沙葱萤叶甲热激蛋白基因GdHsp70的克隆与表达模式分析
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摘要
【目的】本研究旨在克隆沙葱萤叶甲Galeruca daurica热激蛋白Hsp70基因,并对其进行序列和表达模式分析,探讨该基因在沙葱萤叶甲生长发育及响应温度胁迫方面的作用。【方法】采用RT-PCR和RACE技术从沙葱萤叶甲2龄幼虫中克隆Hsp70基因,并进行生物信息学分析;用Wo LF PSORT在线软件进行亚细胞定位预测;采用实时荧光定量PCR检测该基因在沙葱萤叶甲成虫不同组织(头、胸和腹)中、不同发育阶段(卵、1-3龄幼虫、蛹、雌雄成虫)、不同温度(-14,-10,-5,0,5,10,15,20,25和30℃)处理1 h的2龄幼虫及0℃下分别处理0 min,15 min,30min,1 h,1.5 h,2 h和3 h时卵中的相对表达量。【结果】克隆获得一个沙葱萤叶甲Hsp70基因并命名为GdHsp70(GenBank登录号:KY460462),该基因全长2 340 bp,开放阅读框(ORF)1 899 bp,编码632个氨基酸,预测蛋白质分子量为70.12 k D,等电点(p I)为4.79,无跨膜区,无信号肽。蛋白质亚细胞定位预测该蛋白主要位于细胞质内。蛋白质结构域分析表明,GdHsp70有3个功能保守区。同源比对与系统进化分析表明,GdHsp70与分类学关系上较为接近的昆虫的同源蛋白间有较高的相似度。组织特异性和不同发育阶段表达分析表明,GdHsp70在沙葱萤叶甲成虫胸部和卵期表达量最高;高温和低温胁迫均能诱导沙葱萤叶甲2龄幼虫体内GdHsp70的表达,其中-10℃处理1 h表达量最高;0℃低温处理卵15 min至3 h后均能诱导GdHsp70不同程度的表达上调,其中处理1 h上调幅度最大。【结论】沙葱萤叶甲GdHsp70与该虫的生长发育相关,并对高低温胁迫的响应有重要作用。
【Aim】This study aims to clone the heat shock protein Hsp70 gene in Galeruca daurica,and to analyze the gene sequence and mRNA expression profiles so as to explore the function of Hsp70 gene in the development and its responses to thermal stress in G. daurica. 【Methods】The full-length c DNA of a Hsp70 gene was cloned and identified from the 2nd instar larvae of G. daurica using RT-PCR and RACE technique,and the putative amino acid sequence was analyzed by bioinformatics methods. The relative expression levels of the Hsp70 gene in different adult tissues( head,thorax and abdomen),different developmental stages( egg,the 1st-3rd instar larva,pupa and female and male adult),the 2nd instar larvae exposed to different temperatures(-14,-10,-5,0,5,10,15,20,25 and 30℃ for 1 h),and eggs exposed to 0℃ for 0 min,15 min,30 min,1 h,1. 5 h,2 h,and 3 h,respectively,weremeasured using real-time quantitative PCR method. 【Results】A heat shock protein 70 gene was cloned from G. daurica,and named GdHsp70( GenBank accession no. KY460462),which is 2 340 bp in length and contains an open reading frame( ORF) of 1 899 bp,encoding 632 amino acid residues. The putative protein is 70. 12 k D with an isoelectric point(pI) of 4. 79,and has no transmembrane region and signal peptide. Subcellular localization prediction revealed that GdHsp70 is mainly located in the cytoplasm. Protein domain analysis showed that GdHsp70 has three highly conserved domains.Homologous alignment and phylogenetic analysis demonstrated that GdHsp70 has high similarity with the Hsp70 proteins from other insects which are highly close to G. daurica based on taxonomy. Tissue-and developmental stage-specific mRNA expression profiling showed that GdHsp70 had the highest expression levels in the adult thoraxes and eggs. Both heat and cold stress could induce the mRNA expression of GdHsp70 in the 2nd instar larvae with the highest expression level at-10℃ for 1 h. GdHsp70 was upregulated in G. daurica eggs exposed to 0℃ for 15 min to 3 h,with the highest expression level at 1 h after treatment. 【Conclusion】GdHsp70 might be associated with the development and play an important role in response to heat and cold stress in G. daurica.
【Aim】This study aims to clone the heat shock protein Hsp70 gene in Galeruca daurica,and to analyze the gene sequence and mRNA expression profiles so as to explore the function of Hsp70 gene in the development and its responses to thermal stress in G. daurica. 【Methods】The full-length c DNA of a Hsp70 gene was cloned and identified from the 2nd instar larvae of G. daurica using RT-PCR and RACE technique,and the putative amino acid sequence was analyzed by bioinformatics methods. The relative expression levels of the Hsp70 gene in different adult tissues( head,thorax and abdomen),different developmental stages( egg,the 1st-3rd instar larva,pupa and female and male adult),the 2nd instar larvae exposed to different temperatures(-14,-10,-5,0,5,10,15,20,25 and 30℃ for 1 h),and eggs exposed to 0℃ for 0 min,15 min,30 min,1 h,1. 5 h,2 h,and 3 h,respectively,weremeasured using real-time quantitative PCR method. 【Results】A heat shock protein 70 gene was cloned from G. daurica,and named GdHsp70( GenBank accession no. KY460462),which is 2 340 bp in length and contains an open reading frame( ORF) of 1 899 bp,encoding 632 amino acid residues. The putative protein is 70. 12 k D with an isoelectric point(pI) of 4. 79,and has no transmembrane region and signal peptide. Subcellular localization prediction revealed that GdHsp70 is mainly located in the cytoplasm. Protein domain analysis showed that GdHsp70 has three highly conserved domains.Homologous alignment and phylogenetic analysis demonstrated that GdHsp70 has high similarity with the Hsp70 proteins from other insects which are highly close to G. daurica based on taxonomy. Tissue-and developmental stage-specific mRNA expression profiling showed that GdHsp70 had the highest expression levels in the adult thoraxes and eggs. Both heat and cold stress could induce the mRNA expression of GdHsp70 in the 2nd instar larvae with the highest expression level at-10℃ for 1 h. GdHsp70 was upregulated in G. daurica eggs exposed to 0℃ for 15 min to 3 h,with the highest expression level at 1 h after treatment. 【Conclusion】GdHsp70 might be associated with the development and play an important role in response to heat and cold stress in G. daurica.
引文
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Jiang JJ,Huang LF,Chen HS,Yang L,2015.Identification of reference genes and expression analysis of heat shock protein genes in the brown plant hopper,Nilaparvata lugens(Hemiptera:Delphacidae),after exposure to heat stress.Acta Entomol.Sin.,58(5):479-486.
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Li H,Zhou XR,Pang BP,Zhang ZR,Chang J,Shan YM,2015.Effects of low temperature stress on the supercooling capacity and development of Galeruca daurica Joannis larvae(Coleoptera:Chrysomelidae).Chin.J.Appl.Entomol.,52(2):434-439.[李浩,周晓榕,庞保平,张卓然,常静,单艳敏,2015.低温胁迫对沙葱萤叶甲幼虫过冷却能力及生长发育的影响.应用昆虫学报,52(2):434-439]
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Lu MX,Liu ZX,Cui YD,Du YZ,2014.Expression patterns of three heat shock proteins in Chilo suppressalis(Lepidoptera:Pyralidae).Ann.Entomol.Soc.Am.,107(3):667-673.
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Schmid D,Baici A,Gehring H,Christen P,1994.Kinetics of molecular chaperone action.Science,263(5149):971-973.
Sharmas S,Rohilla MS,Tiwari PK,2007.Developmental and hyperthermia-induced expression of the heat shock proteins HSP60and HSP70 in tissues of the housefly Musca domestica:an in vitro study.Genet.Mol.Biol.,30(1):159-168.
Shen HT,Zhu HC,Song MW,Tian YL,Huang YF,Zheng H,Cao RY,Lin J,Bi ZG,Zhong W,2014.A selenosemicarbazone complex with copper efficiently down-regulates the 90-k Da heat shock protein HSP90AA1 and its client proteins in cancer cells.BMC Cancer,14:629.
Shi JH,Fu MJ,Zhao C,Zhou FL,Yang QB,Qiu LH,2016.Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp,Penaeus monodon.Cell Stress Chaperon.,21:295-312.
Si FL,He ZB,Chen B,2016.Cloning and expression profiling of heat shock protein Da HSP23 gene in the winter and summer diapause pupae of the onion maggot,Delia antique(Diptera:Anthomyiidae).Acta Entomol.Sin.,59(4):402-410.[司风玲,何正波,陈斌,2016.葱蝇热激蛋白Da HSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析.昆虫学报,59(4):402-410]
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Tan Y,Zhou XR,Pang P,2016.Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica(Joannis).Bull.Entomol.Res.,107(3):359-368.
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.,359:419-426.
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Boutet I,Tanguy A,Rousseau S,Auffret M,Moraga D,2003.Molecular identification and expression of heat shock cognate 70(hsc70)and heat shock protein 70(hsp70)genes in the Pacific oyster Crassostrea gigas.Cell Stress Chaperon.,8(1):76-85.
Clark MS,Peck LS,2009.Triggers of the HSP70 stress response:environmental responses and laboratory manipulation in an Antarctic marine invertebrate(Nacella concinna).Cell Stress Chaperon.,14(6):649-660.
Daugaard M,Rohde M,JttelM,2007.The heat shock protein 70family:highly homologous proteins with overlapping and distinct functions.FEBS Lett.,581(19):3702-3710.
Feder ME,Hofmann GE,1999.Heat-shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annu.Rev.Physiol.,61(1):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.
Gao JC,Zhou XR,Pang BP,Bao X,Luo JP,Erdeng Q,2015.Effects of low temperature on the survivorship and development of overwintering eggs of Galeruca daurica(Coleoptera:Chrysomelidae).Acta Entomol.Sin.,58(8):881-886.[高靖淳,周晓榕,庞保平,包祥,罗建平,额尔登其木格,2015.低温对沙葱萤叶甲越冬卵存活和发育的影响.昆虫学报,58(8):881-886]
Gkouvitsas T,Kontogiannatos D,Kourti A,2009.Cognate Hsp70 gene is induced during deep larval diapause in the moth Sesamia nonagrioides.Insect Mol.Biol.,18(2):253-264.
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 hsp70and hsc70 mRNA in the Aphis glycines(Hemiptera:Aphididae).Acta Entomol.Sin.,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mRNA表达的影响.昆虫学报,57(4):387-394]
Hao CF,Wang HP,Zhang ZQ,Chang Y,Li ML,2016.Characterization and expression analysis of HSP70 gene in Dastarcus helophoroides.J.Northeast For.Univ.,44(7):108-124.[郝春凤,王化鹏,张正青,常勇,李孟楼,2016.花绒寄甲HSP70基因的特征与表达.东北林业大学学报,44(7):108-124]
Hao X,Zhou XR,Pang BP,Zhang ZR,Bao X,2015.Morphological and biological characteristics of Galeruca daurica Joannis.Acta Agrestia Sin.,23(5):1106-1108.[昊翔,周晓榕,庞保平,张卓然,包祥,2015.沙葱萤叶甲形态特征与生物学特性的研究.草地学报,23(5):1106-1108]
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.,53:1199-1205.
Huang LH,Kang L,2007.Cloning and inter specific altered expression of heat shock protein genes in two leaf miner species in response to thermal stress.Insect Mol.Biol.,16(4):491-500.
Jiang JJ,Huang LF,Chen HS,Yang L,2015.Identification of reference genes and expression analysis of heat shock protein genes in the brown plant hopper,Nilaparvata lugens(Hemiptera:Delphacidae),after exposure to heat stress.Acta Entomol.Sin.,58(5):479-486.
Jing XH,Kang L,2003.Geographical variation in egg cold hardiness:a study on the adaptation strategies of the migratory locust Locusta migratoria L.Ecol.Entomol.,28:151.
Kang L,Chen B,Wei JN,Liu TX,2009.Roles of thermal adaptation and chemical ecology in Liriomyza distribution and control.Annu.Rev.Entomol.,54:127-145.
King AM,MacRae TH,2015.Insect heat shock proteins during stress and diapause.Annu.Rev.Entomol.,60:59-75.
Li H,Zhou XR,Pang BP,Chang J,2014.Supercooling capacity and cold hardiness of Galeruca daurica(Coleoptera:Chrysomelidae).Acta Entomol.Sin.,57(2):212-217.[李浩,周晓榕,庞保平,常静,2014.沙葱萤叶甲的过冷却能力与抗寒性.昆虫学报,57(2):212-217]
Li H,Zhou XR,Pang BP,Zhang ZR,Chang J,Shan YM,2015.Effects of low temperature stress on the supercooling capacity and development of Galeruca daurica Joannis larvae(Coleoptera:Chrysomelidae).Chin.J.Appl.Entomol.,52(2):434-439.[李浩,周晓榕,庞保平,张卓然,常静,单艳敏,2015.低温胁迫对沙葱萤叶甲幼虫过冷却能力及生长发育的影响.应用昆虫学报,52(2):434-439]
Li M,Lu WC,Feng HZ,Yuan L,Wang JJ,He L,2008.Cloning and expression analysis of a heat shock protein 70 gene TCHSP70-4 from Tetranychus cinnabarinus(Acari:Tetranychidae).Acta Entomol.Sin.,51(12):1235-1243.[李明,卢文才,冯宏祖,袁亮,王进军,何林,2008.朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达.昆虫学报,51(12):1235-1243]
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method.Methods,25:402-408.
Lu MX,Liu ZX,Cui YD,Du YZ,2014.Expression patterns of three heat shock proteins in Chilo suppressalis(Lepidoptera:Pyralidae).Ann.Entomol.Soc.Am.,107(3):667-673.
Rinehart JP,Li AQ,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.
Schmid D,Baici A,Gehring H,Christen P,1994.Kinetics of molecular chaperone action.Science,263(5149):971-973.
Sharmas S,Rohilla MS,Tiwari PK,2007.Developmental and hyperthermia-induced expression of the heat shock proteins HSP60and HSP70 in tissues of the housefly Musca domestica:an in vitro study.Genet.Mol.Biol.,30(1):159-168.
Shen HT,Zhu HC,Song MW,Tian YL,Huang YF,Zheng H,Cao RY,Lin J,Bi ZG,Zhong W,2014.A selenosemicarbazone complex with copper efficiently down-regulates the 90-k Da heat shock protein HSP90AA1 and its client proteins in cancer cells.BMC Cancer,14:629.
Shi JH,Fu MJ,Zhao C,Zhou FL,Yang QB,Qiu LH,2016.Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp,Penaeus monodon.Cell Stress Chaperon.,21:295-312.
Si FL,He ZB,Chen B,2016.Cloning and expression profiling of heat shock protein Da HSP23 gene in the winter and summer diapause pupae of the onion maggot,Delia antique(Diptera:Anthomyiidae).Acta Entomol.Sin.,59(4):402-410.[司风玲,何正波,陈斌,2016.葱蝇热激蛋白Da HSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析.昆虫学报,59(4):402-410]
Sonoda S,Ashfaq M,Tsumuki H,2006.Cloning and nucleotide sequencing of three heat shock protein genes(hsp90,hsc70 and hsp19.5)from the diamondback moth,Plutella xylostella(L.)and their expression in relation to developmental stage and temperature.Arch.Insect Biochem.Physiol.,62(2):80-90.
Srensen JG,Kristensen TN,Loeschcke V,2003.The evolutionary and ecological role of heat shock proteins.Ecol.Lett.,6(11):359-353.
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