黄野螟精氨酸激酶基因的鉴定与表达分析
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摘要
为了探索精氨酸激酶(arginine kinase,AK)在昆虫中的功能,经筛选黄野螟(Heortia vitessoides)成虫转录组数据库获得黄野螟精氨酸激酶基因,命名为HvAK(GenBank:MH794282),对其进行生物信息学、各虫态和幼虫组织的定量分析,并通过检测HvAK在高温及低温胁迫条件下的表达水平,揭示HvAK基因在黄野螟生长发育中的作用。结果显示:该序列开放阅读框长1 068 bp,共编码355个氨基酸;同源比对结果表明HvAK与家蚕(Bombyx mori)AK基因同源性最高,为94.10%;实时荧光定量PCR结果显示,黄野螟在所检测的各幼虫组织和发育阶段均有表达,其中在头部的表达量较高,在5龄幼虫阶段到达表达量最高峰;此外,高、低温均诱导HvAK表达量上调,推测HvAK基因在黄野螟生长发育过程中可能参与抵御外界不良环境。
The arginine kinase in Heortia vitessoides was obtained by screening the adult H.vitessoides transcriptome database and named HvAK(GenBank:MH794282).Bioinformatic analyses of HvAK and expression of HvAK at various developmental stages and larval tissues were performed.The role of HvAK gene in the growth and development of H. vitessoides was studied by detecting the expression level of HvAK under high temperature and low temperature stress.The results showed that the HvAK cDNA contains an open reading frame with 1 068 bp,encoding a protein with 355 amino acid.Result of homologous alignment showed that the highest homology between HvAK and AK gene of Bombyx mori was 94.10%.The results of real-time quantitative PCR(RT-qPCR) showed that HvAK was expressed in all larval tissues and developmental stages tested,with the highest expression of HvAK in the head and in the 5 th instar larva.Both high and low temperature induced upregulation of HvAK expression,indicating that HvAK gene may be involved in resisting adverse external environment during H. vitessoides growth and development.
The arginine kinase in Heortia vitessoides was obtained by screening the adult H.vitessoides transcriptome database and named HvAK(GenBank:MH794282).Bioinformatic analyses of HvAK and expression of HvAK at various developmental stages and larval tissues were performed.The role of HvAK gene in the growth and development of H. vitessoides was studied by detecting the expression level of HvAK under high temperature and low temperature stress.The results showed that the HvAK cDNA contains an open reading frame with 1 068 bp,encoding a protein with 355 amino acid.Result of homologous alignment showed that the highest homology between HvAK and AK gene of Bombyx mori was 94.10%.The results of real-time quantitative PCR(RT-qPCR) showed that HvAK was expressed in all larval tissues and developmental stages tested,with the highest expression of HvAK in the head and in the 5 th instar larva.Both high and low temperature induced upregulation of HvAK expression,indicating that HvAK gene may be involved in resisting adverse external environment during H. vitessoides growth and development.
引文
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[13] CHENG J,WANG C Y,LYU Z H,et al.Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera:Crambidae) by antennal transcriptome analysis[J].Comparative biochemistry and physiology part D:genomics and proteomics,2019,29:117-130.
[14] 程杰,吕子豪,林同.黄野螟硫氧还蛋白过氧化物酶Tpx基因的鉴定及表达分析[J].华中农业大学学报,2018,37(2):56-63.
[15] CHENG J,WANG C Y,LYU Z H,et al.Multiple glutathione S-transferase genes in Heortia vitessoides (Lepidoptera:Crambidae):identification and expression patterns.[J].Journal of insect science,2018,18(3):1-10,23.
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[19] WU Q Y,LI F,WANG X Y.Evidence that amino-acid residues are responsible for substrate synergism of locust arginine kinase[J].Insect biochemistry and molecular biology,2008,38(1):59-65.
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[21] WANG H C,ZHANG L,ZHANG L,et al.Arginine kinase:differentiation of gene expression and protein activity in the red imported fire ant,Solenopsis invicta[J].Gene,2009,430(1/2):38-43.
[22] LIU F,WANG X D,ZHAO Y Y,et al.Silencing the HaAK gene by transgenic plant-mediated RNAi impairs larval growth of Helicoverpa armigera[J].International journal of biological sciences,2015,11(1):67-74.
[23] 于雪,曹新茹,高一夫,等.家蝇精氨酸激酶基因克隆及其在害虫防治上的应用[J].河北大学学报(自然科学版),2015,35(1):70-75.
[24] 赵伊英.黄曲条跳甲关键功能基因的克隆及利用RNAi技术控制害虫[D].福州:福建农林大学,2008.
[25] KUCHARSKI R,MALESZKA R.Arginine kinase is highly expressed in the compound eye of the honey bee,Apis mellifera[J].Gene,1998,211(2):343-349.
[26] 王华兵,徐豫松.家蚕精氨酸激酶基因的克隆、基因结构与表达分析[J].中国农业科学,2006,39(11):2354-2361.
[27] LI Y T,ZHAO Q,DUAN X L,et al.Transcription of four Rhopalosiphum padi (L.) heat shock protein genes and their responses to heat stress and insecticide exposure[J].Comparative biochemistry and physiology part A:molecular and integrative physiology,2017,205:48-57.
[28] COLINET H,NGUYEN T T,CLOUTIER C,et al.Proteomic profiling of a parasitic wasp exposed to constant and fluctuating cold exposure[J].Insect biochemistry and molecular biology,2007,37(11):1177-1188.
[29] CLARK M S,THORNE M A S,PURAC J,et al.Surviving the cold:molecular analyses of insect cryoprotective dehydration in the Arctic springtail Megaphorura arctica (Tullberg)[J].BMC genomics,2009,10:328-346.
[2] 张楠,徐贝贝,王建军.精氨酸激酶基因研究进展[J].环境昆虫学报,2017,39(3):730-734.
[3] UDA K,FUJIMOTO N,AKIYAMA Y,et al.Evolution of the arginine kinase genefamily[J].Comparative biochemistry and physiology part D:genomics and proteomics,2006,1(2):209-218.
[4] ZUNIGA F I,OCHOA G H,KELLY,S D,et al.S-crystallin and arginine kinase bind F-actin in light- and dark-adapted octopus retinas[J].Current eye research,2004,28(5):343-350.
[5] ROSENTHAL G A,DAHLMAN D L,ROBINSON G W.L-Arginine kinase from tobacco hornworm,Manduca sexta (L.):purification,properties,and interaction with L-canavanine[J].Journal of biological chemistry,1977,252(11):3679-3683.
[6] 张元臣,安世恒,原国辉.昆虫精氨酸激酶的研究进展[J].应用昆虫学报,2013,50(2):533-538.
[7] CHEN X,YAO P,CHU X,et al.Isolation of arginine kinase from Apis cerana cerana,and its possible involvement in response to adverse stress[J].Cell stress chaperones,2015,20(1):169-183.
[8] ZHU J Y,FANG Q,WANG L,et al.Proteomic analysis of the venom from the endoparasitoid wasp Pteromalus puparum (Hymenoptera:Pteromalidae)[J].Archives of insect biochemistry and physiology,2010,75(1):28-44.
[9] PEREIRA C A,ALONSO G D,IVALDI S,et al.Screening of substrate analogs as potential enzyme inhibitors for the arginine kinase of Trypanosoma cruzi[J].Journal of eukaryotic microbiology,2003,50(2):132-134.
[10] ZHAO Y Y,YANG G,WANG-PRUSKI G,et al.Phyllotreta striolata (Coleoptera:Chrysomelidae):arginine kinase cloning and RNAi-based pest control[J].European journal of entomology,2008,105(5):815-822.
[11] CHENG J,CHEN J,LIN T.De novo assembly and analysis of the Heortia vitessoides transcriptome via high-throughput I llumina sequencing[J].Journal of Asia-Pacific entomology,2017,20(4):1241-1248.
[12] CHENG J,WANG C Y,LYU Z H,et al.Identification and characterization of the catalase gene involved in resistance to thermal stress in Heortia vitessoides using RNA interference[J].Journal of thermal biology,2018,78:114-121.
[13] CHENG J,WANG C Y,LYU Z H,et al.Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera:Crambidae) by antennal transcriptome analysis[J].Comparative biochemistry and physiology part D:genomics and proteomics,2019,29:117-130.
[14] 程杰,吕子豪,林同.黄野螟硫氧还蛋白过氧化物酶Tpx基因的鉴定及表达分析[J].华中农业大学学报,2018,37(2):56-63.
[15] CHENG J,WANG C Y,LYU Z H,et al.Multiple glutathione S-transferase genes in Heortia vitessoides (Lepidoptera:Crambidae):identification and expression patterns.[J].Journal of insect science,2018,18(3):1-10,23.
[16] CARLSON C W,FINK S C,BROSEMER R W.Crystallization of glycerol 3-phosphate dehydrogenase,triosephosphate dehydrogenase,arginine kinase,and cytochrome c from a single extract of honeybees.[J].Archives of biochemistry and biophysics,1971,144(1):107-114.
[17] 赵洁,程婷婷,黄丽娜,等.棉铃虫精氨酸激酶(AK)的表达规律[J].农业生物技术学报,2016,24(3):397-405.
[18] ANAKA K,ICHINARI S,IWANAMI K,et al.Arginine kinase from the beetle Cissites cephalotes (Olivier):molecular cloning,phylogenetic analysis and enzymatic properties[J].Insect biochemistry and molecular biology,2007,37(4):338-345.
[19] WU Q Y,LI F,WANG X Y.Evidence that amino-acid residues are responsible for substrate synergism of locust arginine kinase[J].Insect biochemistry and molecular biology,2008,38(1):59-65.
[20] 王磊,黄体冉,李光栋,等.朱砂叶螨精氨酸激酶基因的克隆与表达特征[J].北京农学院学报,2018,33(3):16-23.
[21] WANG H C,ZHANG L,ZHANG L,et al.Arginine kinase:differentiation of gene expression and protein activity in the red imported fire ant,Solenopsis invicta[J].Gene,2009,430(1/2):38-43.
[22] LIU F,WANG X D,ZHAO Y Y,et al.Silencing the HaAK gene by transgenic plant-mediated RNAi impairs larval growth of Helicoverpa armigera[J].International journal of biological sciences,2015,11(1):67-74.
[23] 于雪,曹新茹,高一夫,等.家蝇精氨酸激酶基因克隆及其在害虫防治上的应用[J].河北大学学报(自然科学版),2015,35(1):70-75.
[24] 赵伊英.黄曲条跳甲关键功能基因的克隆及利用RNAi技术控制害虫[D].福州:福建农林大学,2008.
[25] KUCHARSKI R,MALESZKA R.Arginine kinase is highly expressed in the compound eye of the honey bee,Apis mellifera[J].Gene,1998,211(2):343-349.
[26] 王华兵,徐豫松.家蚕精氨酸激酶基因的克隆、基因结构与表达分析[J].中国农业科学,2006,39(11):2354-2361.
[27] LI Y T,ZHAO Q,DUAN X L,et al.Transcription of four Rhopalosiphum padi (L.) heat shock protein genes and their responses to heat stress and insecticide exposure[J].Comparative biochemistry and physiology part A:molecular and integrative physiology,2017,205:48-57.
[28] COLINET H,NGUYEN T T,CLOUTIER C,et al.Proteomic profiling of a parasitic wasp exposed to constant and fluctuating cold exposure[J].Insect biochemistry and molecular biology,2007,37(11):1177-1188.
[29] CLARK M S,THORNE M A S,PURAC J,et al.Surviving the cold:molecular analyses of insect cryoprotective dehydration in the Arctic springtail Megaphorura arctica (Tullberg)[J].BMC genomics,2009,10:328-346.