桃转录因子PpWRKY11的克隆及表达分析
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摘要
WRKY转录因子能够响应生物及非生物胁迫。本研究基于前期实验室桃花芽休眠解除前后表达量明显下调的Prupe.1G071400基因进行研究,通过定量PCR表明此结果与前期实验结果相符合,证明该基因可能与桃花芽休眠解除密切相关。从桃‘中油四号’叶片克隆了Prupe.1G071400基因,通过生物信息学分析,命名此基因为PpWRKY11; PpWRKY11蛋白编码282个氨基酸,包含特有WRKY结构域。进化树分析发现PpWRKY11与蔷薇科苹果和梨等亲缘关系最为相近;亚细胞定位显示PpWRKY11定位于细胞核中;酵母双杂显示PpWRKY11蛋白没有自主激活活性,进一步对PpWRKY11进行筛库,筛选出互作的蛋白;构建PGEX-PpWRKY11原核表达载体,转化大肠杆菌Rosetta(DE3)诱导出符合大小的蛋白,发现PpWRKY11蛋白主要在沉淀中。
WRKY transcription factors are capable of responding to plant biotic and abiotic stresses. This study was based on the Prupe.1G071400 gene whose expression was significantly down-regulated in the peach flower bud dormancy. The results were consistent with the previous experimental results by quantitative PCR, which proved that the gene may be closely related to the dormancy release of peach flower bud. The Prupe.1G071400 gene was cloned from the leaves of the peach ‘Zhong You 4', and the gene was named PpWRKY11 by bioinformatics analysis. The PpWRKY11 protein encodes 282 amino acids, including the unique WRKY domain. Phylogenetic tree analysis showed that PpWRKY11 was most closely related to Rosaceae apples and pears. Subcellular localization showed that PpWRKY11 was localized in the nucleus; yeast double hybrid showed that PpWRKY11 protein had no autonomous activation activity, and further utilize PpWRKY11 to screen for interaction proteins; The construct PGEX-Pp WRKY11 prokaryotic expression vector transform E. coli Rosetta(DE3)to induce size-matched protein, and found that PpWRKY11 protein is mainly in precipitation. This study laid the foundation for further exploration of the function of the PpWRKY11 gene.
WRKY transcription factors are capable of responding to plant biotic and abiotic stresses. This study was based on the Prupe.1G071400 gene whose expression was significantly down-regulated in the peach flower bud dormancy. The results were consistent with the previous experimental results by quantitative PCR, which proved that the gene may be closely related to the dormancy release of peach flower bud. The Prupe.1G071400 gene was cloned from the leaves of the peach ‘Zhong You 4', and the gene was named PpWRKY11 by bioinformatics analysis. The PpWRKY11 protein encodes 282 amino acids, including the unique WRKY domain. Phylogenetic tree analysis showed that PpWRKY11 was most closely related to Rosaceae apples and pears. Subcellular localization showed that PpWRKY11 was localized in the nucleus; yeast double hybrid showed that PpWRKY11 protein had no autonomous activation activity, and further utilize PpWRKY11 to screen for interaction proteins; The construct PGEX-Pp WRKY11 prokaryotic expression vector transform E. coli Rosetta(DE3)to induce size-matched protein, and found that PpWRKY11 protein is mainly in precipitation. This study laid the foundation for further exploration of the function of the PpWRKY11 gene.
引文
Ali MA,Azeem F,Nawaz MA,et al(2018).Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.J Plant Physiol,226:12-21
Ali MA,Wieczorek K,Kreil DP,et al(2014).The beet cyst nematode Heterodera schachtii modulates the expression of WRKY transcription factors in syncytia to favour its development in Arabidopsis roots.PLoS ONE,9(7):e102360
Chen M,Tan Q,Sun M,et al(2016).Genome-wide identification of WRKY family genes in peach and analysis of WRKY expression during bud dormancy.Mol Genet Genomics,291(3):1319-1332
Chen M,Liu X,Jiang S,et al(2018).Transcriptomic and functional analyses reveal that PpGLK1 regulates chloroplast development in peach(Prunus persica).Front Plant Sci,9:34
Ding ZJ,Yan JY,Li GX,et al(2014).WRKY41 controls Arabidopsis seed dormancy via direct regulation of ABI3transcript levels not downstream of ABA.Plant J,79(5):810-823
Journot-Catalino N,Somssich IE,Roby D,et al(2006).The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana.Plant Cell,18(11):3289-3302
Jiang W,Yu D(2009).Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid.BMC Plant Biol,9(1):96
Li JJ,Han LL,Ma Y,et al(2017).Cloning and expression analysis of transcription factor Pl WRKY40 in peony.Plant Physiol J,53(4):609-618(in Chinese with English abstract)[李俊杰,韩璐璐,马燕等(2017).芍药转录因子PlWRKY40的克隆及表达分析.植物生理学报,53(4):609-618]
Li Z,Reighard GL,Abbott AG,et al(2009).Dormancy-associated MADS genes from the EVG locus of peach[Prunus persica(L.)Batsch]have distinct seasonal and photoperiodic expression patterns.J Exp Bot,60(12):3521-3530
Niu QF,Li JZ,Cai DY,et al(2016).Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear(Pyrus pyrifolia white pear group)flower bud.J Exp Bot,10:1-19
Phukan UJ,Jeena GS,Shukla RK(2016).WRKY transcription factors:molecular regulation and stress responses in plants.Front Plant Sci,7:760
Rushton DL,Tripathi P,Rabara RC,et al(2012).WRKY transcription factors:key components in abscisic acid signalling.Plant Biotechnol J,10(1):2-11
Rushton PJ,Somssich IE,Ringler P,et al(2010).WRKY transcription factors.Trends Plant Sci,15(5):247-258
Takemura Y,Tamura F(2016).Induction of Heat Shock Proteins During the Bud Dormancy Stage in Woody Fruit Plants.In:Asea AAA,Kaur P,Calderwood SK(eds).Heat Shock Proteins and Plants.Cham:Springer,65-67
Tamura K,Stecher G,Peterson D,et al(2013).MEGA6:molecular evolutionary geneti cs analysis version 6.0.Mol Biol Evol,30(12):2725-2729
Saure MC(1985).Dormancy release in deciduous fruit trees.Hortic Rev,7:239-300
Tuan P A,Bai S,Saito T,et al(2017)Dormancy-associated mads-box(Dam)and the abscisic acid pathway regulate pear endodormancy through a feedback mechanism.Plant Cell Physiol,58(8):1378-1390
Wang D,Gao Z,Du P,et al(2016).Expression of ABA metabolism-related genes suggests similarities and differences between seed dormancy and bud dormancy of peach(Prunus persica).Front Plant Sci,6:1248
Yamane H,Ooka T,Jotatsu H,et al(2011).Expressional regulation of PpDAM5 and PpDAM6,peach(Prunus persica)dormancy-associated MADS-box genes,by low temperature and dormancy-breaking reagent treatment.J Exp Bot,62(10):3481-3488
Yang G,Zhang W,Liu Z,et al(2017).Both JrWRKY2 and JrWRKY7 of Juglans regia mediate responses to abiotic stresses and abscisic acid through formation of homodimers and interaction.Plant Biol,19(2):268-278
Zhao TT,Zhang J,Liang LS,et al(2015).Expression and functional analysis of WRKY transcription factors in Chinese wild hazel,Corylus heterophylla Fisch.PLoS ONE,10(8):e0135315
Ali MA,Wieczorek K,Kreil DP,et al(2014).The beet cyst nematode Heterodera schachtii modulates the expression of WRKY transcription factors in syncytia to favour its development in Arabidopsis roots.PLoS ONE,9(7):e102360
Chen M,Tan Q,Sun M,et al(2016).Genome-wide identification of WRKY family genes in peach and analysis of WRKY expression during bud dormancy.Mol Genet Genomics,291(3):1319-1332
Chen M,Liu X,Jiang S,et al(2018).Transcriptomic and functional analyses reveal that PpGLK1 regulates chloroplast development in peach(Prunus persica).Front Plant Sci,9:34
Ding ZJ,Yan JY,Li GX,et al(2014).WRKY41 controls Arabidopsis seed dormancy via direct regulation of ABI3transcript levels not downstream of ABA.Plant J,79(5):810-823
Journot-Catalino N,Somssich IE,Roby D,et al(2006).The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana.Plant Cell,18(11):3289-3302
Jiang W,Yu D(2009).Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid.BMC Plant Biol,9(1):96
Li JJ,Han LL,Ma Y,et al(2017).Cloning and expression analysis of transcription factor Pl WRKY40 in peony.Plant Physiol J,53(4):609-618(in Chinese with English abstract)[李俊杰,韩璐璐,马燕等(2017).芍药转录因子PlWRKY40的克隆及表达分析.植物生理学报,53(4):609-618]
Li Z,Reighard GL,Abbott AG,et al(2009).Dormancy-associated MADS genes from the EVG locus of peach[Prunus persica(L.)Batsch]have distinct seasonal and photoperiodic expression patterns.J Exp Bot,60(12):3521-3530
Niu QF,Li JZ,Cai DY,et al(2016).Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear(Pyrus pyrifolia white pear group)flower bud.J Exp Bot,10:1-19
Phukan UJ,Jeena GS,Shukla RK(2016).WRKY transcription factors:molecular regulation and stress responses in plants.Front Plant Sci,7:760
Rushton DL,Tripathi P,Rabara RC,et al(2012).WRKY transcription factors:key components in abscisic acid signalling.Plant Biotechnol J,10(1):2-11
Rushton PJ,Somssich IE,Ringler P,et al(2010).WRKY transcription factors.Trends Plant Sci,15(5):247-258
Takemura Y,Tamura F(2016).Induction of Heat Shock Proteins During the Bud Dormancy Stage in Woody Fruit Plants.In:Asea AAA,Kaur P,Calderwood SK(eds).Heat Shock Proteins and Plants.Cham:Springer,65-67
Tamura K,Stecher G,Peterson D,et al(2013).MEGA6:molecular evolutionary geneti cs analysis version 6.0.Mol Biol Evol,30(12):2725-2729
Saure MC(1985).Dormancy release in deciduous fruit trees.Hortic Rev,7:239-300
Tuan P A,Bai S,Saito T,et al(2017)Dormancy-associated mads-box(Dam)and the abscisic acid pathway regulate pear endodormancy through a feedback mechanism.Plant Cell Physiol,58(8):1378-1390
Wang D,Gao Z,Du P,et al(2016).Expression of ABA metabolism-related genes suggests similarities and differences between seed dormancy and bud dormancy of peach(Prunus persica).Front Plant Sci,6:1248
Yamane H,Ooka T,Jotatsu H,et al(2011).Expressional regulation of PpDAM5 and PpDAM6,peach(Prunus persica)dormancy-associated MADS-box genes,by low temperature and dormancy-breaking reagent treatment.J Exp Bot,62(10):3481-3488
Yang G,Zhang W,Liu Z,et al(2017).Both JrWRKY2 and JrWRKY7 of Juglans regia mediate responses to abiotic stresses and abscisic acid through formation of homodimers and interaction.Plant Biol,19(2):268-278
Zhao TT,Zhang J,Liang LS,et al(2015).Expression and functional analysis of WRKY transcription factors in Chinese wild hazel,Corylus heterophylla Fisch.PLoS ONE,10(8):e0135315