiosis&Cell Research,1993,10(1-2):35-46
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[34]Meyer E,Weis V M.Study of cnidarian-algal symbiosis in the“omics”age[J].Biology Bulletin,2012,223(1):44-65
[35]Ganot P,Moya A,Magnone V,et al.Adaptations to endosymbiosis in a cnidarian-dinoflagellate association:differential gene expression and specific gene duplications[J].PloS Genetics,2011,7(7):e1002187
[2]Anjum N A,Sharma P,Gill S S,et al.Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants[J].Environmental Science and Pollution Research International,2016,23(19):19002-19029
[3]Sousa R H,Carvalho F E,Ribeiro C W,et al.Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2O2 induced by CAT deficiency in rice[J].Plant Cell and Environment,2015,38(3):499-513
[4]Shigeoka S,Ishikawa T,Tamoi M,et al.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319
[5]Exposito-Rodriguez M,Laissue P P,Yvon-Durocher G,et al.Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism[J].Nature Communication,2017,8(1):49
[6]Smirnoff N,Wheeler G L.Ascorbic acid in plants:biosynthesis and function[J].Critical Reviews in Biochemistry and Molecular Biology,2000,35(4):291-314
[7]Raven E L.Understanding functional diversity and substrate specificity in haem peroxidases:what can we learn from ascorbate peroxidase[J]?Natural Product Reports,2003,20(4):367-381
[8]Sharp K H,Mewies M,Moody P C,et al.Crystal structure of the ascorbate peroxidase-ascorbate complex[J].Nature Structural Biology,2003,10(4):303-307
[9]Karyotou K,Donaldson R P.Ascorbate peroxidase,a scavenger of hydrogen peroxide in glyoxysomal membranes[J].Archives of Biochemistry and Biophysics,2005,434(2):248-257
[10]Smirnoff N.Ascorbic acid:metabolism and functions of a multi-facetted molecule[J].Current Opinion in Plant Biology,2000,3(3):229-235
[11]Sofo A,Scopa A,Nuzzaci M,et al.Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses[J].International Journal of Molecular Sciences,2015,16(6):13561-12578
[12]Mathews M C,Summers C B,Felton G W.Ascorbate peroxidase:A novel antioxidant enzyme in insects[J].Archives of Insect Biochemistry&Physiology,1997,34(1):57-68
[13]Luo Y J,Takeuchi T,Koyanagi R,et al.The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization[J].Nature Communication,2015,6:8301
[14]Chapman J A,Kirkness E F,Simakov O,et al.The dynamic genome of Hydra[J].Nature,2010,464(7288):592-596
[15]Habetha M,Bosch T C.Symbiotic Hydra express a plantlike peroxidase gene during oogenesis[J].Journal of Experimental Biology,2005,208(Pt 11):2157-2165
[16]Pan H C,Yang H Q,Zhao F X,et al.Molecular cloning,sequence analysis,prokaryotic expression,and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain[J].Genetics and Molecular Research,2014,13(3):6610-6622
[17]Larkin M A,Blackshields G,Brown N P,et al.Clustal Wand Clustal X version 2.0[J].Bioinformatics,2007,23(21):2947-2948
[18]Posada D,Buckley T R.Model selection and model averaging in phylogenetics:advantages of akaike information criterion and bayesian approaches over likelihood ratio tests[J].Systematic Biology,2004,53(5):793-808
[19]Jeon H K,Park H,Lee D,et al.Molecular detection of Spirometra decipiens in the United States[J].The Korean Journal of Parasitology,2016,54(4):503-507
[20]Ronquist F,Teslenko M,van der Mark P,et al.MrBayes3.2:efficient Bayesian phylogenetic inference and model choice across a large model space[J].Systematic Biology,2012,61(3):539-542
[21]Tong R,Pan H C,Ma J J,et al.Establishment of prokaryotic construct expressing mouse membrane anti-aging protein klotho gene specific fragment and preparation of polyclonal antibody against klotho[J].Journal of Medical Molecular Biology,2009,6(3):208-213[童睿,潘红春,马静静,等.小鼠膜型抗衰老Klotho蛋白基因特异片段的克隆,原核表达及多克隆抗体制备.医学分子生物学杂志,2009,6(3):208-213]
[22]Pan H C,Song D X,Zhou K Y,et al.Cloning and prokaryotic expression of major ampullate spidroin gene of spider[J].Chinese Journal of Biotechnology,2007,23(3):446-451[潘红春,宋大祥,周开亚,等.蜘蛛大壶状丝蛋白基因的克隆与原核表达.生物工程学报,2007,23(3):446-451]
[23]Koolivand D,Bashir N S,Behjatnia S A,et al.Production of polyclonal antibody against Grapevine fanleaf virus movement protein expressed in Escherichia coli[J].Journal of Plant Pathology,2016,32(5):452-459
[24]Ali-Benali M A,Alary R,Joudrier P,et al.Comparative expression of five Lea genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR[J].Biochimica et Biophysica Acta,2005,1730(1):56-65
[25]Fraune J,Alsheimer M,Volff J N,et al.Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(41):16588-16593
[26]Passardi F,Bakalovic N,Teixeira F K,et al.Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes[J].Genomics,2007,89(5):567-579
[27]Shostak S A.symbiogenetic theory for the origins of cnidocysts in Cnidaria[J].Biosystems,1993,29(1):49-58
[28]Venn A A,Loram J E,Douglas A E.Photosynthetic symbioses in animals[J].Journal of Experimental Botany,2008,59(5):1069-1080
[29]Duroux L,Welinder K G.The peroxidase gene family in plants:a phylogenetic overview[J].Journal of Molecular Evolution,2003,57(4):397-407
[30]Pan H C,Qian X C,Li P,et al.The complete mitochondrial genome of Chinese green hydra,Hydra sinensis(Hydroida:Hydridae)[J].Mitochondrial DNA,2014,25(1):44-45
[31]Huss V A R,Holweg C,Kessler E,et al.There is an ecological basis for host/symbiont specificity in Chlorella/Hydra symbioses[J].Endocytobiosis&Cell Research,1993,10(1-2):35-46
[32]Huss V A R,Frank C,Hartmann E C,et al.Biochemical taxonomy and molecular phylogeny of the Genus Chlorella sensu lato(Chlorophyta)[J].Journal of Phycology,1999,35(3):587-598
[33]Davy S K,Allemand D,Weis V M.Cell biology of cnidarian-dinoflagellate symbiosis[J].Microbiology and Molecular Biology Reviews,2012,76(2):229-261
[34]Meyer E,Weis V M.Study of cnidarian-algal symbiosis in the“omics”age[J].Biology Bulletin,2012,223(1):44-65
[35]Ganot P,Moya A,Magnone V,et al.Adaptations to endosymbiosis in a cnidarian-dinoflagellate association:differential gene expression and specific gene duplications[J].PloS Genetics,2011,7(7):e1002187
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