烟草HD-ZIP Ⅳ转录因子NtPDF2基因的克隆及表达分析
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摘要
为了明确NtPDF2在烟草中的生物学功能及其调控腺毛分化发育的作用机制,同源克隆了烟草NtPDF2基因,并对其序列、基因结构、进化关系以及表达模式进行分析。结果表明:NtPDF2 CDS全长为2 199 bp,编码732个氨基酸残基。烟草PDF2蛋白质序列高度保守,尤其是C端序列,均含有3个保守结构域(Homeobox domain,START domain和SAD domain)和1个保守LZ(Leucine zipper)元件。烟草PDF2基因结构高度保守,均由10个外显子和9个内含子组成。进化分析表明,普通烟草NtPDF2基因是由林烟草ML1基因进化而来。表达分析显示,烟草PDF2基因表达具有明显的时空特异性。干旱、黑暗和磷饥饿胁迫可显著下调NtPDF2基因的表达,而盐胁迫对其没有明显影响;NtPDF2基因的表达在赤霉素(GA)、茉莉酸甲酯(Me JA)、脱落酸(ABA)和打顶处理条件下均显著上调,这暗示烟草NtPDF2基因参与了调控多种非生物胁迫和激素应答过程。
To identify the biological functions of NtPDF2 and its mechanism in regulating trichome development in tobacco, NtPDF2 gene was cloned by homologous cloning strategy. The gene sequence, exon/intron structure, phylogenetic relationship and expression pattern of NtPDF2 were analyzed. The results showed that the full-length CDS sequence of NtPDF2 was 2 199 bp, which encoded 732 amino acids. The putative protein sequences of tobacco PDF2 were highly conserved, especially C-terminal which included three conserved domains(Homeobox domain, START domain and SAD domain) and one conserved LZ(Leucine zipper) motif. The gene structure of NtPDF2 was highly conserved, including 10 exons and 9 introns.Phylogenetic analysis revealed that NtPDF2 was evolved from Nsyl ML1 gene. The expression pattern of NtPDF2 had an obvious spatio-temporal specificity. The transcription level of NtPDF2 significantly decreased under drought, darkness and phosphate starvation, while it did not change obviously under salt stress. Furthermore,GA, ABA, Me JA and topping treatments significantly induced the expression of NtPDF2, which suggested that NtPDF2 might participate in plant responses to various abiotic stresses and phytohormones.
To identify the biological functions of NtPDF2 and its mechanism in regulating trichome development in tobacco, NtPDF2 gene was cloned by homologous cloning strategy. The gene sequence, exon/intron structure, phylogenetic relationship and expression pattern of NtPDF2 were analyzed. The results showed that the full-length CDS sequence of NtPDF2 was 2 199 bp, which encoded 732 amino acids. The putative protein sequences of tobacco PDF2 were highly conserved, especially C-terminal which included three conserved domains(Homeobox domain, START domain and SAD domain) and one conserved LZ(Leucine zipper) motif. The gene structure of NtPDF2 was highly conserved, including 10 exons and 9 introns.Phylogenetic analysis revealed that NtPDF2 was evolved from Nsyl ML1 gene. The expression pattern of NtPDF2 had an obvious spatio-temporal specificity. The transcription level of NtPDF2 significantly decreased under drought, darkness and phosphate starvation, while it did not change obviously under salt stress. Furthermore,GA, ABA, Me JA and topping treatments significantly induced the expression of NtPDF2, which suggested that NtPDF2 might participate in plant responses to various abiotic stresses and phytohormones.
引文
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[32]邹焱,苏以荣.打顶及施用植物生长调节剂对烟草内源激素的影响[J].烟草科技,2008(10):50-52,57.ZOU Yan,SU Yirong.Effects of topping and plant growth regulator applying on endogenous hormones in tobacco[J].Tobacco Science&Technology,2008(10):50-52,57.
[33]李珂.根系响应烟株打顶对烟碱生物合成的影响[D].郑州:河南农业大学,2012.LI Ke.The effect on nicotine biosynthesis in roots responding to tobacco topping[D].Zhengzhou:Henan Agriculture University,2012.
[34]Kosma D K,Bourdenx B,Bernard A,et al.The impact of water deficiency on leaf cuticle lipids of Arabidopsis[J].Plant Physiology,2009,151(4):1918-1929.
[35]Shan C M,Shangguan X X,Zhao B,et al.Control of cotton fibre elongation by a homeodomain transcription factor Gh HOX3[J].Nature Communications,2014,5:5519.
[36]Yang C X,Ye Z B.Trichomes as models for studying plant cell differentiation[J].Cellular and Molecular Life Sciences,2013,70(11):1937-1948.
[2]Nakamura M,Katsumata H,Abe M,et al.Characterization of the class IV homeodomain-leucine zipper gene family in Arabidopsis[J].Plant Physiology,2006,141(4):1363-1375.
[3]Abe M,Katsumata H,Komeda Y,et al.Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis[J].Development,2003,130(4):635-643.
[4]Chew W,Hrmova M,Lopato S.Role of homeodomain leucine zipper(HD-Zip)IV transcription factors in plant development and plant protection from deleterious environmental factors[J].International Journal of Molecular Sciences,2013,14(4):8122-8147.
[5]Nadakuduti S S,Pollard M,Kosma D K,et al.Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[J].Plant Physiology,2012,159(3):945-960.
[6]Schiefelbein J.Cell-fate specification in the epidermis:a common patterning mechanism in the root and shoot[J].Current Opinion in Plant Biology,2003,6(1):74-78.
[7]Serna L,Martin C.Trichomes:different regulatory networks lead to convergent structures[J].Trends in Plant Science,2006,11(6):274-280.
[8]Kubo H,Pereira A,Koornneef M.ANTHOCYANINLESS2,a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis[J].Plant Cell,1999,11(7):1217-1226.
[9]Di Cristina M,Sessa G,Dolan L,et al.The Arabidopsis Athb-10(GLABRA2)is an HD-Zip protein required for regulation of root hair development[J].The Plant Journal,1996,10(3):393-402.
[10]Lin Q,Ohashi Y,Kato M,et al.GLABRA2 directly suppresses basic helix-loop-helix transcription factor genes with diverse functions in root hair development[J].Plant Cell,2015,27(10):2894-2906.
[11]Wang X Y,Wang X L,Hu Q N,et al.Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis[J].The Plant Journal,2015,83(2):300-311.
[12]Guan X Y,Li Q J,Shan C M,et al.The HD-Zip IV gene Ga HOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2[J].Physiologia Plantarum,2008,134(1):174-182.
[13]Yu H,Chen X,Hong Y Y,et al.Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density[J].Plant Cell,2008,20(4):1134-1151.
[14]Javelle M,Vernoud V,Depege N,et al.Over-expression of the epidermis-specific HD-ZIP IV transcription factor OCL1 in maize identifies target genes involved in lipid metabolism and cuticle biosynthesis[J].Plant Physiology,2010,154(1):273-286.
[15]Vernoud V,Laigle G,Rozier F,et al.The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize[J].The Plant Journal,2009,59(6):883-894.
[16]Isaacson T,Kosma D K,Matas A J,et al.Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties,but not transpirational water loss[J].The Plant Journal,2009,60(2):363-377.
[17]Gao Y,Gao S H,Xiong C,et al.Comprehensive analysis and expression profile of the homeodomain leucine zipper IV transcription factor family in tomato[J].Plant Physiology and Biochemistry,2015,96:141-153.
[18]Yang C X,Li H X,Zhang J H,et al.A regulatory gene induces trichome formation and embryo lethality in tomato[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(29):11836-11841.
[19]史宏志,官春云.烟草腺毛分泌物的化学成分及遗传[J].作物研究,1995,9(3):46-49.SHI Hongzhi,GUAN Chunyun.Chemical components and genetics of glandular trichome secretions in tobacco[J].Crop Research,1995,9(3):46-49.
[20]刘燕,孙焕良,李辉,等.烟草腺毛密度和分泌物与烟叶品质的关系[J].作物研究,2012,26(6):737-739,748.LIU Yan,SUN Huanliang,LI Hui,et al.Relationship between the quality of tobacco and density and secretions of glandular trichome in tobacco leaves[J].Crop Research,2012,26(6):737-739,748.
[21]史宏志,刘国顺.烟草香味学[M].北京:中国农业出版社,1998:79-93.SHI Hongzhi,LIU Guoshun.Tobacco flavor[M].Beijing:China Agriculture Press,1998:79-93.
[22]Tso T C.Production,physiology,and biochemistry of tobacco plant[M].Maryland,USA:IDEALS,Inc.,Beltsville,1990.
[23]Li W Z,Cowley A,Uludag M,et al.The EMBL-EBI bioinformatics web and programmatic tools framework[J].Nucleic Acids Research,2015,43(W1):W580-W584.
[24]Wilkins M R,Gasteiger E,Bairoch A,et al.Protein identification and analysis tools in the Ex PASy server[M]//Link A J.2-D Proteome analysis protocols.Totowa,NJ:Humana Press,2005:531-552.
[25]Blom N,Sicheritz-pontén T,Gupta R,et al.Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence[J].Proteomics,2004,4(6):1633-1649.
[26]Letunic I,Doerks T,Bork P.SMART:recent updates,new developments and status in 2015[J].Nucleic Acids Research,2015,43(D1):D257-D260.
[27]Bailey T L,Boden M,Buske F A,et al.MEME Suite:tools for motif discovery and searching[J].Nucleic Acids Research,2009,37(S2):W202-W208.
[28]Finn R D,Attwood T K,Babbitt P C,et al.Inter Pro in2017—beyond protein family and domain annotations[J].Nucleic Acids Research,2017,45(D1):D190-D199.
[29]Hu B,Jin J P,Guo A Y,et al.GSDS 2.0:an upgraded gene feature visualization server[J].Bioinformatics,2014,31(8):1296-1297.
[30]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.
[31]Gómez-Cadenas A,De Ollas C,Manzi M,et al.Phytohormonal crosstalk under abiotic stress[M]//Tran L S,Pal S.Phytohormones:a window to metabolism,signaling and biotechnological applications.New York:Springer,2014.
[32]邹焱,苏以荣.打顶及施用植物生长调节剂对烟草内源激素的影响[J].烟草科技,2008(10):50-52,57.ZOU Yan,SU Yirong.Effects of topping and plant growth regulator applying on endogenous hormones in tobacco[J].Tobacco Science&Technology,2008(10):50-52,57.
[33]李珂.根系响应烟株打顶对烟碱生物合成的影响[D].郑州:河南农业大学,2012.LI Ke.The effect on nicotine biosynthesis in roots responding to tobacco topping[D].Zhengzhou:Henan Agriculture University,2012.
[34]Kosma D K,Bourdenx B,Bernard A,et al.The impact of water deficiency on leaf cuticle lipids of Arabidopsis[J].Plant Physiology,2009,151(4):1918-1929.
[35]Shan C M,Shangguan X X,Zhao B,et al.Control of cotton fibre elongation by a homeodomain transcription factor Gh HOX3[J].Nature Communications,2014,5:5519.
[36]Yang C X,Ye Z B.Trichomes as models for studying plant cell differentiation[J].Cellular and Molecular Life Sciences,2013,70(11):1937-1948.
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