烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析
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摘要
为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。
To investigate the influences of UV-B stress on the expression of Hsp90 gene from green peach aphid Myzus persicae, the full-length cDNA of Hsp90 gene was amplified by the reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE) methods. The characteristics of Hsp90 gene were analyzed by bioinformatic method, and the expression levels of the M. persicae Hsp90 gene under different UV-B treatments were detected by the real-time quantitative PCR. The results showed that the full-length cDNA of M. persicae Hsp90 gene was 2 670 bp, which encoded 728 amino acids, with a molecular weight of 82.6 kD and a theoretical isoelectric point of 4.95. The deduced amino acid sequence of Hsp90 contained one signature sequence of Hsp90 family and a cytoplasmic character sequence(MEEVD) at C-terminal. Therefore, the Hsp90 protein of M. persicae should belong to cytoplasmic heat shock protein. The phylogenetic tree indicated that the protein sequence shared a high homology with Hsp90 proteins from other insect species. The results of real-time quantitative PCR analyses showed that Hsp90 could be induced by UV-B irradiation. The expression of Hsp90 gene increased firstly and then decreased with increasing treatment time. When the treatment time lasted for15, 30, 60, 90 and 120 min, the expression of Hsp90 gene were significantly higher than that of the control, reaching a peak at 60 min, which was 2.05 times that of the control. The expression level of M. persicae Hsp90 gene varied at different time points of UV-B stress, and it might play an important role in the response of the pests to UV-B stress.
To investigate the influences of UV-B stress on the expression of Hsp90 gene from green peach aphid Myzus persicae, the full-length cDNA of Hsp90 gene was amplified by the reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE) methods. The characteristics of Hsp90 gene were analyzed by bioinformatic method, and the expression levels of the M. persicae Hsp90 gene under different UV-B treatments were detected by the real-time quantitative PCR. The results showed that the full-length cDNA of M. persicae Hsp90 gene was 2 670 bp, which encoded 728 amino acids, with a molecular weight of 82.6 kD and a theoretical isoelectric point of 4.95. The deduced amino acid sequence of Hsp90 contained one signature sequence of Hsp90 family and a cytoplasmic character sequence(MEEVD) at C-terminal. Therefore, the Hsp90 protein of M. persicae should belong to cytoplasmic heat shock protein. The phylogenetic tree indicated that the protein sequence shared a high homology with Hsp90 proteins from other insect species. The results of real-time quantitative PCR analyses showed that Hsp90 could be induced by UV-B irradiation. The expression of Hsp90 gene increased firstly and then decreased with increasing treatment time. When the treatment time lasted for15, 30, 60, 90 and 120 min, the expression of Hsp90 gene were significantly higher than that of the control, reaching a peak at 60 min, which was 2.05 times that of the control. The expression level of M. persicae Hsp90 gene varied at different time points of UV-B stress, and it might play an important role in the response of the pests to UV-B stress.
引文
Bacher S,Luder S.2005.Picky predators and the function of the faecal shield of a cassidine larva.Functional Ecology,19(2):263-272
Chen H,Xu XL,Li YP,Wu JX.2014.Characterization of heat shock protein 90,70 and their transcriptional expression patterns on high temperature in adult of Grapholita molesta(Busck).Insect Science,21(4):439-448
Colinet H,Lee SF,Hoffmann AA.2010.Temporal expression of heat shock genes during cold stress and recovery from chill-coma in adult Drosophila melanogaster.FEBS Journal,277(1):174-185
Guo XR,Luo MH,Dang RS.1995.Effects of green peach aphid on the physiology,growth and development of tobacco plants.Acta Agriculturae Boreali-Sinica,10(2):95-99(in Chinese)[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]
Li J.2005.Responses of ecology of aphid under inducement condition.Master Thesis.Yangling:Northwest A&F University(in Chinese)[李军.2005.诱导条件下蚜虫生态学反应.硕士学位论文.杨凌:西北农林科技大学]
Li YT,Zhao Q,Fang B,Chen MH.2017.Molecular cloning and expression analysis of the Hsp90 gene in the bird cherry-oat aphid,Rhopalosiphum padi.Journal of Plant Protection,44(1):16-23(in Chinese)[李玉婷,赵奇,房冰,陈茂华.2017.谷缢管蚜热激蛋白Hsp90基因的克隆和表达分析.植物保护学报,44(1):16-23]
Livak KJ,Schmittgen TD.2001.Analysis or relative gene expression data using real-time quantitative PCR and the 2-△△Ct method.Methods,25:402-408
McKenzie RL,Bj?rn LO,Bais A,Ilyasd M.2007.Changes in biologically-active ultraviolet radiation reaching the earth’s surface.Photochemical&Photobiological Sciences,2:5-15
Picard D.2002.Heat-shock protein 90,a chaperone for folding and regulation.Cellular and Molecular Life Sciences,59(10):1640-1648
Rassow J,Voos W,P fanner N.1995.Partner proteins determine multiple functions of HSP70.Trends in Cell Biology,5:207-212
Ren GW,Zhang LT.2002.Occurrence and control of Myzus Persicae(Sulzer)and Heliothis assulta Guenée.Tobacco Science&Technology,46(5):43-45(in Chinese)[任广伟,张连涛.2002.烟蚜和烟青虫的发生与防治.烟草科技,46(5):43-45]
Rinehart JP,Hayward SAL,Elnitsky MA,Sandro LH,Lee RE Jr.,Denlinger DL.2006.Continuous up-regulation of heat shock proteins in larvae,but not adults,in a polar insect.Proceedings of the National Academy of Sciences of the United States of America,103(38):14223-14227
Sang W,Ma WH,Qiu L,Lei CL.2012.The involvement of heat shock protein and cytochrome P450 genes in response to UV-A exposure in the beetle Tribolium castaneum.Journal of Insect Physiology,58(6):830-836
Shen JR,Li MF,Chen NZ,Wang JJ,Wan FH.2011.Cloning and analysis of the expression characteristics under heat stress of Hsp90gene in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae).Acta Entomologica Sinca,54(11):1236-1248(in Chinese)[申建茹,李明福,陈乃中,王进军,万方浩.2011.苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析.昆虫学报,54(11):1236-1248]
S?rensen JG,Kristensen TN,Loeschcke V.2003.The evolutionary and ecological role of heat shock proteins.Ecology Letters,6(11):1025-1037
Theodoraki MA,Mintzas AC.2006.cDNA cloning,heat shock regulation and developmental expression of the hsp83 gene in the Mediterranean fruit fly Ceratitis capitata.Insect Molecular Biology,15(6):839-852
Verne S,Jaquish B,White R,Ritland C,Ritland K.2011.Global transcriptome analysis of constitutive resistance to the white pine weevil in spruce.Genome Biology and Evolution,3(1):851-867
Wang JY,Ci ZL.2008.Advances in study of heat shock protein.Shanxi Forestry Science and Technology,17(1):27-32(in Chinese)[王建义,慈忠玲.2008.热激蛋白的研究进展.山西林业科技,17(1):27-32]
Wang LJ,Zhou LJ,Zhu ZH,Ma WH,Lei CL.2014.Differential temporal expression profiles of heat shock protein genes in Drosophila melanogaster(Diptera:Drosophilidae)under ultraviolet a radiation stress.Environmental Entomology,43(5):1427-1434
Wang YF.2014.The influence of infection with TMV and CMV in tobacco on the population development,food finding of Myzus persicae and CMV transmission efficiency by aphid.Master Thesis.Chongqing:Southwest University(in Chinese)[王亚锋.2014.TMV与CMV复合侵染烟草对烟蚜的种群增长、取食选择和CMV蚜传效率的影响.硕士学位论文.重庆:西南大学]
Xu CP,Sullivan JH.2010.Reviewing the technical designs for experiments with ultraviolet-B radiation and impact on photosynthesis,DNA and secondary metabolism.Journal of Integrative Plant Biology,52(4):377-387
Yang LH,Dou W,Jiang HB,Niu JZ,Ding TB,Wang JJ.2016.Cloning and expression profiling of heat shock protein gene PcHsp90 in the citrus red mite,Panonychus citri(Acari:Tetranychidae).Acta Entomologica Sinica,59(4):411-420(in Chinese)[杨丽红,豆威,蒋红波,牛金志,丁天波,王进军.2016.柑橘全爪螨热激蛋白基因PcHsp90的克隆及表达模式分析.昆虫学报,59(4):411-420]
Yuan WN,LüN,Sun XL,Liu CZ.2015.Effects of continuous UV-Bstress on biological characteristics of green pea aphid.Chinese Journal of Eco-Agriculture,23(8):1020-1025(in Chinese)[袁伟宁,吕宁,孙小玲,刘长仲.2015.连续UV-B胁迫对绿色型豌豆蚜生物学特性的影响.中国生态农业学报,23(8):1020-1025]
Zhai HF,Jiang XF,Luo LZ.2010.Cloning and expression of HSP90cDNA in larvae of the beet armyworm,Spodoptera exigua(Lepidoptera:Noctuidae)under high temperatures tress.Acta Entomologica Sinca,53(1):20-28(in Chinese)[翟会芳,江幸福,罗礼智.2010.甜菜夜蛾HSP90基因克隆及高温胁迫下其表达量的变化.昆虫学报,53(1):20-28]
Zhang CY.2010.Study on the reproductive compensation of Helicoverpa armigera under UV stress and cloning and expression of Hsps genes.Ph.D Thesis.Wuhan:Huazhong Agricultural University(in Chinese)[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.博士学位论文.武汉:华中农业大学]
Zhang K,Weng QF,Fu HH.2014.Research progress on heat shock protein 90 of insects.Biotechnology Bulletin,30(2):15-23(in Chinese)[张珂,翁群芳,付昊昊.2014.昆虫热激蛋白90的研究进展.生物技术通报,30(2):15-23]
Zhang L.2013.The effects of long-term different intensity of UV-B radiation on the ecological parameters and feeding behavior of Sitobion avenae(Fabricius)(Hemiptera:Aphididae).Master Thesis.Yangling:Northwest A&F University(in Chinese)[张丽.2013.不同强度UV-B长期胁迫对麦长管蚜种群生态学参数和取食行为的影响.硕士学位论文.杨凌:西北农林科技大学]
Zhang Q,Lu MX,Zhu SD.2014.Cloning,sequence analysis and expression profiling of heat shock protein 90 gene in the small brown plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae).Acta Entomologica Sinica,57(7):777-786(in Chinese)[张青,陆明星,祝树德.2014.灰飞虱热激蛋白Hsp90基因的克隆、序列分析与表达模式.昆虫学报,57(7):777-786]
Zhang RG,Zheng YF,He YH,Xiao W.2003.Effects of enhanced UV-Bradiation upon wheat aphids.Journal of Nanjing Institute of Meteorology,26(2):258-262(in Chinese)[张荣刚,郑有飞,何雨红,肖微.2003.紫外UV-B辐射增强对麦田蚜虫的影响.南京气象学院学报,26(2):258-262]
Zhao L,Jones WA.2012.Expression of heat shock protein genes in insect stress responses.Invertebrate Survival Journal,9(1):93-101
Zhou D,Du YM,Yang J,Zhang L,Zhao HY,Hu ZQ,Hu XS.2014.Effects of UV-B radiation generations on the activities of protective enzymes in the grain aphid,Sitobion avenae(Hemiptera:Aphididae).Acta Entomologica Sinica,57(7):762-768(in Chinese)[周冬,杜一民,杨杰,张丽,赵惠燕,胡祖庆,胡想顺.2014.连续多代UV-B照射对麦长管蚜保护酶活性的影响.昆虫学报,57(7):762-768]
Chen H,Xu XL,Li YP,Wu JX.2014.Characterization of heat shock protein 90,70 and their transcriptional expression patterns on high temperature in adult of Grapholita molesta(Busck).Insect Science,21(4):439-448
Colinet H,Lee SF,Hoffmann AA.2010.Temporal expression of heat shock genes during cold stress and recovery from chill-coma in adult Drosophila melanogaster.FEBS Journal,277(1):174-185
Guo XR,Luo MH,Dang RS.1995.Effects of green peach aphid on the physiology,growth and development of tobacco plants.Acta Agriculturae Boreali-Sinica,10(2):95-99(in Chinese)[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]
Li J.2005.Responses of ecology of aphid under inducement condition.Master Thesis.Yangling:Northwest A&F University(in Chinese)[李军.2005.诱导条件下蚜虫生态学反应.硕士学位论文.杨凌:西北农林科技大学]
Li YT,Zhao Q,Fang B,Chen MH.2017.Molecular cloning and expression analysis of the Hsp90 gene in the bird cherry-oat aphid,Rhopalosiphum padi.Journal of Plant Protection,44(1):16-23(in Chinese)[李玉婷,赵奇,房冰,陈茂华.2017.谷缢管蚜热激蛋白Hsp90基因的克隆和表达分析.植物保护学报,44(1):16-23]
Livak KJ,Schmittgen TD.2001.Analysis or relative gene expression data using real-time quantitative PCR and the 2-△△Ct method.Methods,25:402-408
McKenzie RL,Bj?rn LO,Bais A,Ilyasd M.2007.Changes in biologically-active ultraviolet radiation reaching the earth’s surface.Photochemical&Photobiological Sciences,2:5-15
Picard D.2002.Heat-shock protein 90,a chaperone for folding and regulation.Cellular and Molecular Life Sciences,59(10):1640-1648
Rassow J,Voos W,P fanner N.1995.Partner proteins determine multiple functions of HSP70.Trends in Cell Biology,5:207-212
Ren GW,Zhang LT.2002.Occurrence and control of Myzus Persicae(Sulzer)and Heliothis assulta Guenée.Tobacco Science&Technology,46(5):43-45(in Chinese)[任广伟,张连涛.2002.烟蚜和烟青虫的发生与防治.烟草科技,46(5):43-45]
Rinehart JP,Hayward SAL,Elnitsky MA,Sandro LH,Lee RE Jr.,Denlinger DL.2006.Continuous up-regulation of heat shock proteins in larvae,but not adults,in a polar insect.Proceedings of the National Academy of Sciences of the United States of America,103(38):14223-14227
Sang W,Ma WH,Qiu L,Lei CL.2012.The involvement of heat shock protein and cytochrome P450 genes in response to UV-A exposure in the beetle Tribolium castaneum.Journal of Insect Physiology,58(6):830-836
Shen JR,Li MF,Chen NZ,Wang JJ,Wan FH.2011.Cloning and analysis of the expression characteristics under heat stress of Hsp90gene in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae).Acta Entomologica Sinca,54(11):1236-1248(in Chinese)[申建茹,李明福,陈乃中,王进军,万方浩.2011.苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析.昆虫学报,54(11):1236-1248]
S?rensen JG,Kristensen TN,Loeschcke V.2003.The evolutionary and ecological role of heat shock proteins.Ecology Letters,6(11):1025-1037
Theodoraki MA,Mintzas AC.2006.cDNA cloning,heat shock regulation and developmental expression of the hsp83 gene in the Mediterranean fruit fly Ceratitis capitata.Insect Molecular Biology,15(6):839-852
Verne S,Jaquish B,White R,Ritland C,Ritland K.2011.Global transcriptome analysis of constitutive resistance to the white pine weevil in spruce.Genome Biology and Evolution,3(1):851-867
Wang JY,Ci ZL.2008.Advances in study of heat shock protein.Shanxi Forestry Science and Technology,17(1):27-32(in Chinese)[王建义,慈忠玲.2008.热激蛋白的研究进展.山西林业科技,17(1):27-32]
Wang LJ,Zhou LJ,Zhu ZH,Ma WH,Lei CL.2014.Differential temporal expression profiles of heat shock protein genes in Drosophila melanogaster(Diptera:Drosophilidae)under ultraviolet a radiation stress.Environmental Entomology,43(5):1427-1434
Wang YF.2014.The influence of infection with TMV and CMV in tobacco on the population development,food finding of Myzus persicae and CMV transmission efficiency by aphid.Master Thesis.Chongqing:Southwest University(in Chinese)[王亚锋.2014.TMV与CMV复合侵染烟草对烟蚜的种群增长、取食选择和CMV蚜传效率的影响.硕士学位论文.重庆:西南大学]
Xu CP,Sullivan JH.2010.Reviewing the technical designs for experiments with ultraviolet-B radiation and impact on photosynthesis,DNA and secondary metabolism.Journal of Integrative Plant Biology,52(4):377-387
Yang LH,Dou W,Jiang HB,Niu JZ,Ding TB,Wang JJ.2016.Cloning and expression profiling of heat shock protein gene PcHsp90 in the citrus red mite,Panonychus citri(Acari:Tetranychidae).Acta Entomologica Sinica,59(4):411-420(in Chinese)[杨丽红,豆威,蒋红波,牛金志,丁天波,王进军.2016.柑橘全爪螨热激蛋白基因PcHsp90的克隆及表达模式分析.昆虫学报,59(4):411-420]
Yuan WN,LüN,Sun XL,Liu CZ.2015.Effects of continuous UV-Bstress on biological characteristics of green pea aphid.Chinese Journal of Eco-Agriculture,23(8):1020-1025(in Chinese)[袁伟宁,吕宁,孙小玲,刘长仲.2015.连续UV-B胁迫对绿色型豌豆蚜生物学特性的影响.中国生态农业学报,23(8):1020-1025]
Zhai HF,Jiang XF,Luo LZ.2010.Cloning and expression of HSP90cDNA in larvae of the beet armyworm,Spodoptera exigua(Lepidoptera:Noctuidae)under high temperatures tress.Acta Entomologica Sinca,53(1):20-28(in Chinese)[翟会芳,江幸福,罗礼智.2010.甜菜夜蛾HSP90基因克隆及高温胁迫下其表达量的变化.昆虫学报,53(1):20-28]
Zhang CY.2010.Study on the reproductive compensation of Helicoverpa armigera under UV stress and cloning and expression of Hsps genes.Ph.D Thesis.Wuhan:Huazhong Agricultural University(in Chinese)[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.博士学位论文.武汉:华中农业大学]
Zhang K,Weng QF,Fu HH.2014.Research progress on heat shock protein 90 of insects.Biotechnology Bulletin,30(2):15-23(in Chinese)[张珂,翁群芳,付昊昊.2014.昆虫热激蛋白90的研究进展.生物技术通报,30(2):15-23]
Zhang L.2013.The effects of long-term different intensity of UV-B radiation on the ecological parameters and feeding behavior of Sitobion avenae(Fabricius)(Hemiptera:Aphididae).Master Thesis.Yangling:Northwest A&F University(in Chinese)[张丽.2013.不同强度UV-B长期胁迫对麦长管蚜种群生态学参数和取食行为的影响.硕士学位论文.杨凌:西北农林科技大学]
Zhang Q,Lu MX,Zhu SD.2014.Cloning,sequence analysis and expression profiling of heat shock protein 90 gene in the small brown plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae).Acta Entomologica Sinica,57(7):777-786(in Chinese)[张青,陆明星,祝树德.2014.灰飞虱热激蛋白Hsp90基因的克隆、序列分析与表达模式.昆虫学报,57(7):777-786]
Zhang RG,Zheng YF,He YH,Xiao W.2003.Effects of enhanced UV-Bradiation upon wheat aphids.Journal of Nanjing Institute of Meteorology,26(2):258-262(in Chinese)[张荣刚,郑有飞,何雨红,肖微.2003.紫外UV-B辐射增强对麦田蚜虫的影响.南京气象学院学报,26(2):258-262]
Zhao L,Jones WA.2012.Expression of heat shock protein genes in insect stress responses.Invertebrate Survival Journal,9(1):93-101
Zhou D,Du YM,Yang J,Zhang L,Zhao HY,Hu ZQ,Hu XS.2014.Effects of UV-B radiation generations on the activities of protective enzymes in the grain aphid,Sitobion avenae(Hemiptera:Aphididae).Acta Entomologica Sinica,57(7):762-768(in Chinese)[周冬,杜一民,杨杰,张丽,赵惠燕,胡祖庆,胡想顺.2014.连续多代UV-B照射对麦长管蚜保护酶活性的影响.昆虫学报,57(7):762-768]