菜粉蝶HSP20基因的克隆及温度胁迫下表达分析
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摘要
【目的】克隆分析菜粉蝶(Pieris rapae)热激蛋白20基因(HSP20),并检测其在温度胁迫下的表达情况,为探析菜粉蝶适应温度胁迫机制提供参考。【方法】以菜粉蝶为试验材料,采用RT-PCR和RACE克隆获得菜粉蝶HSP20基因cDNA全长,并通过生物学软件分析其序列,明确其基因结构特征和亲缘关系;采用实时荧光定量PCR检测菜粉蝶HSP20基因在低温(-20～0℃)和高温(5～45℃)胁迫下的表达响应。【结果】菜粉蝶HSP20基因全长725 bp,5'-端非编码区105 bp,3'-端非编码区109 bp,开放阅读框531 bp,编码176个氨基酸,预测蛋白分子量19.5 kD,理论等电点6.61;其编码氨基酸序列中含有1个典型的HSP20家族结构域(位于第60～142位氨基酸)、1个α-晶体蛋白(位于第60～157位氨基酸)和1个Allato allatostatin(位于第14～24位氨基酸);基序列相似度分析其与二化螟、家蚕和甘蓝夜蛾HSP20蛋白氨基酸相似度较高并聚为一族。该基因随环境温度的升高或降低发生变化,在-5和40℃诱发该基因高效表达。【结论】低温和高温均能诱导菜粉蝶HSP20基因高效表达。
【Objective】Heat shock protein 20 gene(HSP20)from Pieris rapae was cloned and its expression under temperature stress was detected. The result was helpful for further investigation of the adaptation mechanisms of P. rapae to high and low temperature stresses.【Method】Full length cDNA of HSP20 gene of P. rapae was cloned by RT-PCR and RACE with P. rapae as materials. Its sequence was analyzed by biology software,and its gene structural sequence and genetic relationship were also studied. Response of HSP20 gene to low(-20-0 ℃)and high(5-45 ℃)temperatures were analyzed by fluorescence quantitative PCR.【Result】The full-length of P. rapae HSP20 was 725 bp,and contained 105 bp 5'-noncoding region,109 bp 3'-noncoding region,a 531 bp open reading frame(ORF),encoded 176 amino acids,with an estimated molecular mass of 19.5 kD and an theoretical isoelectric point(pI)of 6.61. In the encoded amino acid sequences,there was one typical HSP20 family domain(located at 60-142 amino acids),one α-crystallin(located at 60-157 amino acids)and a Allato allatostatin(located at 14-24 amino acids). The sequence similarities between P. rapae HSP20 and that of Chilo suppressalis,Bombyx mori,Mamestra brassicae were high and they were clustered into one group. The HSP20 expressions changed with temperature,and high expressions of this gene was observed at-5 and 40 ℃.【Conclusion】Low and high temperatures can lead to the high expression of P. rapae HSP20.
【Objective】Heat shock protein 20 gene(HSP20)from Pieris rapae was cloned and its expression under temperature stress was detected. The result was helpful for further investigation of the adaptation mechanisms of P. rapae to high and low temperature stresses.【Method】Full length cDNA of HSP20 gene of P. rapae was cloned by RT-PCR and RACE with P. rapae as materials. Its sequence was analyzed by biology software,and its gene structural sequence and genetic relationship were also studied. Response of HSP20 gene to low(-20-0 ℃)and high(5-45 ℃)temperatures were analyzed by fluorescence quantitative PCR.【Result】The full-length of P. rapae HSP20 was 725 bp,and contained 105 bp 5'-noncoding region,109 bp 3'-noncoding region,a 531 bp open reading frame(ORF),encoded 176 amino acids,with an estimated molecular mass of 19.5 kD and an theoretical isoelectric point(pI)of 6.61. In the encoded amino acid sequences,there was one typical HSP20 family domain(located at 60-142 amino acids),one α-crystallin(located at 60-157 amino acids)and a Allato allatostatin(located at 14-24 amino acids). The sequence similarities between P. rapae HSP20 and that of Chilo suppressalis,Bombyx mori,Mamestra brassicae were high and they were clustered into one group. The HSP20 expressions changed with temperature,and high expressions of this gene was observed at-5 and 40 ℃.【Conclusion】Low and high temperatures can lead to the high expression of P. rapae HSP20.
引文
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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[J].昆虫学报,54(11):1236-1248.[Shen J R,Li M F,Chen N Z,Wang J J,Wan F H.2011.Cloning and analysis of the expression characteristics under heat stress of Hsp90gene in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae)[J].Acta Entomologica Sinica,54(11):1236-1248.]
夏晓峰,林海兰,郑丹丹,杨广,尤民生.2013.小菜蛾热休克蛋白基因的鉴定及其表达模式分析[J].昆虫学报,56(5):457-464.[Xia X F,Lin H L,Zhen D D,Yang G,You M S.2013.Identification and expression patterns of heat shock protein genes in the diamondback moth,Plutella xylostella(Lepidoptera:Yponomeutidae)[J].Acta Entomologica Sinica,56(5):457-464.]
徐剑文,赵君,刘剑光,肖松华.2017.芝麻全基因组Hsp70基因的鉴定与生物信息学分析[J].江苏农业学报,33(3):493-502.[Xu J W,Zhao J,Liu J G,Xiao S H.2017.Genome-wide identification and bioinformatics analysis of Hsp70 gene family in Sesamum indicum[J].Jiangsu Journal of Agricultural Sciences,33(3):493-502.]
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[J].昆虫学报,57(7):777-786.[Zhang Q,Lu M X,Zhu S D.2014.Cloning,sequence analysis and expression profiling of heat shock protein 90 gene in the small brown plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae)[J].Acta Entomologica Sinica,57(7):777-786.]
张响英,唐现文,陈静波,李腾腾.2017.热应激诱导奶牛乳腺上皮细胞凋亡及其对HSP70基因表达的影响[J].河南农业科学,46(8):142-146.[Zhang X Y,Tang X W,Chen J B,Li T T.2017.Effects of heat stress on apoptosis and expression of HSP70 mRNA in bovine mammary epithelial cells[J].Journal of Henan Agricultural Sciences,46(8):142-146.]
Li Z W,Li X,Yu Q Y.2009.The small heat shock protein(sHSP)genes in silkworm,Bombyx mori and comparative analysis withother insect sHSP genes[J].BMC Evolutionary Biology,9:215.
Lindquist S.1986.The heat-shock response[J].Annual Review of Biochemistry,55(1):1151-1191.
Liu Q N,Liu Y,Zhao Z X,Zhu X Y,Ge B M,Li C F,Wang D,Bian X G,Yang L,Chen L,Tian J W,Zhou C L,Tang B P.2018.A small heat shock protein 21(sHSP21)mediates immune responses in Chinese oak silkworm Antheraea pernyi[J].International Journal of Biological Macromolecules,111:1027-1031.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-△△Ctmethod[J].Methods,25:402-408.
Mehlen P,Schulze-Osthoff K,Arrigo A P.1996.Small stress proteins as novel regulators of apoptosis.Heat shock protein 27 blocks Fas/APO-1-and staurosporine-induced cell death[J].Journal of Biological Chemistry,271(28):16510-16514.
Multhoff G.2007.Heat shock protein 70(Hsp70):Membrane Iloca-tion,export and immunological relevance[J].Methods,43:229-237.
Rinehart J P,Yocum G D,Denlinger D L.2000.Developmental upregulation of inducible hsp70 transcripts,but not the cognate form,during pupal diapause in the flesh fly,Ssarcophaga crassipalpis[J].Insect Biochemistry&Molecular Biology,30(6):515-21.
Shen Y,Gu J,Huang L H,Zheng S C,Liu L,Xu W H,Feng Q L,Kang L.2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura[J].Journal of Insect Physiology,57(7):908-914.
Sonoda S,Ashfaq M,Tsumuki H.2006a.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[J].Archives of Insect Biochemistry&Physiology,62(2):80-90.Sonoda S,Fukumoto K,Izumi Y,Ashfaq M,Yoshida H,Tsumuki H.2006b.A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis[J].Journal of Applied Entomology,130(5):309-313.
Tachibana S,Numata H,Goto S G.2005.Gene expression of heat-shock proteins(Hsp23,Hsp70 and Hsp90)during and after larval diapause in the blow fly Lucilia sericata[J].Journal of Insect Physiology,51(6):641-647.
Wang H,Li K,Zhu J Y,Fang Q,Ye G Y.2012.Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp,pteromalus puparum in response to environmental stresses[J].Archives of Insect Biochemistry and Physiology,79(4-5):247-263.
韩政,赵龙龙,陈光,马瑞燕.2010.热激蛋白与昆虫的耐热性关系研究进展[J].山西农业科学,38(8):92-94.[Han Z,Zhao L L,Chen G,Ma R Y.2010.Research advances of the relationship between heat shock proteins and Insects heat tolerance[J].Journal of Shanxi Agricultural Sciences,38(8):92-94.]
寇利花,吴海花,刘耀明,张育平,张建珍,郭亚平,马恩波.2015.镉急性染毒对中华稻蝗精卵巢小分子热休克蛋白基因表达的影响[J].农业环境科学学报,34(1):7-14.[Kou L H,Wu H H,Liu Y M,Zhang Y P,Zhang JZ,Guo Y P,Ma E B.2015.Expression analysis of OcsHSPgenes in testis and ovary of Oxya chinensis exposed to acute cadmium[J].Journal of Agro-Environment Science,34(1):7-14.]
李斌.2001.家蚕分子连锁图谱的构建和低分子量热激蛋白基因的克隆与特异表达[D].重庆:西南农业大学学报.[Li B.2001.Constructin of the molecular linkage map and isolation,identification and specific expression of small heat-shock genes in silkworm,Bombyx mori[D].Chonɡqing:Southwest Agricultural University.]
任莉萍,曹小汉.2006.热休克蛋白研究进展[J].安徽农业科学,34(10):2040-2042.[Ren L P,Cao X H.2006.Study on heat shock protein[J].Journal of Anhui Agricultural Sciences,34(10):2040-2042.]
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[J].昆虫学报,54(11):1236-1248.[Shen J R,Li M F,Chen N Z,Wang J J,Wan F H.2011.Cloning and analysis of the expression characteristics under heat stress of Hsp90gene in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae)[J].Acta Entomologica Sinica,54(11):1236-1248.]
夏晓峰,林海兰,郑丹丹,杨广,尤民生.2013.小菜蛾热休克蛋白基因的鉴定及其表达模式分析[J].昆虫学报,56(5):457-464.[Xia X F,Lin H L,Zhen D D,Yang G,You M S.2013.Identification and expression patterns of heat shock protein genes in the diamondback moth,Plutella xylostella(Lepidoptera:Yponomeutidae)[J].Acta Entomologica Sinica,56(5):457-464.]
徐剑文,赵君,刘剑光,肖松华.2017.芝麻全基因组Hsp70基因的鉴定与生物信息学分析[J].江苏农业学报,33(3):493-502.[Xu J W,Zhao J,Liu J G,Xiao S H.2017.Genome-wide identification and bioinformatics analysis of Hsp70 gene family in Sesamum indicum[J].Jiangsu Journal of Agricultural Sciences,33(3):493-502.]
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[J].昆虫学报,57(7):777-786.[Zhang Q,Lu M X,Zhu S D.2014.Cloning,sequence analysis and expression profiling of heat shock protein 90 gene in the small brown plant hopper,Laodelphax striatellus(Hemiptera:Delphacidae)[J].Acta Entomologica Sinica,57(7):777-786.]
张响英,唐现文,陈静波,李腾腾.2017.热应激诱导奶牛乳腺上皮细胞凋亡及其对HSP70基因表达的影响[J].河南农业科学,46(8):142-146.[Zhang X Y,Tang X W,Chen J B,Li T T.2017.Effects of heat stress on apoptosis and expression of HSP70 mRNA in bovine mammary epithelial cells[J].Journal of Henan Agricultural Sciences,46(8):142-146.]
Li Z W,Li X,Yu Q Y.2009.The small heat shock protein(sHSP)genes in silkworm,Bombyx mori and comparative analysis withother insect sHSP genes[J].BMC Evolutionary Biology,9:215.
Lindquist S.1986.The heat-shock response[J].Annual Review of Biochemistry,55(1):1151-1191.
Liu Q N,Liu Y,Zhao Z X,Zhu X Y,Ge B M,Li C F,Wang D,Bian X G,Yang L,Chen L,Tian J W,Zhou C L,Tang B P.2018.A small heat shock protein 21(sHSP21)mediates immune responses in Chinese oak silkworm Antheraea pernyi[J].International Journal of Biological Macromolecules,111:1027-1031.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-△△Ctmethod[J].Methods,25:402-408.
Mehlen P,Schulze-Osthoff K,Arrigo A P.1996.Small stress proteins as novel regulators of apoptosis.Heat shock protein 27 blocks Fas/APO-1-and staurosporine-induced cell death[J].Journal of Biological Chemistry,271(28):16510-16514.
Multhoff G.2007.Heat shock protein 70(Hsp70):Membrane Iloca-tion,export and immunological relevance[J].Methods,43:229-237.
Rinehart J P,Yocum G D,Denlinger D L.2000.Developmental upregulation of inducible hsp70 transcripts,but not the cognate form,during pupal diapause in the flesh fly,Ssarcophaga crassipalpis[J].Insect Biochemistry&Molecular Biology,30(6):515-21.
Shen Y,Gu J,Huang L H,Zheng S C,Liu L,Xu W H,Feng Q L,Kang L.2011.Cloning and expression analysis of six small heat shock protein genes in the common cutworm,Spodoptera litura[J].Journal of Insect Physiology,57(7):908-914.
Sonoda S,Ashfaq M,Tsumuki H.2006a.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[J].Archives of Insect Biochemistry&Physiology,62(2):80-90.Sonoda S,Fukumoto K,Izumi Y,Ashfaq M,Yoshida H,Tsumuki H.2006b.A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis[J].Journal of Applied Entomology,130(5):309-313.
Tachibana S,Numata H,Goto S G.2005.Gene expression of heat-shock proteins(Hsp23,Hsp70 and Hsp90)during and after larval diapause in the blow fly Lucilia sericata[J].Journal of Insect Physiology,51(6):641-647.
Wang H,Li K,Zhu J Y,Fang Q,Ye G Y.2012.Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp,pteromalus puparum in response to environmental stresses[J].Archives of Insect Biochemistry and Physiology,79(4-5):247-263.
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