烟草甲两个羧酸酯酶基因的克隆及表达模式
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摘要
为了深入了解羧酸酯酶(Carboxylesterase,Car E)基因在烟草甲Lasioderma serricorne生长发育及响应CO_2气调胁迫方面的作用,在转录组测序基础上,利用RT-PCR技术克隆了烟草甲2个Car E基因c DNA全序列,对其分子特性和系统发育进行分析,并利用实时定量PCR技术检测其在不同发育阶段及不同φ(CO_2)气调胁迫条件下的表达模式。获得了烟草甲2个Car E基因完整的开放阅读框,其长度为1 698 bp和1 695 bp,编码565和564个氨基酸。氨基酸同源性和系统进化树分析表明,它们均具有羧酸酯酶典型的保守结构域,且属于β家族,分别命名为Ls Car EB1和Ls Car EB2(Gen Bank登录号:MG189601和MG189602)。Ls Car EB1和Ls Car EB2在烟草甲的各发育阶段均有所表达,其中在高龄幼虫期的表达量较高,显著高于低龄幼虫、蛹和成虫期的表达水平。LC10、LC30和LC503种φ(CO_2)处理6 h后,Ls Car EB1和Ls Car EB2的mRNA表达量随胁迫φ(CO_2)升高而上调且显著高于对照组。推测Ls Car EB1和Ls Car EB2不仅对烟草甲的生长发育具有重要作用,而且是其响应CO_2气调胁迫的重要机制之一。
In order to better understand the roles of carboxylesterase( Car E) genes in development and response to CO_2 exposure in the cigarette beetle,Lasioderma serricorne,we identified and cloned two fulllength c DNAs of Car E genes using transcriptome data and RT-PCR technique. We analyzed their molecular characterization and phylogenetic relationship,and detected their expression profiles in different developmental stages and under different CO_2 stress conditions by using quantitative real-time PCR( q PCR).The open reading frames of two genes were 1 698 bp and 1 695 bp,encoding proteins comprised of 565 and 564 amino acid residues,respectively. The amino acid sequence homology and phylogenetic tree analysis indicated that both of them have typical conserved motifs of Car E protein and belong to β subfamily,and termed as Ls Car EB1 and Ls Car EB2( Gen Bank accession no: MG189601 and MG189602),respectively. The q PCR results showed that two genes were both expressed in all tested developmental stages,and the relative mRNA expression levels were significantly higher in lately larvae than those in early larvae,pupae and adults. In L. serricorne larvae exposed to CO_2 for 6 h,the mRNA levels of Ls Car EB1 and Ls Car EB2 were significantly increased at different dose treatments compared to that of the control.We speculated that Ls Car EB1 and Ls Car EB2 may be involved in development and play important roles in response to CO_2 stress in L. serricorne.
In order to better understand the roles of carboxylesterase( Car E) genes in development and response to CO_2 exposure in the cigarette beetle,Lasioderma serricorne,we identified and cloned two fulllength c DNAs of Car E genes using transcriptome data and RT-PCR technique. We analyzed their molecular characterization and phylogenetic relationship,and detected their expression profiles in different developmental stages and under different CO_2 stress conditions by using quantitative real-time PCR( q PCR).The open reading frames of two genes were 1 698 bp and 1 695 bp,encoding proteins comprised of 565 and 564 amino acid residues,respectively. The amino acid sequence homology and phylogenetic tree analysis indicated that both of them have typical conserved motifs of Car E protein and belong to β subfamily,and termed as Ls Car EB1 and Ls Car EB2( Gen Bank accession no: MG189601 and MG189602),respectively. The q PCR results showed that two genes were both expressed in all tested developmental stages,and the relative mRNA expression levels were significantly higher in lately larvae than those in early larvae,pupae and adults. In L. serricorne larvae exposed to CO_2 for 6 h,the mRNA levels of Ls Car EB1 and Ls Car EB2 were significantly increased at different dose treatments compared to that of the control.We speculated that Ls Car EB1 and Ls Car EB2 may be involved in development and play important roles in response to CO_2 stress in L. serricorne.
引文
[1]OMAE Y,FUCHIKAWA T,NAKAYAMA S,et al.Life history and mating behavior of a black-bodied strain of the cigarette beetle Lasioderma serricorne(Coleoptera:Anobiidae)[J].Applied Entomology and Zoology,2012,47(2):157-163.
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[5]BELL C H.A review of insect responses to variations encountered in the managed storage environment[J].Journal of Stored Products Research,2014,59:260-274.
[6]TANAKA F,CHO R,HATA S,et al.Mortality modeling of adult Tribolium confusum,(Jacquelin du Val)exposed to different concentrations of carbon dioxide in a mixture with nitrogen[J].Journal of Stored Products Research,2016,69:113-118.
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[8]NAVARRO S,DIAS R,DONEHAYE E.Induced tolerance of Sitophilus oryzae adults to carbon dioxide[J].Journal of Stored Products Research,1985,21(4):207
[9]MAHBUB H M,AIKINS M J,WES S,et al.Efficacy of controlled atmosphere treatments to manage arthropod pests of dry-cured hams[J].Insects,2016,7(3):44.
[10]GUNASEKARAM N,RAJENDRAN S.Toxicity of carbon dioxide to drugstore beetle Stegobium paniceum and cigarette beetle Lasioderma serricorne[J].Journal of Stored Products Research,2005,41(3):283-294.
[11]MARSHALL S D,PUTTERILL J J,PLUMMERl K M,et al.The carboxylesterase gene family from Arabidopsis thaliana[J].Journal of Molecular Evolution,2003,57(5):487-500.
[12]YAN S,CUI F,QIAO C.Structure,function and applications of carboxylesterases from insects for insecticide resistance[J].Protein and Peptide Letters,2009,16(10):1181-1188.
[13]JEON J H,KIM S J,LEE H S,et al.Novel metagenome-derived carboxylesterase that hydrolyzesβ-lactam antibiotics[J].Applied and Environmental Microbiology,2011,77(21):7830-7836.
[14]LI X,SCHULER M A,BERENBAUM M R.Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics[J].Annual Review of Entomology,2007,52:231-253.
[15]PAN Y O,GUO H L,GAO X W.Carboxylesterase activity,c DNA sequence,and gene expression in malathion susceptible and resistant strains of the cotton aphid,Aphis gossypii[J].Comparative Biochemistry and Physiology(Part B),2009,152(3):266-270.
[16]MERLIN C,ROSELL G,CAROTSANS G,et al.Antennal esterase c DNAs from two pest moths,Spodoptera littoralis,potentially involved in odourant degradation[J].Insect Molecular Biology,2007,16(1):73-81.
[17]BISWAS S,REINHARD J,OAKESHOTT J,et al.Sensory regulation of Neuroligins and Neurexin 1 in the honeybee brain[J].PLo S ONE,2010,5(2):e9133.
[18]LI C,LI Z Z,CAO Y,et al.Partial characterization of stress-induced carboxylesterase from adults of Stegobium paniceum and Lasioderma serricorne(Coleoptera:Anobiidae)subjected to CO2-enriched atmosphere[J].Journal of Pest Science,2009,82(1):7-11.
[19]李灿,李子忠.气调胁迫下3种中药材储藏期害虫谷胱甘肽转移酶活性研究[J].植物保护,2009,35(2):91-94.LI C,LI Z Z.GSTs activity of three pests in stored Chinese medicinal materials[J].Plant Protection,2009,35(2):91-94.
[20]TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
[21]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[22]ADAMS M D,CELNIKER S E,HOLT R A.The genome sequence of Drosophila melanogaster[J].Science,2000,287(5461):2185-2195.
[23]RANSON H,CLAUDIANOS C,ORTELLI F,et al.Evolution of supergene families associated with insecticide resistance[J].Science,2002,298(5591):179-181.
[24]YU Q Y,LU C,LI W L,et al.Annotation and expression of carboxylesterases in the silkworm,Bombyx mori[J].BMC Genomics,2009,10(1):553.
[25]CLAUDIANOS C,RANSON H,JOHNSON R M,et al.A deficit of detoxification enzymes:pesticide sensitivity and environmental response in the honeybee[J].Insect Molecular Biology,2006,15(5):615-636.
[26]CRISTOFOLETTI P T,RIBERIO A F,DERAISON C,et al.Midgut adaption and digestive enzyme distribution in a phloem feeding insect,the pea aphid Acyrthosiphon pisum[J].Journal of Insect Physiology,2003,49(1):11-24.
[27]KARATOLOS N,PAUCHET Y,WILKINSON P,et al.Pyrosequencing the transcriptome of the greenhouse whitefly,Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes[J].BMC Genomics,2011,12(1):56.
[28]TSUBOTA T,SHIOTSUKI T.Genomic analysis of carboxyl/cholinesterase genes in the silkworm Bombyx mori[J].BMC Genomics,2010,11(1):377.
[29]刘娇,张建珍,李大琪,等.中华稻蝗羧酸酯酶家族基因生物信息学及组织表达特异性分析[J].中国农业科学,2015,48(21):4272-4284.LIU J,ZHANG J Z,LI D Q,et al.Bioinformatics and tissue-specific expression analysis of carboxylesterase genes from Oxya chinensis[J].Scientia Agricultura Sinica,2015,48(21):4272-4284.
[30]WANG L L,HUANG Y,LU X P,et al.Overexpression of twoα-esterase genes mediates metabolic resistance to malathion in the oriental fruit fly,Bactrocera dorsalis(Hendel)[J].Insect Molecular Biology,2015,24(4):467-479.
[31]OAKESHOTT J G,CLAUDIANOS C,CAMPBELL P M,et al.Biochemical genetics and genomics of insect esterases[M]∥GILBERT L I,IATROU K,GILL S.Comprehensive molecular insect science(Vol.5).Oxford,UK:Elsevier,2005:309-381.
[32]张建琴,葛娉婷,李大琪,等.飞蝗羧酸酯酶基因Lm Ces F1的时空表达及与杀虫剂耐受性的关系[J].中国农业科学,2014,47(8):1522-1530.ZHANG J Q,GE P T,LI D Q,et al.Spatio-temporal expression and insecticide tolerance analysis of carboxylesterase gene Lm Ces F1 from Locusta migratoria[J].Scientia Agricultura Sinica,2014,47(8):1522-1530.
[33]申光茂,豆威,王进军.橘小实蝇羧酸酯酶基因BdCAREB1的克隆及其表达模式解析[J].中国农业科学,2014,47(10):1947-1955.SHEN G M,DOU W,WANG J J.Cloning and expression analysis of a carboxylesterase gene Bd CAREB1 from Bactrocera dorsalis[J].Scientia Agricultura Sinica,2014,47(10):1947-1955.
[2]IMAI T,MASUDA R.Insecticidal activities of methyleugenol andβ-asarone,from the herbal medicines Saishin and Sekishōkon,and other alkoxy-propenyl-benzene derivatives against the cigarette beetle Lasioderma serricorne,(Coleoptera:Anobiidae)[J].Applied Entomology and Zoology,2017,52(2):183-188.
[3]PIMENTEL M A G,FARONILR D A,TOTOLA M R,et al.Phosphine resistance,respiration rate and witness consequences in stored-product insects[J].Pest Management Science,2007,63(9):876-881.
[4]HORI M,KASAISHI Y.Effects of the conditions of the cigarette beetle,Lasioderma serricorne(Fabricius)(Coleoptera:Anobiidae),on the knockdown time by phosphine fumigation[J].Applied Entomology and Zoology,2005,40(4):557-561.
[5]BELL C H.A review of insect responses to variations encountered in the managed storage environment[J].Journal of Stored Products Research,2014,59:260-274.
[6]TANAKA F,CHO R,HATA S,et al.Mortality modeling of adult Tribolium confusum,(Jacquelin du Val)exposed to different concentrations of carbon dioxide in a mixture with nitrogen[J].Journal of Stored Products Research,2016,69:113-118.
[7]HUSAIN M,SUKIRNO S,MEHMOOD K,et al.Effectiveness of carbon dioxide against different developmental stages of Cadra cautella and Tribolium castaneum[J].Environmental Science and Pollution Research,2017,24(14):12787-12795.
[8]NAVARRO S,DIAS R,DONEHAYE E.Induced tolerance of Sitophilus oryzae adults to carbon dioxide[J].Journal of Stored Products Research,1985,21(4):207
[9]MAHBUB H M,AIKINS M J,WES S,et al.Efficacy of controlled atmosphere treatments to manage arthropod pests of dry-cured hams[J].Insects,2016,7(3):44.
[10]GUNASEKARAM N,RAJENDRAN S.Toxicity of carbon dioxide to drugstore beetle Stegobium paniceum and cigarette beetle Lasioderma serricorne[J].Journal of Stored Products Research,2005,41(3):283-294.
[11]MARSHALL S D,PUTTERILL J J,PLUMMERl K M,et al.The carboxylesterase gene family from Arabidopsis thaliana[J].Journal of Molecular Evolution,2003,57(5):487-500.
[12]YAN S,CUI F,QIAO C.Structure,function and applications of carboxylesterases from insects for insecticide resistance[J].Protein and Peptide Letters,2009,16(10):1181-1188.
[13]JEON J H,KIM S J,LEE H S,et al.Novel metagenome-derived carboxylesterase that hydrolyzesβ-lactam antibiotics[J].Applied and Environmental Microbiology,2011,77(21):7830-7836.
[14]LI X,SCHULER M A,BERENBAUM M R.Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics[J].Annual Review of Entomology,2007,52:231-253.
[15]PAN Y O,GUO H L,GAO X W.Carboxylesterase activity,c DNA sequence,and gene expression in malathion susceptible and resistant strains of the cotton aphid,Aphis gossypii[J].Comparative Biochemistry and Physiology(Part B),2009,152(3):266-270.
[16]MERLIN C,ROSELL G,CAROTSANS G,et al.Antennal esterase c DNAs from two pest moths,Spodoptera littoralis,potentially involved in odourant degradation[J].Insect Molecular Biology,2007,16(1):73-81.
[17]BISWAS S,REINHARD J,OAKESHOTT J,et al.Sensory regulation of Neuroligins and Neurexin 1 in the honeybee brain[J].PLo S ONE,2010,5(2):e9133.
[18]LI C,LI Z Z,CAO Y,et al.Partial characterization of stress-induced carboxylesterase from adults of Stegobium paniceum and Lasioderma serricorne(Coleoptera:Anobiidae)subjected to CO2-enriched atmosphere[J].Journal of Pest Science,2009,82(1):7-11.
[19]李灿,李子忠.气调胁迫下3种中药材储藏期害虫谷胱甘肽转移酶活性研究[J].植物保护,2009,35(2):91-94.LI C,LI Z Z.GSTs activity of three pests in stored Chinese medicinal materials[J].Plant Protection,2009,35(2):91-94.
[20]TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
[21]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[22]ADAMS M D,CELNIKER S E,HOLT R A.The genome sequence of Drosophila melanogaster[J].Science,2000,287(5461):2185-2195.
[23]RANSON H,CLAUDIANOS C,ORTELLI F,et al.Evolution of supergene families associated with insecticide resistance[J].Science,2002,298(5591):179-181.
[24]YU Q Y,LU C,LI W L,et al.Annotation and expression of carboxylesterases in the silkworm,Bombyx mori[J].BMC Genomics,2009,10(1):553.
[25]CLAUDIANOS C,RANSON H,JOHNSON R M,et al.A deficit of detoxification enzymes:pesticide sensitivity and environmental response in the honeybee[J].Insect Molecular Biology,2006,15(5):615-636.
[26]CRISTOFOLETTI P T,RIBERIO A F,DERAISON C,et al.Midgut adaption and digestive enzyme distribution in a phloem feeding insect,the pea aphid Acyrthosiphon pisum[J].Journal of Insect Physiology,2003,49(1):11-24.
[27]KARATOLOS N,PAUCHET Y,WILKINSON P,et al.Pyrosequencing the transcriptome of the greenhouse whitefly,Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes[J].BMC Genomics,2011,12(1):56.
[28]TSUBOTA T,SHIOTSUKI T.Genomic analysis of carboxyl/cholinesterase genes in the silkworm Bombyx mori[J].BMC Genomics,2010,11(1):377.
[29]刘娇,张建珍,李大琪,等.中华稻蝗羧酸酯酶家族基因生物信息学及组织表达特异性分析[J].中国农业科学,2015,48(21):4272-4284.LIU J,ZHANG J Z,LI D Q,et al.Bioinformatics and tissue-specific expression analysis of carboxylesterase genes from Oxya chinensis[J].Scientia Agricultura Sinica,2015,48(21):4272-4284.
[30]WANG L L,HUANG Y,LU X P,et al.Overexpression of twoα-esterase genes mediates metabolic resistance to malathion in the oriental fruit fly,Bactrocera dorsalis(Hendel)[J].Insect Molecular Biology,2015,24(4):467-479.
[31]OAKESHOTT J G,CLAUDIANOS C,CAMPBELL P M,et al.Biochemical genetics and genomics of insect esterases[M]∥GILBERT L I,IATROU K,GILL S.Comprehensive molecular insect science(Vol.5).Oxford,UK:Elsevier,2005:309-381.
[32]张建琴,葛娉婷,李大琪,等.飞蝗羧酸酯酶基因Lm Ces F1的时空表达及与杀虫剂耐受性的关系[J].中国农业科学,2014,47(8):1522-1530.ZHANG J Q,GE P T,LI D Q,et al.Spatio-temporal expression and insecticide tolerance analysis of carboxylesterase gene Lm Ces F1 from Locusta migratoria[J].Scientia Agricultura Sinica,2014,47(8):1522-1530.
[33]申光茂,豆威,王进军.橘小实蝇羧酸酯酶基因BdCAREB1的克隆及其表达模式解析[J].中国农业科学,2014,47(10):1947-1955.SHEN G M,DOU W,WANG J J.Cloning and expression analysis of a carboxylesterase gene Bd CAREB1 from Bactrocera dorsalis[J].Scientia Agricultura Sinica,2014,47(10):1947-1955.