苹果MdWRKY53基因克隆和功能鉴定
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摘要
利用拟南芥WRKY53蛋白序列在苹果基因组中进行比对得到一个得分最高、同源性最高的序列,将其命名为MdWRKY53 (MDP0000299114)。根据MdWRKY53基因序列设计引物,从‘皇家嘎啦’(Malus×domestica)苹果苗上克隆得到一个约1000 bp的基因条带,测序发现该基因包含长为1056 bp的完整开放阅读框,编码351个氨基酸,和苹果基因组预测序列相符。进化树分析表明苹果WRKY53与白梨WRKY53亲缘关系最近,且发现MdWRKY53含有一个保守的WRKY结构域。荧光定量PCR分析表明, MdWRKY53在苹果的不同组织中均有表达,并且在果实中表达量最高,茎中表达量最低,并且MdWRKY53能够响应低温、盐胁迫、脱落酸、水杨酸、赤霉素和生长素信号。
The Arabidopsis WRKY53 protein sequence was used as a query to search its homologue in the apple genome. We obtained a sequence with the highest score and highest homology, which was named as MdWRKY53(MDP0000299114). The MdWRKY53 gene contained a complete open reading frame of 1056 bp in length and encoded 351 amino acids, which was consistent with the predicted sequence of the apple genome.Phylogenetic analysis showed that the sequence of MdWRKY53 exhibited the closest relationship with PbWRKY53 in pear. The MdWRKY53 contained a conserved WRKY domain. Quantitative real-time PCR analysis showed that MdWRKY53 was expressed in different apple tissues, and the expression was the highest in apple fruit and the lowest in stem. At the same time, it was found that MdWRKY53 could respond to low temperature, salt stress, abscisic acid, salicylic acid, gibberellin, and auxin signal.
The Arabidopsis WRKY53 protein sequence was used as a query to search its homologue in the apple genome. We obtained a sequence with the highest score and highest homology, which was named as MdWRKY53(MDP0000299114). The MdWRKY53 gene contained a complete open reading frame of 1056 bp in length and encoded 351 amino acids, which was consistent with the predicted sequence of the apple genome.Phylogenetic analysis showed that the sequence of MdWRKY53 exhibited the closest relationship with PbWRKY53 in pear. The MdWRKY53 contained a conserved WRKY domain. Quantitative real-time PCR analysis showed that MdWRKY53 was expressed in different apple tissues, and the expression was the highest in apple fruit and the lowest in stem. At the same time, it was found that MdWRKY53 could respond to low temperature, salt stress, abscisic acid, salicylic acid, gibberellin, and auxin signal.
引文
Assmann SM(2003).OPEN STOMATAl opens the door to ABA signaling in Arabidopsis guard cells.Trends Plant Sci,8(4):151-153
Chen JN,Nolan TM,Ye HX,et al(2017a).Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses.Plant Cell,29(6):1425-1439
Chen LG,Xiang SY,Chen YL,et al(2017b).Arabidopsis WRKY45 interacts with the DELLA protein RGL1 to positively regulate age-triggered leaf senescence.Mol Plant,10(9):1174-1189
Dai WH,Wang M,Gong XQ,et al(2018).The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs.New Phytol,219(3):972-989
De Storme N,Geelen D(2014).The impact of environmental stress on male reproductive development in plants:biological processes and molecular mechanisms.Plant Cell Environ,37(1):1-18
Ding WW,Fang WB,Shi SY,et al(2016).Wheat WRKY type transcription factor gene TaWRKY1 is essential in mediating drought tolerance associated with an ABA-dependent pathway.Plant Mol Biol Rep,34(6):1111-1126
Eulgem T,Rushton PJ,Robatzek S,et al(2000).The WRKYsuperfamily of plant transcription factors.Trends Plant Sci,5(5):199-206
Fan HK,Wang F,Gao H,et al(2011).Pathogen-induced MdWRKY1 in‘Qinguan’apple enhances disease resistance.JPlant Biol,54(3):150-158
Fan J,Hill L,Crooks C,et al(2009).Abscisic acid has a key role in modulating diverse plant-pathogen interactions.Plant Physiol,150(4):1750-1761
Hu DG,Sun MH,Sun CH,et al(2015).Conserved vacuolar H+-ATPase subunit B1 improves salt stress tolerance in apple calli and tomato plants.Sci Hortic,197:107-116
Hu YR,Dong QY,Yu DQ(2012).Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae.Plant Sci,185:288-297
Huang Y,Feng CZ,Ye Q,et al(2016).Arabidopsis WRKY6transcription factor acts as a positive regulator of abscisic acid signaling during seed germination and early seedling development.PLoS Genet,12(2):e1005833
Jiang YJ,Yu DQ(2016).WRKY57 regulates JAZ genes transcriptionally to compromise Botrytis cinerea resistance in Arabidopsis thaliana.Plant Physiol,doi:10.1104/pp.16.00747
Jin WH,Zhou Q,Wei YF,et al(2017).NtWRKY-R1,a novel transcription factor,integrates IAA and JA signal pathway by topping damage in Nicotiana tabacum.Front Plant Sci,8:2263-2273
Kim CY,Vo KTX,Nguyen CD,et al(2016).Functional analysis of a cold-responsive rice WRKY gene,OsWRKY71.Plant Biotechnol Rep,10(1):13-23
LagacéM,Matton DP(2004).Characterization of a WRKYtranscription factor expressed in late torpedo-stage embryos of Solanum chacoense.Planta,219(1):185-189
Li SJ,Fu QT,Chen LG,et al(2011).Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance.Planta,233(6):1237-1252
Li W,Wang HP,Yu DQ(2016).Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions.Mol Plant,9(11):1492-1503
Liu S,Kracher B,Ziegler J,et al(2015).Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100.eLife,4:e07295
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25(4):402-408
Popko J,H?nsch R,Mendel RR,et al(2010).The role of abscisic acid and auxin in the response of poplar to abiotic stress.Plant Biol,12(2):242-258
Robert-Seilaniantz A,Navarro L,Bari R,et al(2007).Pathological hormone imbalances.Curr Opin Plant Biol,10(4):372-379
Suzuki N,Rivero RM,Shulaev V,et al(2014).Abiotic and biotic stress combinations.New Phytol,203(1):32-43
Ton J,Flors V,Mauch-Mani B(2009).The multifaceted role of ABA in disease resistance.Trends Plant Sci,14(6):310-317
Wang Y,Shu Z,Wang W,et al(2016).CsWRKY2,a novel WRKY gene from Camellia sinensis,is involved in cold and drought stress responses.Biol Plantarum,60(3):443-451
Yan HR,Jia HH,Chen XB,et al(2014).The cotton WRKYtranscription factor Gh WRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production.Plant Cell Physiol,55(12):2060-2076
Yokotani N,Sato Y,Tanabe S,et al(2013).WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.J Exp Bot,64(16):5085-5097
Zhang LY,Gu LK,Ringler P,et al(2015).Three WRKYtranscription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.Plant Sci,236:214-222
Zheng XD,Zhao Y,Shan DQ,et al(2018).MdWRKY9overexpression confers intensive dwarfing in the M26rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression.New Phytol,217(3):1086-1098
Zhu D,Hou LX,Xiao PL,et al(2018).VvWRKY30,a grape WRKY transcription factor,plays a positive regulatory role under salinity stress.Plant Sci,280:132-142
Chen JN,Nolan TM,Ye HX,et al(2017a).Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses.Plant Cell,29(6):1425-1439
Chen LG,Xiang SY,Chen YL,et al(2017b).Arabidopsis WRKY45 interacts with the DELLA protein RGL1 to positively regulate age-triggered leaf senescence.Mol Plant,10(9):1174-1189
Dai WH,Wang M,Gong XQ,et al(2018).The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs.New Phytol,219(3):972-989
De Storme N,Geelen D(2014).The impact of environmental stress on male reproductive development in plants:biological processes and molecular mechanisms.Plant Cell Environ,37(1):1-18
Ding WW,Fang WB,Shi SY,et al(2016).Wheat WRKY type transcription factor gene TaWRKY1 is essential in mediating drought tolerance associated with an ABA-dependent pathway.Plant Mol Biol Rep,34(6):1111-1126
Eulgem T,Rushton PJ,Robatzek S,et al(2000).The WRKYsuperfamily of plant transcription factors.Trends Plant Sci,5(5):199-206
Fan HK,Wang F,Gao H,et al(2011).Pathogen-induced MdWRKY1 in‘Qinguan’apple enhances disease resistance.JPlant Biol,54(3):150-158
Fan J,Hill L,Crooks C,et al(2009).Abscisic acid has a key role in modulating diverse plant-pathogen interactions.Plant Physiol,150(4):1750-1761
Hu DG,Sun MH,Sun CH,et al(2015).Conserved vacuolar H+-ATPase subunit B1 improves salt stress tolerance in apple calli and tomato plants.Sci Hortic,197:107-116
Hu YR,Dong QY,Yu DQ(2012).Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae.Plant Sci,185:288-297
Huang Y,Feng CZ,Ye Q,et al(2016).Arabidopsis WRKY6transcription factor acts as a positive regulator of abscisic acid signaling during seed germination and early seedling development.PLoS Genet,12(2):e1005833
Jiang YJ,Yu DQ(2016).WRKY57 regulates JAZ genes transcriptionally to compromise Botrytis cinerea resistance in Arabidopsis thaliana.Plant Physiol,doi:10.1104/pp.16.00747
Jin WH,Zhou Q,Wei YF,et al(2017).NtWRKY-R1,a novel transcription factor,integrates IAA and JA signal pathway by topping damage in Nicotiana tabacum.Front Plant Sci,8:2263-2273
Kim CY,Vo KTX,Nguyen CD,et al(2016).Functional analysis of a cold-responsive rice WRKY gene,OsWRKY71.Plant Biotechnol Rep,10(1):13-23
LagacéM,Matton DP(2004).Characterization of a WRKYtranscription factor expressed in late torpedo-stage embryos of Solanum chacoense.Planta,219(1):185-189
Li SJ,Fu QT,Chen LG,et al(2011).Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance.Planta,233(6):1237-1252
Li W,Wang HP,Yu DQ(2016).Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions.Mol Plant,9(11):1492-1503
Liu S,Kracher B,Ziegler J,et al(2015).Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100.eLife,4:e07295
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25(4):402-408
Popko J,H?nsch R,Mendel RR,et al(2010).The role of abscisic acid and auxin in the response of poplar to abiotic stress.Plant Biol,12(2):242-258
Robert-Seilaniantz A,Navarro L,Bari R,et al(2007).Pathological hormone imbalances.Curr Opin Plant Biol,10(4):372-379
Suzuki N,Rivero RM,Shulaev V,et al(2014).Abiotic and biotic stress combinations.New Phytol,203(1):32-43
Ton J,Flors V,Mauch-Mani B(2009).The multifaceted role of ABA in disease resistance.Trends Plant Sci,14(6):310-317
Wang Y,Shu Z,Wang W,et al(2016).CsWRKY2,a novel WRKY gene from Camellia sinensis,is involved in cold and drought stress responses.Biol Plantarum,60(3):443-451
Yan HR,Jia HH,Chen XB,et al(2014).The cotton WRKYtranscription factor Gh WRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production.Plant Cell Physiol,55(12):2060-2076
Yokotani N,Sato Y,Tanabe S,et al(2013).WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance.J Exp Bot,64(16):5085-5097
Zhang LY,Gu LK,Ringler P,et al(2015).Three WRKYtranscription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.Plant Sci,236:214-222
Zheng XD,Zhao Y,Shan DQ,et al(2018).MdWRKY9overexpression confers intensive dwarfing in the M26rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression.New Phytol,217(3):1086-1098
Zhu D,Hou LX,Xiao PL,et al(2018).VvWRKY30,a grape WRKY transcription factor,plays a positive regulatory role under salinity stress.Plant Sci,280:132-142