粘虫hsc70克隆及其密度胁迫下的表达模式
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摘要
【目的】昆虫种群密度的升高往往成为一种胁迫因子。粘虫在低密度下形成散居型,而在高密度下则形成群居型。本研通过比较hsc70在两型粘虫中的表达模式,究旨在探索hsc70在粘虫Mythimna separata响应密度胁迫中的作用。【方法】采用RT-PCR和RACE技术克隆粘虫hsc70cDNA全长序列,利用生物信息学软件分析该基因及其编码蛋白质的序列特性;运用实时荧光定量PCR技术(qRT-PCR)分析该基因在两型粘虫不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、表皮、前肠、中肠、后肠和马氏管)以及群居和分离处理后mRNA表达量的变化。【结果】克隆得到的HSC70基因命名为Mshsc70(GenBank登录号:MH669276),全长cDNA长2 184 bp,开放阅读框为1 965 bp,编码653个氨基酸。序列分析发现MsHSC70具有HSP70家族典型的结构域,即IDLGTTYS(11-18aa),IFDLGGGTFDVSLL (198-211 aa)和IVLVGGSTRIPKVQQ (341-355 aa)。序列比对结果显示,MsHSC70与斜纹夜蛾Spodopteralitura,棉铃虫Helicoverpaarmigera,大螟Sesamiainferens、甘蓝夜蛾Mamestra brassicae HSC70的相似性高达99%;基于HSC70的系统发育分析显示,粘虫与夜蛾科(Noctuidae)昆虫的亲缘关系最近。Mshsc70在两型粘虫的各个发育阶段均表达,hsc70的表达量在1龄和5龄中的表达量较散居型显著上调,而在2-3龄中的表达量显著下调。除表皮和前肠外,群居型粘虫的其他组织中hsc70的表达量显著高于散居型。散居型粘虫经群聚饲养36h后,hsc70显著上调表达,表达量是对照的3.17倍,而群居型粘虫经分离饲养36 h后,hsc70的表达量与对照无显著差异。【结论】粘虫的种群密度是决定粘虫hsc70表达的一个重要因子。
[Objectives] High population density causes stress to insect populations. Mythimna separata has solitary and gregarious phases that occur at low and high population densities, respectively. This study aims to explore the role of hsc70 under crowding stress by comparing the expression profiles of hsc70 in the two phases of the M. separata. [Methods] The complete cDNA sequence of hsc70 was cloned by RT-PCR and RACE. Bioinformatics programs were used to analyze the sequence characteristics of the gene and its amino acid sequences. The expression levels of hsc70 in different developmental stages(egg, 1 st-6 th instar larva, pupa and adult) and tissues of 5 th instar larvae(head, epidermis, fore-gut, mid-gut, hindgut and Malpighian tubules) from different phases(solitary and gregarious) were detected by Quantitative Real-time PCR(qRT-PCR). [Results] The complete cDNA of hsc70 obtained from M. separata(named as Mshsc70) was 2 184 bp in length(GenBank accession no.: MH669276), including a 1 965 bp opening reading frame(ORF) encoding 653 amino acids. Sequence analysis indicates that MsHSC70 has typical motifs of the HSP70 family, including IDLGTTYS(11-18 aa), IFDLGGGTFDVSLL(198-211 aa) and IVLVGGSTRIPKVQQ(341-355 aa). Phylogenetic analysis indicates that HSC70 s from Noctuid moths could be assigned to one well-supported cluster. Mshsc70 was expressed in all developmental stages of both phases, its expression in 1 st and 5 th instar larvae of gregarious individuals was significantly up-regulated compared to solitary individuals, whereas its expression in 2 nd and 3 rd instar larvae was obviously down-regulated. The differential expression of hsc70 in different tissues was higher in gregarious than in solitary individuals, except in the epidermis and foregut. hsc70 transcripts were significantly up-regulated(3.17 times of that the control) in solitary M. separata larvae after these were kept in crowded conditions for 36 h, but transcript abundance did not change in gregarious larvae that were kept isolated for 36 h. [Conclusion] Population density may be an important factor determining hsc70 expression in M. separata
[Objectives] High population density causes stress to insect populations. Mythimna separata has solitary and gregarious phases that occur at low and high population densities, respectively. This study aims to explore the role of hsc70 under crowding stress by comparing the expression profiles of hsc70 in the two phases of the M. separata. [Methods] The complete cDNA sequence of hsc70 was cloned by RT-PCR and RACE. Bioinformatics programs were used to analyze the sequence characteristics of the gene and its amino acid sequences. The expression levels of hsc70 in different developmental stages(egg, 1 st-6 th instar larva, pupa and adult) and tissues of 5 th instar larvae(head, epidermis, fore-gut, mid-gut, hindgut and Malpighian tubules) from different phases(solitary and gregarious) were detected by Quantitative Real-time PCR(qRT-PCR). [Results] The complete cDNA of hsc70 obtained from M. separata(named as Mshsc70) was 2 184 bp in length(GenBank accession no.: MH669276), including a 1 965 bp opening reading frame(ORF) encoding 653 amino acids. Sequence analysis indicates that MsHSC70 has typical motifs of the HSP70 family, including IDLGTTYS(11-18 aa), IFDLGGGTFDVSLL(198-211 aa) and IVLVGGSTRIPKVQQ(341-355 aa). Phylogenetic analysis indicates that HSC70 s from Noctuid moths could be assigned to one well-supported cluster. Mshsc70 was expressed in all developmental stages of both phases, its expression in 1 st and 5 th instar larvae of gregarious individuals was significantly up-regulated compared to solitary individuals, whereas its expression in 2 nd and 3 rd instar larvae was obviously down-regulated. The differential expression of hsc70 in different tissues was higher in gregarious than in solitary individuals, except in the epidermis and foregut. hsc70 transcripts were significantly up-regulated(3.17 times of that the control) in solitary M. separata larvae after these were kept in crowded conditions for 36 h, but transcript abundance did not change in gregarious larvae that were kept isolated for 36 h. [Conclusion] Population density may be an important factor determining hsc70 expression in M. separata
引文
Bai YY,2010.Effects of larval density on growth,development and defense capabilitiesof oriental armyworm,Mythimna separata.Master's dissertation.Wuhan:Huazhong Agricultural University.[白毓昕,2010.幼虫密度对粘虫生长发育和防御能力的影响.硕士学位论文.武汉:华中农业大学.]
Castillo-Davis CI,Mekhedov SL,Hartl DL,Koonin EV,Kondrashov FA,2002.Selection for short introns in highly expressed genes.Nature Genetics,31(4):415-418.
Chen B,Li S,Ren Q,Tong X,Zhang X,Kang L,2015.Paternal epigenetic effects of population density on locust phase-related characteristics associated with heat-shock protein expression.Molecular Ecology,24(4):851-862.
Clegg NJ,Honda BM,Whitehead IP,Grigliatti TA,Wakimoto B,Brock HW,Lloyd VK,Sinclair DA,1998.Suppressors of position-effect variegation in Drosophila melanogaster affect expression of the heterochromatic gene light in the absence of a chromosome rearrangement.Genome,41(4):495-503.
Collett M,Despland E,Simpson SJ,Krakauer D,1998.Spatial scales of desert locust gregarization.Proceedings of the National Academy of Sciences of the United States of America,95(22):13052-13055.
Denlinger DL,Rinehart JP,Yocumb GD,2001.Stress Proteins:ARole in Insect Diapause?Insect Timing:Circadian Rhythmicity to Seasonality.Amsteridam:Elsevier.155-171.
Feder ME,Hofmann GE,1999.Heat shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annual Review of Physiology,61:243-282.
Gething MJ,Sambrook J,1992.Protein folding in the cell.Nature,355(6355):33-45.
Han LL,Zhu MH,Dong TY,Zhao KJ,Qu ZC,Lv Y,Han XX,2014.Effects of heat shock and imidacloprid on the expressions of hsp70 and hsc70 m RNA in the Aphis glycines(Hemiptera.Aphididae).Acta Entomologica Sinica,57(4):387-394.[韩岚岚,朱明贺,董天宇,赵奎军,曲忠诚,吕洋,韩晓旭,2014.热应激和吡虫啉对大豆蚜hsp70和hsc70基因m RNA表达的影响.昆虫学报,57(4):387-394.]
Henry Y,Renault D,Colinet H,2018.Hormesis-like effect of mild larval crowding on thermotolerance in Drosophila flies.Journal of Experimental Biology,221(Pt 2):jeb.169342.
Jiang XF,Zhang L,Chen YX,Luo LZ,2014.Novel features,occurrence trends and economic impact of the oriental armyworm,Mythimna separata(Walker)in China.Chinese Journal of Applied Entomology,51(6):1444-1449.[江幸福,张蕾,程云霞,罗礼智,2014.我国粘虫发生危害新特点及趋势分析.应用昆虫学报,51(6):1444-1449.]
Jiang YY,Li CG,Zeng J,Liu J,2014.Population dynamics of the armyworm in China:A review of the past 60 years’research.Chinese Journal of Applied Entomology,51(4):890-898.[姜玉英,李春广,曾娟,刘杰,2014.我国粘虫发生概况:60年回顾.应用昆虫学报,51(4):890-898.]
Kregel KC,2002.Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance.Journal of Applied Physiology,192(5):2177-2186.
Li HB,Dai CG,Zhang CR,He YF,Ran HY,Chen SH,2018.Screening potential reference genes for quantitative real-time PCR analysis in the oriental armyworm,Mythimna separata.PLoS ONE,13(4):e0195096.
Luo LZ,Li GB,Hu Y,1993.Effects of larval density on development and phase change of the oriental armyworm,Mythimna separata.Agricultural Science,1:239-244[罗礼智,李光博,胡毅,1995.粘虫幼虫密度对其生长发育及变型的影响.农业科学集刊,1:239-244.]
Pener M,1991.Locust phase polymorphism and its endocrine relations.Advance of Insect Physiology,23:1-79.
Qin W,Tyshenko MG,Wu BS,Walker VK,Robertson RM,2003.Cloning and characterization of a member of the Hsp70 gen family from Locusta migratoria,a highly thermotolerant insect.Cell Stress Chaperones,8(2):144-152.
S?ensen JG,2010.Application of heat shock protein expression for detecting natural adaptation and exposure to stress in natural populations.Current Zoology,56(6):703-713.
S?rensen JG,Loeschcke V,2001.Larval crowding in Drosophila melanogaster induces Hsp70 expression,and leads to increased adult longevity and adult thermal stress resistance.Journal of Insect Physiology,47(11):1301-1307.
Tang QY,Zhang CX,2013.Data Processing System(DPS)software with experimental design,statistical analysis and data mining developed for use in entomological research.Insect Science,20(2):254-260.
Wang HH,Lei ZR,2005.Current developments of heat shock proteins in insect.Scientia Agricultura Sinica,38(10):2023-2034.[王海鸿,雷仲仁,2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034.]
Wang HS,Wang XH,Zhou CS,Huang LH,Zhang SF,Guo W,Kang L,2007.cDNA cloning of heat shock proteins and their expression in the two phases of the migratory locust.Insect Molecular Biology,16(2):207-219.
Wang Y,Yang P,Cui F,Kang L,2013.Altered immunity in crowded locust reduced fungal(Metarhizium anisopliae)pathogenesis.PLoS Pathogens,9(1):e1003102.
Wilson K,Reeson AF,1998.Density-dependent prophylaxis:evidence from Lepidoptera-baculovirus interactions?Ecological Entomology,23(1):100-101.
Wilson K,Thomas MB,Blanford S,Doggett M,Simpson SJ,Moore SL,2002.Coping with crowds:density-dependent disease resistance in desert locusts.Proceedings of the National Academy of Sciences of the United States of America,99(8):5471-5475.
Wilson K,Cotter SC,Reeson AF,Pell JK,2001.Melanism and disease resistance in insects.Ecology Letters,4(6):637-649.
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 repeat containing proteins.Biochemical Journal,359(Pt2):419-426.
Xu K,Niu QS,Liu YL,Chen DH,Yang S,Zhao HT,Jiang YS,2017.Expression characteristcs of HSC70-4 gene in Apis cerana(Hymenoptera:Apidae).Journal of Environmental Entomology,39(1):55-61.[徐凯,牛庆生,刘玉玲,陈东海,杨爽,赵慧婷,姜玉锁,2017.中华蜜蜂HSC70-4基因表达特性的研究.环境昆虫学报,39(1):55-61.]
Zhang YH,Zhang Z,Jiang YY,Zeng J,Gao YB,Cheng DF,2012.Preliminary analysis of the outbreak of the third-generation army worm Mythimna separata in China in 2012.Plant Protection,38(5):1-8.[张云慧,张智,姜玉英,曾娟,高月波,程登发,2012.2012年三代黏虫大发生原因初步分析.植物保护,38(5):1-8.]
Castillo-Davis CI,Mekhedov SL,Hartl DL,Koonin EV,Kondrashov FA,2002.Selection for short introns in highly expressed genes.Nature Genetics,31(4):415-418.
Chen B,Li S,Ren Q,Tong X,Zhang X,Kang L,2015.Paternal epigenetic effects of population density on locust phase-related characteristics associated with heat-shock protein expression.Molecular Ecology,24(4):851-862.
Clegg NJ,Honda BM,Whitehead IP,Grigliatti TA,Wakimoto B,Brock HW,Lloyd VK,Sinclair DA,1998.Suppressors of position-effect variegation in Drosophila melanogaster affect expression of the heterochromatic gene light in the absence of a chromosome rearrangement.Genome,41(4):495-503.
Collett M,Despland E,Simpson SJ,Krakauer D,1998.Spatial scales of desert locust gregarization.Proceedings of the National Academy of Sciences of the United States of America,95(22):13052-13055.
Denlinger DL,Rinehart JP,Yocumb GD,2001.Stress Proteins:ARole in Insect Diapause?Insect Timing:Circadian Rhythmicity to Seasonality.Amsteridam:Elsevier.155-171.
Feder ME,Hofmann GE,1999.Heat shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annual Review of Physiology,61:243-282.
Gething MJ,Sambrook J,1992.Protein folding in the cell.Nature,355(6355):33-45.
Han LL,Zhu MH,Dong TY,Zhao KJ,Qu ZC,Lv Y,Han XX,2014.Effects of heat shock and imidacloprid on the expressions of hsp70 and hsc70 m RNA in the Aphis glycines(Hemiptera.Aphididae).Acta Entomologica Sinica,57(4):387-394.[韩岚岚,朱明贺,董天宇,赵奎军,曲忠诚,吕洋,韩晓旭,2014.热应激和吡虫啉对大豆蚜hsp70和hsc70基因m RNA表达的影响.昆虫学报,57(4):387-394.]
Henry Y,Renault D,Colinet H,2018.Hormesis-like effect of mild larval crowding on thermotolerance in Drosophila flies.Journal of Experimental Biology,221(Pt 2):jeb.169342.
Jiang XF,Zhang L,Chen YX,Luo LZ,2014.Novel features,occurrence trends and economic impact of the oriental armyworm,Mythimna separata(Walker)in China.Chinese Journal of Applied Entomology,51(6):1444-1449.[江幸福,张蕾,程云霞,罗礼智,2014.我国粘虫发生危害新特点及趋势分析.应用昆虫学报,51(6):1444-1449.]
Jiang YY,Li CG,Zeng J,Liu J,2014.Population dynamics of the armyworm in China:A review of the past 60 years’research.Chinese Journal of Applied Entomology,51(4):890-898.[姜玉英,李春广,曾娟,刘杰,2014.我国粘虫发生概况:60年回顾.应用昆虫学报,51(4):890-898.]
Kregel KC,2002.Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance.Journal of Applied Physiology,192(5):2177-2186.
Li HB,Dai CG,Zhang CR,He YF,Ran HY,Chen SH,2018.Screening potential reference genes for quantitative real-time PCR analysis in the oriental armyworm,Mythimna separata.PLoS ONE,13(4):e0195096.
Luo LZ,Li GB,Hu Y,1993.Effects of larval density on development and phase change of the oriental armyworm,Mythimna separata.Agricultural Science,1:239-244[罗礼智,李光博,胡毅,1995.粘虫幼虫密度对其生长发育及变型的影响.农业科学集刊,1:239-244.]
Pener M,1991.Locust phase polymorphism and its endocrine relations.Advance of Insect Physiology,23:1-79.
Qin W,Tyshenko MG,Wu BS,Walker VK,Robertson RM,2003.Cloning and characterization of a member of the Hsp70 gen family from Locusta migratoria,a highly thermotolerant insect.Cell Stress Chaperones,8(2):144-152.
S?ensen JG,2010.Application of heat shock protein expression for detecting natural adaptation and exposure to stress in natural populations.Current Zoology,56(6):703-713.
S?rensen JG,Loeschcke V,2001.Larval crowding in Drosophila melanogaster induces Hsp70 expression,and leads to increased adult longevity and adult thermal stress resistance.Journal of Insect Physiology,47(11):1301-1307.
Tang QY,Zhang CX,2013.Data Processing System(DPS)software with experimental design,statistical analysis and data mining developed for use in entomological research.Insect Science,20(2):254-260.
Wang HH,Lei ZR,2005.Current developments of heat shock proteins in insect.Scientia Agricultura Sinica,38(10):2023-2034.[王海鸿,雷仲仁,2005.昆虫热休克蛋白的研究进展.中国农业科学,38(10):2023-2034.]
Wang HS,Wang XH,Zhou CS,Huang LH,Zhang SF,Guo W,Kang L,2007.cDNA cloning of heat shock proteins and their expression in the two phases of the migratory locust.Insect Molecular Biology,16(2):207-219.
Wang Y,Yang P,Cui F,Kang L,2013.Altered immunity in crowded locust reduced fungal(Metarhizium anisopliae)pathogenesis.PLoS Pathogens,9(1):e1003102.
Wilson K,Reeson AF,1998.Density-dependent prophylaxis:evidence from Lepidoptera-baculovirus interactions?Ecological Entomology,23(1):100-101.
Wilson K,Thomas MB,Blanford S,Doggett M,Simpson SJ,Moore SL,2002.Coping with crowds:density-dependent disease resistance in desert locusts.Proceedings of the National Academy of Sciences of the United States of America,99(8):5471-5475.
Wilson K,Cotter SC,Reeson AF,Pell JK,2001.Melanism and disease resistance in insects.Ecology Letters,4(6):637-649.
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 repeat containing proteins.Biochemical Journal,359(Pt2):419-426.
Xu K,Niu QS,Liu YL,Chen DH,Yang S,Zhao HT,Jiang YS,2017.Expression characteristcs of HSC70-4 gene in Apis cerana(Hymenoptera:Apidae).Journal of Environmental Entomology,39(1):55-61.[徐凯,牛庆生,刘玉玲,陈东海,杨爽,赵慧婷,姜玉锁,2017.中华蜜蜂HSC70-4基因表达特性的研究.环境昆虫学报,39(1):55-61.]
Zhang YH,Zhang Z,Jiang YY,Zeng J,Gao YB,Cheng DF,2012.Preliminary analysis of the outbreak of the third-generation army worm Mythimna separata in China in 2012.Plant Protection,38(5):1-8.[张云慧,张智,姜玉英,曾娟,高月波,程登发,2012.2012年三代黏虫大发生原因初步分析.植物保护,38(5):1-8.]