小桐子14-3-3基因家族的鉴定及低温胁迫应答
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摘要
14-3-3蛋白家族在真核生物中广泛存在且高度保守,以同源/异源二聚体形式与2个靶蛋白或1个靶蛋白的两个磷酸化Ser/Thr模体结构域结合来发挥其调控作用。以拟南芥14-3-3蛋白为探针序列,对小桐子蛋白质数据库进行搜索,共鉴定到9个14-3-3基因,其中ε类4个、非ε类5个,并对其理化性质、系统进化、基因结构、顺式作用元件及低温胁迫表达等进行了分析。结果显示,小桐子14-3-3基因在进化上较为保守,存在该家族中典型的9段α-螺旋结构,ε类基因结构包含7个外显子,非ε类包含4~5个外显子。另外,在9个14-3-3基因上游调控区都鉴定到多个激素与逆境胁迫应答元件。qRT-PCR分析结果显示,包含有低温应答元件的Jc14-3-3b与Jc14-3-3h基因在小桐子的各组织器官中都有表达,但存在组织表达特异性,都在茎中表达量较高,其次是根,而在叶中表达量相对较低。同时,Jc14-3-3b与Jc14-3-3h基因都属于低温诱导表达基因,分别在茎与根中低温胁迫0.5~3.0h达到最大表达量,与小桐子的抗冷性形成直接相关。本研究为小桐子14-3-3基因家族的克隆与功能验证提供了参考。
The family of 14-3-3 proteins,which acts as homodimers or heterodimers,exists in all eukaryotic organisms with highly conserved sequence. The regulatory function of 14-3-3 proteins is mainly mediated by phosphoerine/phosphothreonine motifs with two targeting proteins or two different domains in one targeting protien simultaneously. Based on the conserved domains of Arabidopsis thaliana 14-3-3 protein as probe sequences,nine 14-3-3 genes including 4 ε-type and 5 non-ε type were identified from the Jatropha curcas protein database,and the physical and chemical characteristics,evolutionary relationship,gene structure,cis-elements,and chilling stress response were systematically analyzed. The result showed that 14-3-3 protein in J. curcas were highly conserved and classical 9-α-helix were found in all 14-3-3 proteins' secondary structures. Seven exons were included in ε-type gene structure and 4-5 exons were in non-ε type. Multiple ciselements responsive to different homones and environmental stresses were found in upstream sequences of each 14-3-3 genes in J. curcas. Quantitative RT-PCR revealed that Jc14-3-3 b and Jc14-3-3 h were expressed differently in different tissues,abundant in stem and root,scarce in leaf. Chilling responsive genes Jc14-3-3 b and Jc14-3-3 h reached the highest expression level in stem and root after 0.5-3. 0 h chilling stress induction.
The family of 14-3-3 proteins,which acts as homodimers or heterodimers,exists in all eukaryotic organisms with highly conserved sequence. The regulatory function of 14-3-3 proteins is mainly mediated by phosphoerine/phosphothreonine motifs with two targeting proteins or two different domains in one targeting protien simultaneously. Based on the conserved domains of Arabidopsis thaliana 14-3-3 protein as probe sequences,nine 14-3-3 genes including 4 ε-type and 5 non-ε type were identified from the Jatropha curcas protein database,and the physical and chemical characteristics,evolutionary relationship,gene structure,cis-elements,and chilling stress response were systematically analyzed. The result showed that 14-3-3 protein in J. curcas were highly conserved and classical 9-α-helix were found in all 14-3-3 proteins' secondary structures. Seven exons were included in ε-type gene structure and 4-5 exons were in non-ε type. Multiple ciselements responsive to different homones and environmental stresses were found in upstream sequences of each 14-3-3 genes in J. curcas. Quantitative RT-PCR revealed that Jc14-3-3 b and Jc14-3-3 h were expressed differently in different tissues,abundant in stem and root,scarce in leaf. Chilling responsive genes Jc14-3-3 b and Jc14-3-3 h reached the highest expression level in stem and root after 0.5-3. 0 h chilling stress induction.
引文
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[18]VAN DEN WIGNGAARD P W,SINNIGE M P,ROOBEEK I,et al.Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root[J].Plant J,2005,41(1):43-55.
[19]GAMPALA S S,KIM T W,HE J X,et al.An essential role for14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis[J].Dev Cell,2007,13(2):177-189.
[20]RYU H,CHO H,KIM K,et al.Phosphorylation dependent nucleocytoplasmic shuttling of BES1 is a key regulatory event in brassinosteroid signaling[J].Molecular Cells,2010,29(3):283-290.
[21]SOLANO R,ECKER J R.Ethylene gas:perception,signaling and response[J].Curr Opin Plant Biol,1998,1(5):393-398.
[22]FOLTA K M,PAUL A L,MAYFIELD J D,et al.14-3-3 isoforms participate in red light signaling and photoperiodic flowering[J].Plant Signal Behav,2008,3(5):304-306.
[23]HAYASHI M,INOUE S,TAKAHASHI K,et al.Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells[J].Plant Cell Physiol,2011,52(7):1238-1248.
[24]PIGNOCCHI C,DOONAN J H.Interaction of a 14-3-3 protein with the plant microtubule-associated protein EDE1[J].Ann Bot,2011,107(7):1103-1109.
[25]KANCZEWSKA J,MARCO S,VANDERRMEEREN C,et al.Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 protein converts a dimmer into a hexamer[J].P Natl Acad Sci USA,2005,102(33):11675-11680.
[26]SHIN R,ALVAREZ S,BURCH A Y,et al.Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermentinglike kinase SnRK2.8 reveals a connection to metabolic processes[J].P Natl Acad Sci USA,2007,104(15):6460-6465.
[27]KIDOU S I,UMEDA M,KATO A,et al.Isolation and characterization of a rice c DNA similar to the bovine brain-specific 14-3-3protein gene[J].Plant Molar Biol,1993,21(1):191-194.
[28]YAO Y,DU Y,JIANG L,et al.Molecular analysis and expression patterns of the 14-3-3 gene family from Oryza sativa[J].J Biochem Mol Biol,2007,40(3):349-357.
[29]LI X Y,DHAUBHADEL S.Soybean 14-3-3 gene family:identification and molecular characterization[J].Planta,2011,322(3):569-582.
[30]XU W F,SHI W M.Expression profiling of the 14-3-3 gene family in response to salt and potassium and iron deficiencies in young tomato(Solanum lycopersicum)roots:analysis by real-time RT-PCR[J].Ann Bot,2006,98(5):965-974.
[31]李忠光,龚明.不同化学消毒剂对小桐子种子萌发和幼苗生长的影响[J].种子,2010,30(2):4-7,12.
[32]SUN G,XIE F,ZHANG B.Transcriptome-wide identification and stress properties of the 14-3-3 gene family in cotton(Gossypium hirsutum L.)[J].Funct Integr Genomics,2011,11(4):627-636.
[33]YAN J Q,HE C X,WANG J,et al.Overexpression of the Arabidopsis 14-3-3 protein GF14λin cotton leads to a‘Stay-Green’phenotype andimproves stress tolerance under moderate drought conditions[J].Plant Cell Physiol,2004,45(8):1007-1014.
[34]MAGNUS A,PAUL C,SEHNKE,et al.Plasma membrane H+-ATPase and 14-3-3 isoforms of Arabidopsis leaves:evidence for isoform specificity in the 14-3-3/H+-ATPase interaction[J].Plant Cell Physiol,2004,45(9):1202-1210.
[35]JAHN T,FUGLSANG A T,OLSSON A,et al.The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H+-ATPase[J].Plant Cell,1997,9(10):1805-1814.
[36]OLSSON A,SVENNELID F,EK B,et al.A phosphothreonine residue at the C-terminal end of the plasma membrane H+-ATPase is protected by fusicoccin-induced 14-3-3 binding[J].Plant Physiol,1998,118(2):551-555.
[37]YANG Z M,NIAN H,SIVAGURU M,et al.Characterization of aluminum-induced citrate secretion in aluminum tolerant soybean(Glycine max L.)plants[J].Physiol Plantarum,2001,113(1):64-71.
[2]YANG C Y,FANG Z,LI B,et al.Review and prospects of Jatropha biodiesel industry in China[J].Renew Sust Energ Rev,2012,16(4):2178-2190.
[3]LIN J,ZHOU X,TANG K X,et al.A survey of the studies on the resources of Jatropha curcas L.[J].J Trop Subtrop Bot,2004,12(3):285-290.
[4]何璐,虞泓,范源洪,等.麻疯树(Jatropha curcas L.)植物学研究进展[J].长江流域资源与环境,2010,19(S1):120-127.
[5]王海燕,文明富,刘石生,等.麻疯树生物学研究进展及其开发利用[J].热带作物学报,2010,31(4):670-675.
[6]PAULETTE M.14-3-3 proteins-an update[J].Cell Res,2005,15(4):228-236.
[7]MOORE B W,PEREZ V J.Specific acidic proteins of the nervous system[M].Englewood Cliffs:Prentice Hall,1968:343-359.
[8]RANDT J,THORDAL-CHRISTENSEN H,VAD K,et al.A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator[J].P lant J,1992,2(5):815-820.
[9]WU K,ROONEY M F,FERL R J.The Arabidopsis 14-3-3 multigene family[J].Plant physiol,1997,114(4):1421-1431.
[10]YANG X W,LEE W H,SOBOTT F,et al.Structural basis for protein-protein interactions in the 14-3-3 protein family[J].P Natl Acad Sci USA,2006,103(46):17237-17242.
[11]GANGULY S,WELLER J L,HO A,et al.Melatonin synthesis:14-3-3-dependent activation and inhibition of arylalkylamine Nacetyltransferase mediated by phosphoserine-205[J].P Natl Acad Sci USA,2005,102(4):1222-1227.
[12]COBLITZ B,WU M,SHIKANO S,et al.C-terminal binding:an expanded repertoire and function of 14-3-3 proteins[J].FEBS Lett,2006,580(6):1531-1535.
[13]ANDREWS R K,HARRIS S J,MCNALLY T,et al.Binding of purified 14-3-3ζsignaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex[J].Biochem,1998,37(2):638-647.
[14]PETOSA C,MASTERS S C,BANKSTON L A,et al.14-3-3ζbinds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove[J].J Biol Chemy,1998,273(26):16305-16310.
[15]COMPAROT S,LINGIGH G,MARTIN T.Function and specificity of 14-3-3 proteins in the regulation of carbohydrate and nitrogen metabolism[J].J Exp Bot,2003,54(382):595-604.
[16]ISHIDA S,FUKAZAWA J,YUASA T,et al.Involvement of 14-3-3 signaling protein binding in the functional regulation of the transcriptional activator repression of shoot growth by gibberellins[J].Plant Cell,2004,16(10):2641-2651.
[17]DEL VISO F,CASARETTO J A,QUATRANO R S.14-3-3 proteins are components of the transcription complex of the ATEM1promoter in Arabidopsis[J].Planta,2007,227(1):167-175.
[18]VAN DEN WIGNGAARD P W,SINNIGE M P,ROOBEEK I,et al.Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root[J].Plant J,2005,41(1):43-55.
[19]GAMPALA S S,KIM T W,HE J X,et al.An essential role for14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis[J].Dev Cell,2007,13(2):177-189.
[20]RYU H,CHO H,KIM K,et al.Phosphorylation dependent nucleocytoplasmic shuttling of BES1 is a key regulatory event in brassinosteroid signaling[J].Molecular Cells,2010,29(3):283-290.
[21]SOLANO R,ECKER J R.Ethylene gas:perception,signaling and response[J].Curr Opin Plant Biol,1998,1(5):393-398.
[22]FOLTA K M,PAUL A L,MAYFIELD J D,et al.14-3-3 isoforms participate in red light signaling and photoperiodic flowering[J].Plant Signal Behav,2008,3(5):304-306.
[23]HAYASHI M,INOUE S,TAKAHASHI K,et al.Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells[J].Plant Cell Physiol,2011,52(7):1238-1248.
[24]PIGNOCCHI C,DOONAN J H.Interaction of a 14-3-3 protein with the plant microtubule-associated protein EDE1[J].Ann Bot,2011,107(7):1103-1109.
[25]KANCZEWSKA J,MARCO S,VANDERRMEEREN C,et al.Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 protein converts a dimmer into a hexamer[J].P Natl Acad Sci USA,2005,102(33):11675-11680.
[26]SHIN R,ALVAREZ S,BURCH A Y,et al.Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermentinglike kinase SnRK2.8 reveals a connection to metabolic processes[J].P Natl Acad Sci USA,2007,104(15):6460-6465.
[27]KIDOU S I,UMEDA M,KATO A,et al.Isolation and characterization of a rice c DNA similar to the bovine brain-specific 14-3-3protein gene[J].Plant Molar Biol,1993,21(1):191-194.
[28]YAO Y,DU Y,JIANG L,et al.Molecular analysis and expression patterns of the 14-3-3 gene family from Oryza sativa[J].J Biochem Mol Biol,2007,40(3):349-357.
[29]LI X Y,DHAUBHADEL S.Soybean 14-3-3 gene family:identification and molecular characterization[J].Planta,2011,322(3):569-582.
[30]XU W F,SHI W M.Expression profiling of the 14-3-3 gene family in response to salt and potassium and iron deficiencies in young tomato(Solanum lycopersicum)roots:analysis by real-time RT-PCR[J].Ann Bot,2006,98(5):965-974.
[31]李忠光,龚明.不同化学消毒剂对小桐子种子萌发和幼苗生长的影响[J].种子,2010,30(2):4-7,12.
[32]SUN G,XIE F,ZHANG B.Transcriptome-wide identification and stress properties of the 14-3-3 gene family in cotton(Gossypium hirsutum L.)[J].Funct Integr Genomics,2011,11(4):627-636.
[33]YAN J Q,HE C X,WANG J,et al.Overexpression of the Arabidopsis 14-3-3 protein GF14λin cotton leads to a‘Stay-Green’phenotype andimproves stress tolerance under moderate drought conditions[J].Plant Cell Physiol,2004,45(8):1007-1014.
[34]MAGNUS A,PAUL C,SEHNKE,et al.Plasma membrane H+-ATPase and 14-3-3 isoforms of Arabidopsis leaves:evidence for isoform specificity in the 14-3-3/H+-ATPase interaction[J].Plant Cell Physiol,2004,45(9):1202-1210.
[35]JAHN T,FUGLSANG A T,OLSSON A,et al.The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H+-ATPase[J].Plant Cell,1997,9(10):1805-1814.
[36]OLSSON A,SVENNELID F,EK B,et al.A phosphothreonine residue at the C-terminal end of the plasma membrane H+-ATPase is protected by fusicoccin-induced 14-3-3 binding[J].Plant Physiol,1998,118(2):551-555.
[37]YANG Z M,NIAN H,SIVAGURU M,et al.Characterization of aluminum-induced citrate secretion in aluminum tolerant soybean(Glycine max L.)plants[J].Physiol Plantarum,2001,113(1):64-71.