柳枝稷PvbZIP8基因的克隆与表达分析
|
摘要
bZIP转录因子在植物非生物逆境胁迫的响应中发挥着重要作用,本研究通过同源克隆在柳枝稷(Panicum virgatum L.)中获得PvbZIP8基因,并对该基因进行了初步的生物信息学分析,同时利用荧光定量PCR技术进行了非生物胁迫下基因表达模式分析以及组织特异性表达分析。结果显示:基因的开放阅读框长度为468bp,编码155个氨基酸,分子式为C_(761)H_(1248)N_(24)O_(23)S_8,分子量为17.81kDa,为亲水性蛋白。系统进化分析表明该蛋白与哈氏黍(Panicum hallii)、谷子(Setaria italica)和糜子(Panicum miliaceum)相似性较高,并且具有典型的bZIP保守结构域,属于bZIP转录因子家族成员。定量PCR结果显示,PvbZIP8基因在盐、干旱、高温和低温胁迫下上调表达,在柳枝稷抗逆过程中发生作用。组织特异性表达分析表明,PvbZIP8在多个组织或器官均有表达,其中在根、茎、叶中表达量较高。本研究初步确定柳枝稷PvbZIP8基因响应抗逆性反应,并为进一步研究柳枝稷PvbZIP8基因的生物学功能奠定基础。
bZIP transcription factors play significant roles in regulating expression of functional genes in response to growth and development as well as environmental stress signaling.In this paper,the PvbZIP8gene was obtained by homologous cloning from switchgrass(Panicum virgatum L.).Subsequently,the bioinformatics analysis,tissue-specific expression analysis and gene expression patterns analysis under abiotic stress were carried out.The results showed that the length of open reading frame of PvbZIP8gene was 468 bp,encoding 155amino acids.In addition,PvbZIP8was predicted as a hydrophilic protein,and the molecular formula was C_(761)H_(1248)N_(24)O_(23)S_8and the molecular weight was 17.81k Da.Phylogenetic analysis indicated that the protein sequence was highly similar to Panicum hallii,Setaria italicaand Panicum miliaceum,and it contained the typical conserved bZIP domain and belonged to the bZIP transcription factor family.Quantitative PCR results showed that PvbZIP8gene was up-regulated under salt,drought,high temperature and low temperature stress,indicating key roles in response to abiotic stress in switchgrass.Tissue-specific expression analysis showed that PvbZIP8 was detected in different tissues and organs,with significantly high expressions in roots,stems and leaves.The investigation determined PvbZIP8 gene was related to abiotic stress tolerance and laid the foundation for further study of PvbZIP8 gene function.
bZIP transcription factors play significant roles in regulating expression of functional genes in response to growth and development as well as environmental stress signaling.In this paper,the PvbZIP8gene was obtained by homologous cloning from switchgrass(Panicum virgatum L.).Subsequently,the bioinformatics analysis,tissue-specific expression analysis and gene expression patterns analysis under abiotic stress were carried out.The results showed that the length of open reading frame of PvbZIP8gene was 468 bp,encoding 155amino acids.In addition,PvbZIP8was predicted as a hydrophilic protein,and the molecular formula was C_(761)H_(1248)N_(24)O_(23)S_8and the molecular weight was 17.81k Da.Phylogenetic analysis indicated that the protein sequence was highly similar to Panicum hallii,Setaria italicaand Panicum miliaceum,and it contained the typical conserved bZIP domain and belonged to the bZIP transcription factor family.Quantitative PCR results showed that PvbZIP8gene was up-regulated under salt,drought,high temperature and low temperature stress,indicating key roles in response to abiotic stress in switchgrass.Tissue-specific expression analysis showed that PvbZIP8 was detected in different tissues and organs,with significantly high expressions in roots,stems and leaves.The investigation determined PvbZIP8 gene was related to abiotic stress tolerance and laid the foundation for further study of PvbZIP8 gene function.
引文
[1]Zhao SX,Ming C,Lian CL,et al.Functions and application of the AP2/ERF transcription factor family in crop improvement[J].Journal of Integrative Plant Biology,2011,53(7):570-585
[2]Landschulz WH,Johnson PF,Mcknight SL.The leucine zipper:a hypothetical structure common to a new class of DNAbinding proteins[J].Science,1988,240(4860):1759-1764
[3]杨颖,高世庆,唐益苗,等.植物bZIP转录因子的研究进展[J].麦类作物学报,2009,29(4):730-737
[4]曹红利,岳川,王新超,等.bZIP转录因子与植物抗逆性研究进展[J].南方农业学报,2012,43(8):1094-1100
[5]Zou M,Guan Y,Ren H,et al.A bZIP transcription factor,OsABI5,is involved in rice fertility and stress tolerance[J].Plant Molecular Biology,2008,66(6):675-683
[6]Rook F,Gerrits N,Kortstee A,et al.Sucrose-specific signalling represses translation of the Arabidopsis ATB2bZIP transcription factor gene[J].Plant Journal,2010,15(2):253-263
[7]Martinez-Garcia JF,Moyano E,Alcocer MJC,et al.Two bZIPproteins from Antirrhinum flowers preferentially bind a hybrid C-box/G-box motif and help to define a new sub-family of bZIPtranscription factors[J].Plant Journal,1998,13(4):489-505
[8]Kusano T,Berberich T,Harada M,et al.A maize DNA-binding factor with a bZIP motif is induced by low temperature[J].Molecular and General Genetics,1995,248(5):507-517
[9]Chen H,Chen W,Zhou J,et al.Basic leucine zipper transcription factor OsbZIP16positively regulates drought resistance in rice[J].Plant Science,2012,193-194(3):8-17
[10]Liu C,Mao B,Ou S,et al.OsbZIP71,a bZIP transcription factor,confers salinity and drought tolerance in rice[J].Plant Molecular Biology,2014,84(1-2):19-36
[11]杜菲,陈新,杨春华,等.NaCl胁迫对不同柳枝稷材料种子萌发与幼苗生长的影响[J].草地学报,2011,19(6):1018-1024
[12]于晓丹,杜菲,张蕴薇.盐胁迫对柳枝稷种子萌发和幼苗生长的影响[J].草地学报,2010,18(6):810-815
[13]Fike JH,Parrish DJ,Wolf DD,et al.Long-term yield potential of switchgrass-for-biofuel systems[J].Biomass&Bioenergy,2006,30(3):198-206
[14]Kim S,Rayburn AL,Voigt T,et al.Salinity Effects on Germination and Plant Growth of Prairie Cordgrass and Switchgrass[J].Bioenergy Research,2012,5(1):225-235
[15]Barney JN,Mann JJ,Kyser GB,et al.Tolerance of switchgrass to extreme soil moisture stress:Ecological implications[J].Plant Science,2009,177(6):724-732
[16]朱毅,范希峰,侯新村,等.中性盐胁迫对柳枝稷苗期生长和生理特性的影响[J].草地学报,2015,23(2):1476-1480
[17]赵晓晓,谢坤良,贾冬冬,等.柳枝稷幼穗分化过程及其分期[J].草地学报,2019,27(1):170-177
[18]Panicum virgatum v1.0,DOE-JGI,http://phytozome.jgi.doe.gov/
[19]Gasteiger E,Hoogland C,Gattiker A,et al.Protein Identification and Analysis Tools on the ExPASy Server[J].The proteomics protocols handbook,2005
[20]Deléage G.ALIGNSEC:viewing protein secondary structure predictions within large multiple sequence alignments[J].Bioinformatics,2017,33(24):3991-3992
[21]Waterhouse A,Bertoni M,Bienert S,et al.SWISS-MODEL:homology modelling of protein structures and complexes[J].Nucleic Acids Research,2018,46(Web Server issue):W296-W303
[22]Nielsen H.Predicting Secretory Proteins with SignalP[J].Methods in Molecular Biology,2017,1611:59-73
[23]Paul H,Keun-Joon P,Takeshi O,et al.WoLF PSORT:protein localization predictor[J].Nucleic Acids Research,2007,35(Web Server):W585-W587
[24]Marchlerbauer A,Bo Y,Han L,et al.CDD/SPARCLE:functional classification of proteins via subfamily domain architectures[J].Nucleic Acids Research,2017,45(Database issue):D200-D203
[25]Thompson JD,Gibson TJ,Plewniak F,et al.The ClustalXwindows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Research[J].1997,25(24):4876-4882
[26]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
[27]Bailey TL,Mikael B,Buske FA,et al.MEME SUITE:tools for motif discovery and searching[J].Nucleic Acids Research,2009,37(Web Server issue):W202-W208
[28]Jacinta G,Nicholas E,Allen VD,et al.Selection and validation of reference genes for gene expression analysis in switchgrass(Panicum virgatum)using quantitative real-time RT-PCR[J].Plos One,2014,9(3):e91474
[29]Livaka KJ,Schmittgenb TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408
[30]Jakoby M,Weisshaar B,Droge-Laser W,et al.bZIP transcription factors in Arabidopsis[J].Trends in Plant Science,2002,7(3):106-111
[31]Nijhawan A,Jain M,Tyagi AK,et al.Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice[J].Plant Physiology,2008,146(2):333-350
[32]Wei KF,Chen J,Wang YM,et al.Genome-wide analysis of bZIP-encoding genes in maize[J].DNA Research,2012,19(6):463-476
[33]Wang JZ,Zhou JX,Zhang BL,et al.Genome-wide expansion and expression divergence of the basic leucine zipper transcription factors in higher plants with an emphasis on sorghum[J].Journal of Integrative Plant Biology,2011,53(3):212-231
[34]Zhang M,Liu YH,Shi H,et al.Evolutionary and expression analyses of soybean basic Leucine zipper transcription factor family[J].BMC Genomics,2018,19(1):159
[2]Landschulz WH,Johnson PF,Mcknight SL.The leucine zipper:a hypothetical structure common to a new class of DNAbinding proteins[J].Science,1988,240(4860):1759-1764
[3]杨颖,高世庆,唐益苗,等.植物bZIP转录因子的研究进展[J].麦类作物学报,2009,29(4):730-737
[4]曹红利,岳川,王新超,等.bZIP转录因子与植物抗逆性研究进展[J].南方农业学报,2012,43(8):1094-1100
[5]Zou M,Guan Y,Ren H,et al.A bZIP transcription factor,OsABI5,is involved in rice fertility and stress tolerance[J].Plant Molecular Biology,2008,66(6):675-683
[6]Rook F,Gerrits N,Kortstee A,et al.Sucrose-specific signalling represses translation of the Arabidopsis ATB2bZIP transcription factor gene[J].Plant Journal,2010,15(2):253-263
[7]Martinez-Garcia JF,Moyano E,Alcocer MJC,et al.Two bZIPproteins from Antirrhinum flowers preferentially bind a hybrid C-box/G-box motif and help to define a new sub-family of bZIPtranscription factors[J].Plant Journal,1998,13(4):489-505
[8]Kusano T,Berberich T,Harada M,et al.A maize DNA-binding factor with a bZIP motif is induced by low temperature[J].Molecular and General Genetics,1995,248(5):507-517
[9]Chen H,Chen W,Zhou J,et al.Basic leucine zipper transcription factor OsbZIP16positively regulates drought resistance in rice[J].Plant Science,2012,193-194(3):8-17
[10]Liu C,Mao B,Ou S,et al.OsbZIP71,a bZIP transcription factor,confers salinity and drought tolerance in rice[J].Plant Molecular Biology,2014,84(1-2):19-36
[11]杜菲,陈新,杨春华,等.NaCl胁迫对不同柳枝稷材料种子萌发与幼苗生长的影响[J].草地学报,2011,19(6):1018-1024
[12]于晓丹,杜菲,张蕴薇.盐胁迫对柳枝稷种子萌发和幼苗生长的影响[J].草地学报,2010,18(6):810-815
[13]Fike JH,Parrish DJ,Wolf DD,et al.Long-term yield potential of switchgrass-for-biofuel systems[J].Biomass&Bioenergy,2006,30(3):198-206
[14]Kim S,Rayburn AL,Voigt T,et al.Salinity Effects on Germination and Plant Growth of Prairie Cordgrass and Switchgrass[J].Bioenergy Research,2012,5(1):225-235
[15]Barney JN,Mann JJ,Kyser GB,et al.Tolerance of switchgrass to extreme soil moisture stress:Ecological implications[J].Plant Science,2009,177(6):724-732
[16]朱毅,范希峰,侯新村,等.中性盐胁迫对柳枝稷苗期生长和生理特性的影响[J].草地学报,2015,23(2):1476-1480
[17]赵晓晓,谢坤良,贾冬冬,等.柳枝稷幼穗分化过程及其分期[J].草地学报,2019,27(1):170-177
[18]Panicum virgatum v1.0,DOE-JGI,http://phytozome.jgi.doe.gov/
[19]Gasteiger E,Hoogland C,Gattiker A,et al.Protein Identification and Analysis Tools on the ExPASy Server[J].The proteomics protocols handbook,2005
[20]Deléage G.ALIGNSEC:viewing protein secondary structure predictions within large multiple sequence alignments[J].Bioinformatics,2017,33(24):3991-3992
[21]Waterhouse A,Bertoni M,Bienert S,et al.SWISS-MODEL:homology modelling of protein structures and complexes[J].Nucleic Acids Research,2018,46(Web Server issue):W296-W303
[22]Nielsen H.Predicting Secretory Proteins with SignalP[J].Methods in Molecular Biology,2017,1611:59-73
[23]Paul H,Keun-Joon P,Takeshi O,et al.WoLF PSORT:protein localization predictor[J].Nucleic Acids Research,2007,35(Web Server):W585-W587
[24]Marchlerbauer A,Bo Y,Han L,et al.CDD/SPARCLE:functional classification of proteins via subfamily domain architectures[J].Nucleic Acids Research,2017,45(Database issue):D200-D203
[25]Thompson JD,Gibson TJ,Plewniak F,et al.The ClustalXwindows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Research[J].1997,25(24):4876-4882
[26]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
[27]Bailey TL,Mikael B,Buske FA,et al.MEME SUITE:tools for motif discovery and searching[J].Nucleic Acids Research,2009,37(Web Server issue):W202-W208
[28]Jacinta G,Nicholas E,Allen VD,et al.Selection and validation of reference genes for gene expression analysis in switchgrass(Panicum virgatum)using quantitative real-time RT-PCR[J].Plos One,2014,9(3):e91474
[29]Livaka KJ,Schmittgenb TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408
[30]Jakoby M,Weisshaar B,Droge-Laser W,et al.bZIP transcription factors in Arabidopsis[J].Trends in Plant Science,2002,7(3):106-111
[31]Nijhawan A,Jain M,Tyagi AK,et al.Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice[J].Plant Physiology,2008,146(2):333-350
[32]Wei KF,Chen J,Wang YM,et al.Genome-wide analysis of bZIP-encoding genes in maize[J].DNA Research,2012,19(6):463-476
[33]Wang JZ,Zhou JX,Zhang BL,et al.Genome-wide expansion and expression divergence of the basic leucine zipper transcription factors in higher plants with an emphasis on sorghum[J].Journal of Integrative Plant Biology,2011,53(3):212-231
[34]Zhang M,Liu YH,Shi H,et al.Evolutionary and expression analyses of soybean basic Leucine zipper transcription factor family[J].BMC Genomics,2018,19(1):159