松墨天牛小热激蛋白基因的克隆、表达谱及对温度胁迫的响应
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摘要
【目的】松墨天牛Monochamus alternatus是我国南方松林中的重要蛀干害虫,也是林业检疫性病害——松材线虫病的主要媒介昆虫,其分布范围较广,对温度的适应性强,本研究旨在初步探讨松墨天牛对温度胁迫适应性的分子机制。【方法】采用RT-PCR与RACE技术克隆松墨天牛小热激蛋白(small heat shock protein,sHSP)基因的全长cDNA,结合生物信息学方法分析小热激蛋白的结构特征;利用qPCR技术测定该基因在松墨天牛不同发育阶段、4龄幼虫不同组织及不同低温和高温胁迫下4龄幼虫中的表达谱,并用geNorm,NormFinder和BestKeeper软件对不同温度下内参基因稳定性进行评价。【结果】获得松墨天牛sHSP基因的cDNA全长序列,命名为MaltHSP21.20(GenBank登录号:MH091811),全长为871 bp,编码187个氨基酸,信号肽预测表明其N末端含有18个氨基酸的信号肽,成熟蛋白预测分子质量为21.20 kD,等电点为8.65。结构域预测符合小热激蛋白家族的特征,共含有10个β折叠片,在α结构域内含有7个β折叠片。进化树分析表明该蛋白氨基酸序列与光肩星天牛Anoplophora glabripennis的sHSP有较高的同源性。不同软件分析得到最稳定的内参基因存在差异,结合3种方法评价得出RPL10最为稳定。MaltHSP21.20在不同发育阶段的松墨天牛体内均有表达,滞育幼虫中表达量最高,卵期及蛹期表达量次之;在4龄幼虫不同组织中均有分布,脂肪体中表达量最高;4龄幼虫MaltHSP21.20对低温诱导无响应,其表达量在35℃时显著上调,45℃处理2和3 h最高,50℃时下降。【结论】RPL10是不同温度胁迫下较稳定的内参基因。相比其他热激蛋白基因,MaltHSP21.20对低温敏感性较低,推测其在幼虫滞育越冬及抵抗高温中发挥重要作用。
【Aim】Monochamus alternatus,an important stem borer in pine forests in southern China,is a major vector of pine wilt disease. It has a wide range of distribution and strong resistance to temperature stress. The study aims to better understand the molecular mechanism of resistance to temperature stress inM. alternatus. 【Methods】The full-length cDNA encoding small heat shock protein( sHSP) was cloned from M. alternatus by RT-PCR and RACE techniques. The sequence characteristics of this sHSP were analyzed by bioinformatics methods. The expression patterns of this gene in different developmental stages,various tissues of the 4 th instar larvae and the 4 th instar larvae stressed under different low and high temperatures were detected by qPCR, and the stability of the reference genes at different temperatures was analyzed by geNorm,NormFinder and BestKeeper tools. 【Results】The complete cDNA of the gene encoding sHSP was obtained from M. alternatus,and named MaltHSP21. 20( GenBank accession no. : MH091811). The complete cDNA( 871 bp) encodes a protein of 187 amino acids,with a signal peptide of 18 amino acids in N-terminus,the molecular weight of 21. 20 kD and the theoretical p I of 8. 65. The domain prediction conforms to the characteristics of the sHSP family,containing 10 β-sheets and 7 β-sheets in the α-domain. Phylogenetic tree analysis showed that the sHSP sequence shares high homology with sHSP of Anoplophora glabripennis. The most stable reference gene was different identified by different methods,and assessed by three methods the most stable reference gene was RPL10. MaltHSP21. 20 was expressed in all developmental stages of M. alternatus,with the highest expression level in diapause larvae,and moderate in egg and pupal stages. MaltHSP21. 20 was expressed in different tissues of the 4 th instar larvae,with the highest expression level in fat body. The expression of MaltHSP21. 20 in the 4 th instar larvae showed no response to low temperature,while was up-regulated significantly at 35℃,reached the maximum after exposure to 45℃ for 2 and 3 h,and down-regulated at50℃. 【Conclusion】RPL10 is a more stable reference gene under different temperature stress. Compared to other heat shock protein genes,MaltHSP21. 20 is less sensitive to low temperature and presumably plays an important role in larval diapause during overwintering and the resistance to high temperature.
【Aim】Monochamus alternatus,an important stem borer in pine forests in southern China,is a major vector of pine wilt disease. It has a wide range of distribution and strong resistance to temperature stress. The study aims to better understand the molecular mechanism of resistance to temperature stress inM. alternatus. 【Methods】The full-length cDNA encoding small heat shock protein( sHSP) was cloned from M. alternatus by RT-PCR and RACE techniques. The sequence characteristics of this sHSP were analyzed by bioinformatics methods. The expression patterns of this gene in different developmental stages,various tissues of the 4 th instar larvae and the 4 th instar larvae stressed under different low and high temperatures were detected by qPCR, and the stability of the reference genes at different temperatures was analyzed by geNorm,NormFinder and BestKeeper tools. 【Results】The complete cDNA of the gene encoding sHSP was obtained from M. alternatus,and named MaltHSP21. 20( GenBank accession no. : MH091811). The complete cDNA( 871 bp) encodes a protein of 187 amino acids,with a signal peptide of 18 amino acids in N-terminus,the molecular weight of 21. 20 kD and the theoretical p I of 8. 65. The domain prediction conforms to the characteristics of the sHSP family,containing 10 β-sheets and 7 β-sheets in the α-domain. Phylogenetic tree analysis showed that the sHSP sequence shares high homology with sHSP of Anoplophora glabripennis. The most stable reference gene was different identified by different methods,and assessed by three methods the most stable reference gene was RPL10. MaltHSP21. 20 was expressed in all developmental stages of M. alternatus,with the highest expression level in diapause larvae,and moderate in egg and pupal stages. MaltHSP21. 20 was expressed in different tissues of the 4 th instar larvae,with the highest expression level in fat body. The expression of MaltHSP21. 20 in the 4 th instar larvae showed no response to low temperature,while was up-regulated significantly at 35℃,reached the maximum after exposure to 45℃ for 2 and 3 h,and down-regulated at50℃. 【Conclusion】RPL10 is a more stable reference gene under different temperature stress. Compared to other heat shock protein genes,MaltHSP21. 20 is less sensitive to low temperature and presumably plays an important role in larval diapause during overwintering and the resistance to high temperature.
引文
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Hayes D,Napoli V,Mazurkie A,Stafford WF,Graceffa P,2009.Phosphorylation dependence of Hsp27 multimeric size and molecular chaperone function.J.Biol.Chem.,284(28):18801-18807.
Hayward SA,Pavlides SC,Tammariello SP,Rinehart JP,Denlinger DL,2005.Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence.J.Insect Physiol.,51(6):631-640.
Hellemans J,Mortier G,Paepe AD,Speleman F,Vandesompele J,2007.q Base relative quantification framework and software for management and automated analysis of real-time quantitative PCRdata.Genome Biol.,8(2):R19.
Huang LH,Kang L,2007.Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress.Insect Mol.Biol.,16(4):491-500.
Huang LH,Wang CZ,Kang L,2009.Cloning and expression of five heat shock protein genes in relation to cold hardening and development in the leafminer,Liriomyza sativa.J.Insect Physiol.,55(3):279-285.
King AM,Macrae TH,2015.Insect heat shock proteins during stress and diapause.Annu.Rev.Entomol.,60(1):59-75.
Kong WN,Wang H,Li J,Zhao F,Ma RY,2006.The effects of temperature and humidity on the longevity of Monochamus alternatus Hope(Coleoptera:Cerambycidae).Shanxi Agric.Univ.(Nat.Sci.Ed.),26(3):294-295.[孔维娜,王慧,李捷,赵飞,马瑞燕,2006.温湿度对松墨天牛越冬幼虫寿命的影响.山西农业大学学报(自然科学版),26(3):294-295]
Kostenko S,Moens U,2009.Heat shock protein 27 phosphorylation:kinases,phosphatases,functions and pathology.Cell.Mol.Life Sci.,66(20):3289-3307.
Lin T,Cai Z,Wu H,2015.Transcriptome analysis of the Japanese pine sawyer beetle,Monochamus alternatus(Coleoptera:Cerambycidae)by high-throughput Illumina sequencing.J.Asia-Pacific Entomol.,18(3):439-445.
Liu QN,Zhu BJ,Dai LS,Fu WW,Lin KZ,Liu CL,2013.Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress.J.Insect Physiol.,59(8):848-854.
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.
Lu Y,Yuan M,Gao X,Kang T,Zhan S,Hu W,Li JH,2013.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLo S ONE,8(7):e68059.
Luo LL,Wu HJ,Lin T,2015.Characterization and expression of subunit D gene of ATP synthase from Monochamus alternatus.Sci.Silv.Sin.,51(7):60-68.[罗淋淋,吴华俊,林同,2015.松墨天牛ATP合成酶D亚基基因的鉴定与表达.林业科学,51(7):60-68]
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Pan DD,Lu MX,Li QY,Du YZ,2018.Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from Chilo suppressalis.Cell Stress Chaperones,23(1):55-64.
Pfaffl MW,Tichopad A,Prgomet C,Neuvians TP,2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:bestkeeper-excel-based tool using pair-wise correlations.J.Biotechnol.Lett.,26(6):509-515.
Qian K,Feng B,Wu XL,Lao C,Shen YL,Du YJ,2015.Cloning,sequence analysis,tissue expression profiling and prokaryotic expression of odorant binding protein genes Malt OBP2 and Malt OBP6 from Monochamus alternatus(Coleoptera:Cerambycidae).Acta Entomol.Sin.,58(5):496-506.[钱凯,冯波,巫诩亮,劳冲,沈幼莲,杜永均,2015.松墨天牛OBP基因Malt OBP2和Malt OBP6的克隆、序列分析及组织表达谱和原核表达研究.昆虫学报,58(5):496-506]
Quan G,Duan J,Ladd T,Krell PJ,2018.Identification and expression analysis of multiple small heat shock protein genes in spruce budworm,Choristoneura fumiferana(L.).Cell Stress Chaperones,23(1):141-154.
Rinehart JP,Li A,Yocum GD,Robich RM,Hayward SA,Denlinger DL,2007.Up-regulation of heat shock proteins is essential for cold survival during insect diapause.Proc.Natl.Acad.Sci.USA,104(27):11130-11137.
Shen Y,Gu J,Huang LH,Zheng SC,Liu L,Xu WH,Feng QL,Kang L,2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura.J.Insect Physiol.,57(7):908-914.
Sonoda S,Fukumoto K,Izumi Y,Ashfaq M,Yoshida H,Tsumuki H,2006.A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis.J.Appl.Entomol.,130(5):309-313.
S?rensen JG,Kristensen TN,Loeschcke V,2003.The evolutionary and ecological role of heat shock proteins.Ecol.Lett.,6(11):1025-1037.
Takahashi KH,Rako L,Takano-Shimizu T,Hoffmann AA,Lee SF,2010.Effects of small Hsp genes on developmental stability and microenvironmental canalization.BMC Evol.Biol.,10(1):1-11.
van Montfort RLM,Basha E,Friedrich KL,Slingsby C,Vierling E,2001.Crystal structure and assembly of a eukaryotic small heat shock protein.Nat.Struct.Biol.,8(12):1025-1030.
Vandesompele J,Preter KD,Pattyn F,Poppe B,Roy NV,Paepe AD,Speleman F,2002.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol.,3(7):1-12.
Wang H,Fang Y,Bao Z,Jin X,Zhu WJ,Wang L,Liu T,Ji HP,Wang HY,Xu SQ,Sima YH,2014.Identification of a Bombyx mori,gene encoding small heat shock protein Bm Hsp27.4 expressed in response to high-temperature stress.Gene,538(1):56-62.
Wang HJ,Shi ZK,Shen QD,Xu CD,Wang B,Meng ZJ,Wang SG,Tang B,Wang S,2017.Molecular cloning and induced expression of six small heat shock proteins mediating cold-hardiness in Harmonia axyridis(Coleoptera:Coccinellidae).Front.Physiol.,8:60-74.
Wu QY,1981.Role of fat body in metabolism in insects.Entomol.Knowl.,18(2):92-95.[吴秋雁,1981.昆虫脂肪体的代谢作用.昆虫知识,18(2):92-95]
Zhang YY,Liu YL,Guo XL,Li YL,Gao HR,Guo XQ,Xu BH,2014.s Hsp22.6,an intronless small heat shock protein gene,is involved in stress defence and development in Apis cerana cerana.Insect Biochem.Molec.Biol.,53(2):1-12.
Zhao L,Jones WA,2012.Expression of heat shock protein genes in insect stress responses.Invert.Surv.J.,9(1):93-101.
Zhu JR,Chai XM,He ZH,2001.Effects of temperature and light on the growth of Monochamus alternatus.J.Zhejiang Sci.Tech.,21(4):27-29.[朱锦茹,柴希民,何志华,2001.温度和光照对松褐天牛生长的影响.浙江林业科技,21(4):27-29]
Bagneris C,Bateman OA,Naylor CE,Cronin N,Boelens WC,Keep NH,Slingsby C,2009.Crystal structures ofα-crystallin domain dimers ofαB-crystallin and Hsp20.J.Mol.Biol.,392(5):1242-1252.
Basha E,Friedrich KL,Vierling E,2006.The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity.J.Biol.Chem.,281(52):39943-39952.
Basha E,O’Neill H,Vierling E,2012.Small heat shock proteins andα-crystallins:dynamic proteins with flexible functions.Trends Biochem.Sci.,37(3):106-117.
Cai Z,Chen J,Cheng J,Lin T,2017.Overexpression of three heat shock proteins protects Monochamus alternatus(Coleoptera:Cerambycidae)from thermal stress.J.Insect Sci.,17(6):1-11.
Chen RX,Wang LJ,Lin T,Wei ZQ,Wang Y,Hao DJ,2017.Rearing techniques of Monochamus alternatus Hope(Coleoptera:Cerambycidae)on artificial diets.J.Nanjing For.Univ.(Nat.Sci.Ed.),41(1):199-202.[陈瑞旭,王露洁,林涛,韦志强,王焱,郝德君,2017.松墨天牛的人工饲育技术研究.南京林业大学学报(自然科学版),41(1):199-202]
Enayati AA,Ranson H,Hemingway J,2005.Insect glutathione transferases and insecticide resistance.Insect Mol.Biol.,14(1):3-8.
Feng B,Guo QS,Mao BP,Du YJ,2016.Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus(Coleoptera:Cerambycidae)by RT-q PCR.Acta Entomol.Sin.,59(4):427-437.[冯波,郭前爽,毛必鹏,杜永均,2016.松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选.昆虫学报,59(4):427-437]
Garrido C,Paul C,Seigneuric R,Kampinga HH,2012.The small heat shock proteins family:the long forgotten chaperones.Int.J.Biochem.Cell Biol.,44(10):1588-1592.
Gkouvitsas T,Kontogiannatos D,Kourti A,2008.Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides(Lef.).J.Insect Physiol.,54(12):1503-1510.
Hayes D,Napoli V,Mazurkie A,Stafford WF,Graceffa P,2009.Phosphorylation dependence of Hsp27 multimeric size and molecular chaperone function.J.Biol.Chem.,284(28):18801-18807.
Hayward SA,Pavlides SC,Tammariello SP,Rinehart JP,Denlinger DL,2005.Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence.J.Insect Physiol.,51(6):631-640.
Hellemans J,Mortier G,Paepe AD,Speleman F,Vandesompele J,2007.q Base relative quantification framework and software for management and automated analysis of real-time quantitative PCRdata.Genome Biol.,8(2):R19.
Huang LH,Kang L,2007.Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress.Insect Mol.Biol.,16(4):491-500.
Huang LH,Wang CZ,Kang L,2009.Cloning and expression of five heat shock protein genes in relation to cold hardening and development in the leafminer,Liriomyza sativa.J.Insect Physiol.,55(3):279-285.
King AM,Macrae TH,2015.Insect heat shock proteins during stress and diapause.Annu.Rev.Entomol.,60(1):59-75.
Kong WN,Wang H,Li J,Zhao F,Ma RY,2006.The effects of temperature and humidity on the longevity of Monochamus alternatus Hope(Coleoptera:Cerambycidae).Shanxi Agric.Univ.(Nat.Sci.Ed.),26(3):294-295.[孔维娜,王慧,李捷,赵飞,马瑞燕,2006.温湿度对松墨天牛越冬幼虫寿命的影响.山西农业大学学报(自然科学版),26(3):294-295]
Kostenko S,Moens U,2009.Heat shock protein 27 phosphorylation:kinases,phosphatases,functions and pathology.Cell.Mol.Life Sci.,66(20):3289-3307.
Lin T,Cai Z,Wu H,2015.Transcriptome analysis of the Japanese pine sawyer beetle,Monochamus alternatus(Coleoptera:Cerambycidae)by high-throughput Illumina sequencing.J.Asia-Pacific Entomol.,18(3):439-445.
Liu QN,Zhu BJ,Dai LS,Fu WW,Lin KZ,Liu CL,2013.Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress.J.Insect Physiol.,59(8):848-854.
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.
Lu Y,Yuan M,Gao X,Kang T,Zhan S,Hu W,Li JH,2013.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLo S ONE,8(7):e68059.
Luo LL,Wu HJ,Lin T,2015.Characterization and expression of subunit D gene of ATP synthase from Monochamus alternatus.Sci.Silv.Sin.,51(7):60-68.[罗淋淋,吴华俊,林同,2015.松墨天牛ATP合成酶D亚基基因的鉴定与表达.林业科学,51(7):60-68]
Marionnet C,Bernerd F,Dumas A,Verrecchia F,Mollier K,Compan D,Bernard D,Lahfa M,Leclaire J,Medaisko C,Mehul B,Seite S,Mauviel A,Dubertret L,2003.Modulation of gene expression induced in human epidermis by environmental stress in vivo.J.Invest.Dermatol.,121(6):1447-1458.
Pan DD,Lu MX,Li QY,Du YZ,2018.Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from Chilo suppressalis.Cell Stress Chaperones,23(1):55-64.
Pfaffl MW,Tichopad A,Prgomet C,Neuvians TP,2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:bestkeeper-excel-based tool using pair-wise correlations.J.Biotechnol.Lett.,26(6):509-515.
Qian K,Feng B,Wu XL,Lao C,Shen YL,Du YJ,2015.Cloning,sequence analysis,tissue expression profiling and prokaryotic expression of odorant binding protein genes Malt OBP2 and Malt OBP6 from Monochamus alternatus(Coleoptera:Cerambycidae).Acta Entomol.Sin.,58(5):496-506.[钱凯,冯波,巫诩亮,劳冲,沈幼莲,杜永均,2015.松墨天牛OBP基因Malt OBP2和Malt OBP6的克隆、序列分析及组织表达谱和原核表达研究.昆虫学报,58(5):496-506]
Quan G,Duan J,Ladd T,Krell PJ,2018.Identification and expression analysis of multiple small heat shock protein genes in spruce budworm,Choristoneura fumiferana(L.).Cell Stress Chaperones,23(1):141-154.
Rinehart JP,Li A,Yocum GD,Robich RM,Hayward SA,Denlinger DL,2007.Up-regulation of heat shock proteins is essential for cold survival during insect diapause.Proc.Natl.Acad.Sci.USA,104(27):11130-11137.
Shen Y,Gu J,Huang LH,Zheng SC,Liu L,Xu WH,Feng QL,Kang L,2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura.J.Insect Physiol.,57(7):908-914.
Sonoda S,Fukumoto K,Izumi Y,Ashfaq M,Yoshida H,Tsumuki H,2006.A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis.J.Appl.Entomol.,130(5):309-313.
S?rensen JG,Kristensen TN,Loeschcke V,2003.The evolutionary and ecological role of heat shock proteins.Ecol.Lett.,6(11):1025-1037.
Takahashi KH,Rako L,Takano-Shimizu T,Hoffmann AA,Lee SF,2010.Effects of small Hsp genes on developmental stability and microenvironmental canalization.BMC Evol.Biol.,10(1):1-11.
van Montfort RLM,Basha E,Friedrich KL,Slingsby C,Vierling E,2001.Crystal structure and assembly of a eukaryotic small heat shock protein.Nat.Struct.Biol.,8(12):1025-1030.
Vandesompele J,Preter KD,Pattyn F,Poppe B,Roy NV,Paepe AD,Speleman F,2002.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol.,3(7):1-12.
Wang H,Fang Y,Bao Z,Jin X,Zhu WJ,Wang L,Liu T,Ji HP,Wang HY,Xu SQ,Sima YH,2014.Identification of a Bombyx mori,gene encoding small heat shock protein Bm Hsp27.4 expressed in response to high-temperature stress.Gene,538(1):56-62.
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