烟蚜热激蛋白基因MpHsp70的克隆及在UV-B胁迫下的表达分析
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摘要
【目的】为探讨烟蚜Myzus persicae适应UV-B胁迫的分子机制。【方法】采用RT-PCR和RACE技术克隆了烟蚜热激蛋白基因Hsp70的全长,利用生物信息学方法分析了其特征;采用实时荧光定量PCR检测了不同时长(0,15,30,60,90和120 min) UV-B胁迫下烟蚜成虫中该基因的相对表达量。【结果】克隆获得烟蚜Hsp70基因并命名为MpHsp70(GenBank登录号:MF509827),该基因全长为2 221 bp,开放阅读框(ORF) 1 965 bp,编码654个氨基酸,蛋白相对分子量为71. 41kD,等电点(p I)为5. 34,末端高度保守序列EEVD显示该蛋白属于胞质热激蛋白。系统进化关系分析表明,MpHsp70与多种昆虫的Hsp70的同源性较高,表现出Hsp70基因的高度保守性。实时荧光定量PCR分析表明,随着UV-B照射时间的延长,烟蚜成虫体内MpHsp70基因的表达量先上升后下降,当照射时间为30 min时表达量最大。【结论】烟蚜MpHsp70基因可以响应UV-B的胁迫,它可能在烟蚜适应UV-B胁迫过程中起到重要作用。
【Aim 】 To explore the molecular mechanism of Myzus persicae to adapt to UV-B stress.【Methods】The full-length cDNA of the heat shock protein gene Hsp70 was cloned from M. persicae by using RT-PCR and RACE techniques,and its sequence characteristics were analyzed by bioinformatics methods. The relative expression levels of this gene in M. persicae adults exposed to UV-B stress for different time(0,15,30,60,90 and 120 min) were detected by qPCR. 【Results】The cDNA of a Hsp70 gene was cloned from M. persicae and named as MpHsp70(GenBank accession no. : MF509827).The full length of the gene is 2 221 bp and the open reading frame(ORF) is 1 965 bp,encoding 654 amino acids. The relative molecular weight of its encoded protein is 71. 41 k D,and the isoelectric point(pI) is 5. 34. The highly conserved sequence EEVD shows that this protein belongs to the cytoplasmic heat shock protein(Hsp). The phylogenetic relationship analysis showed that Mp Hsp70 is highly homologous to Hsp70 s of various insects and shows the high conservation characteristic of Hsp70 gene.The qPCR results showed that the relative expression level of MpHsp70 in M. persicae adults increased first,achieved the highest at 30 min post exposure,and then decreased with the prolonged UV-B irradiation time. 【Conclusion】MpHsp70 gene in M. persicae can respond to UV-B stress and may play an important role in the adaptation of M. persicae to UV-B stress.
【Aim 】 To explore the molecular mechanism of Myzus persicae to adapt to UV-B stress.【Methods】The full-length cDNA of the heat shock protein gene Hsp70 was cloned from M. persicae by using RT-PCR and RACE techniques,and its sequence characteristics were analyzed by bioinformatics methods. The relative expression levels of this gene in M. persicae adults exposed to UV-B stress for different time(0,15,30,60,90 and 120 min) were detected by qPCR. 【Results】The cDNA of a Hsp70 gene was cloned from M. persicae and named as MpHsp70(GenBank accession no. : MF509827).The full length of the gene is 2 221 bp and the open reading frame(ORF) is 1 965 bp,encoding 654 amino acids. The relative molecular weight of its encoded protein is 71. 41 k D,and the isoelectric point(pI) is 5. 34. The highly conserved sequence EEVD shows that this protein belongs to the cytoplasmic heat shock protein(Hsp). The phylogenetic relationship analysis showed that Mp Hsp70 is highly homologous to Hsp70 s of various insects and shows the high conservation characteristic of Hsp70 gene.The qPCR results showed that the relative expression level of MpHsp70 in M. persicae adults increased first,achieved the highest at 30 min post exposure,and then decreased with the prolonged UV-B irradiation time. 【Conclusion】MpHsp70 gene in M. persicae can respond to UV-B stress and may play an important role in the adaptation of M. persicae to UV-B stress.
引文
Becker J,Craig EA,1994.Heat-shock proteins as molecular chaperones.Eur.J.Biochem.,219(1-2):11-23.
Chen HZ,Han R,2015.Research progress of plant response to UV-Bradiation.Chin.Bull.Bot.,50(6):790-801.[陈慧泽,韩榕,2015.植物响应UV-B辐射的研究进展.植物学报,50(6):790-801]
Chen MS,Xu C,Song XC,Liu XQ,Xing XM,Zhao JP,2016.Advances on heat-shock protein.J.Econ.Anim.,20(1):44-53.[陈明帅,徐超,宋兴超,刘学庆,邢秀梅,赵家平,2016.热休克蛋白的研究进展.经济动物学报,20(1):44-53]
Cui XH,Xie M,Wan FH,2007.Changes in expression level of heatshock protein 70 gene in Bemisia tabaci B-biotype(Homoptera:Aleyrodidae)under high temperature stress.Acta Entomol.Sin.,50(11):1087-1091.[崔旭红,谢明,万方浩,2007.高温胁迫下B型烟粉虱热激蛋白基因hsp70表达量的变化.昆虫学报,50(11):1087-1091]
Didomenico BJ,Bugaisky GE,Lindquist S,1982.The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels.Cell,31(3):593-603.
Du EX,Zhao HY,Guo JW,2007.UV-induced ecological response and COⅠ-Ⅱmutation of Myzus persicae.J.Northwest A&F Univ.(Nat.Sci.Ed.),35(09):123-126.[都二霞,赵惠燕,郭剑文,2007.紫外线诱导对桃蚜生态学及线粒体基因COⅠ-Ⅱ突变的影响.西北农林科技大学学报(自然科学版),35(9):123-126]
Feder ME,Hofmann GE,1999.Heat-shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annu.Rev.Physiol.,61:243-282.
Franzellitti S,Fabbri E,2005.Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors.Biochem.Biophys.Res.Commun.,336(4):1157-1163.
Guo XR,Luo MH,Dang RS,1995.Effects of Myzus persicae on the physiology,growth and development of tobacco plants.Acta Agric.Boreali-Sin.,10(2):95-99.[郭线茹,罗梅浩,党润生,1995.烟蚜危害对烟草生理及生长发育的影响.华北农学报,10(2):95-99]
Huang LH,Chen B,Kang L,2007.Impact of mild temperature hardening on thermotolerance,fecundity,and Hsp gene expression in Liriomyza huidobrensis.J.Insect Physiol.,53(12):1199-1205.
Jia D,Liu YH,Han Z,Zhao LL,Lv XY,Ma RY,2014.Cloning of heat-shock protein gene hsp70 in Agasicles hygrophila and its expression in relation to high temperatures.Acta Agric.Nucl.Sin.,28(6):970-977.[贾栋,刘艳红,韩政,赵龙龙,吕向阳,马瑞燕,2014.莲草直胸跳甲Hsp70基因克隆及高温表达分析.核农学报,28(6):970-977]
Jiang JJ,Wang FY,Huang LF,Chen HS,Li L,Yang L,2015.Cloning and sequence analysis of heat-shock protein 70 gene(Hsp70)from Bactrocera cucurbitae Coquillett.J.South.Agric.,46(5):800-805.[姜建军,王凤英,黄立飞,陈红松,李磊,杨朗,2015.瓜实蝇热激蛋白基因Hsp70的克隆及序列分析.南方农业学报,46(5):800-805]
Jurkiewicz BA,Buettner GR,1994.Ultraviolet light-induced free radical formation in skin:an electron paramagnetic resonance study.Photochem.Photobiol.,59(1):1-4.
Li J,2005.Responses of Ecology of Aphid under Inducement Condition.MSc Thesis,Northwest A&F University,Yangling,Shaanxi.[李军,2005.诱导条件下蚜虫生态学反应.陕西杨凌:西北农林科技大学硕士学位论文]
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod.Methods,25(4):402-408.
Lopezmartinez G,Elnitsky MA,Benoit JB,Lee REJr,Denlinger DL,2008.High resistance to oxidative damage in the Antarctic midge Belgica antarctica,and developmentally linked expression of genes encoding superoxide dismutase,catalase and heat shock proteins.Insect Biochem.Molec.Biol.,38(8):796-804.
Lowe DG,Fulford WD,Moran LA,1983.Mouse and Drosophila genes encoding the major heat-shock protein(hsp70)are highly conserved.Mol.Cell.Biol.,3(8):1540-1543.
Mc Kay DB,Wibanks SM,Flaherty KM,Ha JH,O'Brien MC,Shirvanee LL,1994.Stress-70 proteins and their interaction with nucleotides.In:Morimoto RI,Tissieres A,Georgopoulos C eds.The Biology of Heat Shock Proteins and Molecular Chaperones.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY.153-177.
Morimoto RI,1993.Cells in stress:transcriptional activation of heatshock genes.Science,259(5100):1409-1410.
Pu LQ,Wang FH,Huo MP,2008.Research progress of heat-shock proteins.J.Yan'an Univ.,27(1):72-75.[蒲力群,王逢会,霍满鹏,2008.热休克蛋白的研究进展.延安大学学报,27(1):72-75]
Schauen M,Hornig-Do HT,Schomberg S,Herrmann G,Wiesner RJ,2007.Mitochondrial electron transport chain activity is not involved in ultraviolet A(UVA)-induced cell death.Free Radical Biol.Med.,42(4):499-509.
Schmid D,Baici A,Gehring H,Christen P,1994.Kinetics of molecular chaperone action.Science,263(5149):971-973.
Vile GF,Tyrrell RM,1995.UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen.Free Radical Biol.Med.,18(4):721-730.
Wang GH,2015.Cloning and Expression of Heat-shock Protein Gene(hsp90,hsp70)in Lithocolletis ringoniella.MSc Thesis,Northwest A&F University,Yangling,Shaanxi.[王冠华,2015.金纹细蛾热激蛋白HSP90和HSP70基因的克隆与表达.陕西杨凌:西北农林科技大学硕士学位论文]
Wang HH,2005.Molecular Cloning and Bacterial Expression of HeatShock Protein and Their Relationship with Stress Tolerance in Bemisia tabaci(B Biotype).PhD Dissertation,Chinese Academy of Agricultural Sciences,Beijing.[王海鸿,2005.B型烟粉虱热休克蛋白基因的克隆和表达及其与胁迫耐受性关系的研究.北京:中国农业科学院博士学位论文]
Wang HH,Lei ZR,2005.Current developments of heat-shock proteins in insect.Sci.Agric.Sin.,38(10):2023-2034.[王海鸿,雷忠仁,2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034]
Wang W,Han LL,Zhao KJ,2009.Research advance of heat-shock protein 70 in entomology.J.Northeast Agric.Univ.,40(11):129-132.[王薇,韩岚岚,赵奎军,2009.昆虫热休克蛋白Hsp70的研究进展.东北农业大学学报,40(11):129-132]
Wu SJ,Liu FH,Hu SM,Wang C,2001.Different combinations of the heat-shock cognate protein 70(hsc70)C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeatcontaining proteins.Biochem.J.,359(2):419-426.
Xiao CX,Du YZ,Qiang CK,2006.Present situation on heat-shock proteins of insects.Guangdong Agric.Sci.,45(5):110-112.[肖春霞,杜予州,强承魁,2006.昆虫热休克蛋白研究概况.广东农业科学,45(5):110-112]
Zhang CY,2010.Study on the Reproductive Compensation of Helicoverpa armigera under UV Stress and Cloning and Cxpression of Hsps Genes.Ph D Dissertation,Huazhong Agricultural University,Wuhan.[张长禹,2010.UV胁迫下棉铃虫生殖补偿研究及Hsps基因的克隆与表达.武汉:华中农业大学博士学位论文]
Zhang Q,Lu MX,Zhu SD,2015.Cloning,sequence analysis and expression profiling of two heat-shock protein 70 genes in small plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae).Jiangsu J.Agric.Sci.,31(6):1257-1264.[张青,陆明星,祝树德,2015.灰飞虱2种热激蛋白基因Hsp70的克隆、分析.江苏农业学报,31(6):1257-1264]
Zhao HY,Du EX,Guo JW,Zhang GS,Wang CP,Hu ZQ,Hu XS,2010.Preliminary study on molecular ecology of gene expression of Myzus persicae under UV-B stress.J.Northwest A&F Univ.(Nat.Sci.Ed.),38(3):132-138.[赵惠燕,都二霞,郭剑文,张改生,王春平,胡祖庆,胡想顺,2010.UV-B辐射胁迫下桃蚜差异基因表达的分子生态初步研究.西北农林科技大学学报(自然科学版),38(3):132-138]
Chen HZ,Han R,2015.Research progress of plant response to UV-Bradiation.Chin.Bull.Bot.,50(6):790-801.[陈慧泽,韩榕,2015.植物响应UV-B辐射的研究进展.植物学报,50(6):790-801]
Chen MS,Xu C,Song XC,Liu XQ,Xing XM,Zhao JP,2016.Advances on heat-shock protein.J.Econ.Anim.,20(1):44-53.[陈明帅,徐超,宋兴超,刘学庆,邢秀梅,赵家平,2016.热休克蛋白的研究进展.经济动物学报,20(1):44-53]
Cui XH,Xie M,Wan FH,2007.Changes in expression level of heatshock protein 70 gene in Bemisia tabaci B-biotype(Homoptera:Aleyrodidae)under high temperature stress.Acta Entomol.Sin.,50(11):1087-1091.[崔旭红,谢明,万方浩,2007.高温胁迫下B型烟粉虱热激蛋白基因hsp70表达量的变化.昆虫学报,50(11):1087-1091]
Didomenico BJ,Bugaisky GE,Lindquist S,1982.The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels.Cell,31(3):593-603.
Du EX,Zhao HY,Guo JW,2007.UV-induced ecological response and COⅠ-Ⅱmutation of Myzus persicae.J.Northwest A&F Univ.(Nat.Sci.Ed.),35(09):123-126.[都二霞,赵惠燕,郭剑文,2007.紫外线诱导对桃蚜生态学及线粒体基因COⅠ-Ⅱ突变的影响.西北农林科技大学学报(自然科学版),35(9):123-126]
Feder ME,Hofmann GE,1999.Heat-shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annu.Rev.Physiol.,61:243-282.
Franzellitti S,Fabbri E,2005.Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors.Biochem.Biophys.Res.Commun.,336(4):1157-1163.
Guo XR,Luo MH,Dang RS,1995.Effects of Myzus persicae on the physiology,growth and development of tobacco plants.Acta Agric.Boreali-Sin.,10(2):95-99.[郭线茹,罗梅浩,党润生,1995.烟蚜危害对烟草生理及生长发育的影响.华北农学报,10(2):95-99]
Huang LH,Chen B,Kang L,2007.Impact of mild temperature hardening on thermotolerance,fecundity,and Hsp gene expression in Liriomyza huidobrensis.J.Insect Physiol.,53(12):1199-1205.
Jia D,Liu YH,Han Z,Zhao LL,Lv XY,Ma RY,2014.Cloning of heat-shock protein gene hsp70 in Agasicles hygrophila and its expression in relation to high temperatures.Acta Agric.Nucl.Sin.,28(6):970-977.[贾栋,刘艳红,韩政,赵龙龙,吕向阳,马瑞燕,2014.莲草直胸跳甲Hsp70基因克隆及高温表达分析.核农学报,28(6):970-977]
Jiang JJ,Wang FY,Huang LF,Chen HS,Li L,Yang L,2015.Cloning and sequence analysis of heat-shock protein 70 gene(Hsp70)from Bactrocera cucurbitae Coquillett.J.South.Agric.,46(5):800-805.[姜建军,王凤英,黄立飞,陈红松,李磊,杨朗,2015.瓜实蝇热激蛋白基因Hsp70的克隆及序列分析.南方农业学报,46(5):800-805]
Jurkiewicz BA,Buettner GR,1994.Ultraviolet light-induced free radical formation in skin:an electron paramagnetic resonance study.Photochem.Photobiol.,59(1):1-4.
Li J,2005.Responses of Ecology of Aphid under Inducement Condition.MSc Thesis,Northwest A&F University,Yangling,Shaanxi.[李军,2005.诱导条件下蚜虫生态学反应.陕西杨凌:西北农林科技大学硕士学位论文]
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod.Methods,25(4):402-408.
Lopezmartinez G,Elnitsky MA,Benoit JB,Lee REJr,Denlinger DL,2008.High resistance to oxidative damage in the Antarctic midge Belgica antarctica,and developmentally linked expression of genes encoding superoxide dismutase,catalase and heat shock proteins.Insect Biochem.Molec.Biol.,38(8):796-804.
Lowe DG,Fulford WD,Moran LA,1983.Mouse and Drosophila genes encoding the major heat-shock protein(hsp70)are highly conserved.Mol.Cell.Biol.,3(8):1540-1543.
Mc Kay DB,Wibanks SM,Flaherty KM,Ha JH,O'Brien MC,Shirvanee LL,1994.Stress-70 proteins and their interaction with nucleotides.In:Morimoto RI,Tissieres A,Georgopoulos C eds.The Biology of Heat Shock Proteins and Molecular Chaperones.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY.153-177.
Morimoto RI,1993.Cells in stress:transcriptional activation of heatshock genes.Science,259(5100):1409-1410.
Pu LQ,Wang FH,Huo MP,2008.Research progress of heat-shock proteins.J.Yan'an Univ.,27(1):72-75.[蒲力群,王逢会,霍满鹏,2008.热休克蛋白的研究进展.延安大学学报,27(1):72-75]
Schauen M,Hornig-Do HT,Schomberg S,Herrmann G,Wiesner RJ,2007.Mitochondrial electron transport chain activity is not involved in ultraviolet A(UVA)-induced cell death.Free Radical Biol.Med.,42(4):499-509.
Schmid D,Baici A,Gehring H,Christen P,1994.Kinetics of molecular chaperone action.Science,263(5149):971-973.
Vile GF,Tyrrell RM,1995.UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen.Free Radical Biol.Med.,18(4):721-730.
Wang GH,2015.Cloning and Expression of Heat-shock Protein Gene(hsp90,hsp70)in Lithocolletis ringoniella.MSc Thesis,Northwest A&F University,Yangling,Shaanxi.[王冠华,2015.金纹细蛾热激蛋白HSP90和HSP70基因的克隆与表达.陕西杨凌:西北农林科技大学硕士学位论文]
Wang HH,2005.Molecular Cloning and Bacterial Expression of HeatShock Protein and Their Relationship with Stress Tolerance in Bemisia tabaci(B Biotype).PhD Dissertation,Chinese Academy of Agricultural Sciences,Beijing.[王海鸿,2005.B型烟粉虱热休克蛋白基因的克隆和表达及其与胁迫耐受性关系的研究.北京:中国农业科学院博士学位论文]
Wang HH,Lei ZR,2005.Current developments of heat-shock proteins in insect.Sci.Agric.Sin.,38(10):2023-2034.[王海鸿,雷忠仁,2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034]
Wang W,Han LL,Zhao KJ,2009.Research advance of heat-shock protein 70 in entomology.J.Northeast Agric.Univ.,40(11):129-132.[王薇,韩岚岚,赵奎军,2009.昆虫热休克蛋白Hsp70的研究进展.东北农业大学学报,40(11):129-132]
Wu SJ,Liu FH,Hu SM,Wang C,2001.Different combinations of the heat-shock cognate protein 70(hsc70)C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeatcontaining proteins.Biochem.J.,359(2):419-426.
Xiao CX,Du YZ,Qiang CK,2006.Present situation on heat-shock proteins of insects.Guangdong Agric.Sci.,45(5):110-112.[肖春霞,杜予州,强承魁,2006.昆虫热休克蛋白研究概况.广东农业科学,45(5):110-112]
Zhang CY,2010.Study on the Reproductive Compensation of Helicoverpa armigera under UV Stress and Cloning and Cxpression of Hsps Genes.Ph D Dissertation,Huazhong Agricultural University,Wuhan.[张长禹,2010.UV胁迫下棉铃虫生殖补偿研究及Hsps基因的克隆与表达.武汉:华中农业大学博士学位论文]
Zhang Q,Lu MX,Zhu SD,2015.Cloning,sequence analysis and expression profiling of two heat-shock protein 70 genes in small plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae).Jiangsu J.Agric.Sci.,31(6):1257-1264.[张青,陆明星,祝树德,2015.灰飞虱2种热激蛋白基因Hsp70的克隆、分析.江苏农业学报,31(6):1257-1264]
Zhao HY,Du EX,Guo JW,Zhang GS,Wang CP,Hu ZQ,Hu XS,2010.Preliminary study on molecular ecology of gene expression of Myzus persicae under UV-B stress.J.Northwest A&F Univ.(Nat.Sci.Ed.),38(3):132-138.[赵惠燕,都二霞,郭剑文,张改生,王春平,胡祖庆,胡想顺,2010.UV-B辐射胁迫下桃蚜差异基因表达的分子生态初步研究.西北农林科技大学学报(自然科学版),38(3):132-138]
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