文心兰OnRR9基因克隆及表达
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摘要
利用RACE技术从文心兰花瓣中克隆到RR基因,将其命名为OnRR9(登录号:MH758789)。OnRR9基因全长为627 bp,编码208个氨基酸。对其进行蛋白质2级结构分析,构建进化树,并进行实时荧光定量分析。结果表明:OnRR9蛋白为亲水性的分泌蛋白,定位于细胞核中。同源序列比对显示,OnRR9基因属于Yes N/Ara C家族,氨基酸序列中含有保守的REC结构域,可能参与双组分信号转导途径。构建进化树表明,OnRR9蛋白与拟南芥中A型ARRs亲缘关系接近,OnRR9基因可能作为细胞分裂素信号传导的成分。实时荧光定量分析结果表明,OnRR9基因在盛开期前后表达模式发生逆转,表明它可能参与文心兰花瓣衰老启动过程。
A gene of response regulator,OnRR9 gene( GenBank accession number: MH758789),was cloned from the petals of Oncidium Gower Ramsey flowers with RACE technology. The OnRR9 gene is 627 bp in length and encodes a protein of 208 amino acids in size. The secondary structure analysis of the protein showed that OnRR9 protein is a hydrophilic secreted protein localized in the nucleus. The homologous sequence alignment revealed that the OnRR9 gene belongs to the YesN/AraC family and contains a conserved REC domain in the amino acid sequence,which may be involved in the two-component signal transduction pathway. The construction of the phylogenetic tree demonstrated that the OnRR9 protein is closely related to the type A ARRs in Arabidopsis thaliana,suggesting that the OnRR9 may be involved in the response of cytokinin signaling. Real-time fluorescence quantitative analysis indicated that the OnRR9 gene had the maximum expression in the full blossom flower but was low expressed before and after full blossom,suggesting that this gene might play a role in the iniitation of petal senescence of Oncidium Gower Ramsey flowers.
A gene of response regulator,OnRR9 gene( GenBank accession number: MH758789),was cloned from the petals of Oncidium Gower Ramsey flowers with RACE technology. The OnRR9 gene is 627 bp in length and encodes a protein of 208 amino acids in size. The secondary structure analysis of the protein showed that OnRR9 protein is a hydrophilic secreted protein localized in the nucleus. The homologous sequence alignment revealed that the OnRR9 gene belongs to the YesN/AraC family and contains a conserved REC domain in the amino acid sequence,which may be involved in the two-component signal transduction pathway. The construction of the phylogenetic tree demonstrated that the OnRR9 protein is closely related to the type A ARRs in Arabidopsis thaliana,suggesting that the OnRR9 may be involved in the response of cytokinin signaling. Real-time fluorescence quantitative analysis indicated that the OnRR9 gene had the maximum expression in the full blossom flower but was low expressed before and after full blossom,suggesting that this gene might play a role in the iniitation of petal senescence of Oncidium Gower Ramsey flowers.
引文
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[15]YANG G-H,LIU J-P. Isolation of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey[J].Genetics and Molecular Research,2014,13(4):8480-8488.
[16]杨光华,刘进平.文心兰ACC氧化酶基因On ACO1克隆与表达分析[J].热带作物学报,2014,35(4):693-699.
[17]SHI L-S,LIU J-P. Molecular cloning and expression analysis of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey[J]. Biochemical and Biophysical Research Communications,2016,469:203-209.
[18]石乐松,刘进平.文心兰RNA不同提取方法比较[J].生物技术,2012,22(6):42-45.
[19]SCOTTO-LAVINO E,Du G,Frohman M A. 5'end c DNA amplification using classic RACE[J]. Nature protocols,2006,1(6):2555.
[20]石乐松,孙慧,田晓岩,等.文心兰南茜种花发育进程与分期[J].热带生物学报,2016,7(4):440-443,449.
[21]VERMA V,SIVARAMAN J,SRIVASTAVA A K,et al. Destabilization of interaction between cytokinin signaling intermediates AHP 1 and ARR 4 modulates Arabidopsis development[J]. New Phytologist,2015,206(2):726-737.
[22]OSAKABE Y,MIYATA S,URAO T,et al. Overexpression of Arabidopsis response regulators,ARR4/ATRR1/IBC7 and ARR8/ATRR3,alters cytokinin responses differentially in the shoot and in callus formation[J]. Biochemical and biophysical research communications,2002,293(2):806-815.
[23]HOEBERICHTS F A,VAN DOORN W G,VORST O,et al. Sucrose prevents up-regulation of senescence-associated genes in carnation petals[J]. Journal of Experimental Botany,2007,58(11):2873-2885.
[24]CHANG H,JONES M L,BANOWETZ G M,et al. Overproduction of cytokinins in petunia flowers transformed with PSAG12-IPT delays corolla senescence and decreases sensitivity to ethylene[J]. Plant Physiology,2003,132(4):2174-2183.
[25]VAN STADEN J,DIMALLA G G. The effect of silver thiosulphate preservative on the physiology of cut carnations II. Influence on endogenous cytokinins[J]. Zeitschrift für Pflanzenphysiologie,1980,99(1):19-26.
[2]MIZUNO T. Plant response regulators implicated in signal transduction and circadian rhythm[J]. Current opinion in plant biology,2004,7(5):499-505.
[3]HWANG I,CHEN H C,SHEEN J. Two-component signal transduction pathways in Arabidopsis[J]. Plant Physiology,2002,129(2):500-515.
[4]HWANG I,SHEEN J. Two-component circuitry in Arabidopsis cytokinin signal transduction[J]. Nature,2001,413(6854):383.
[5]SALOMP A,TO J P C,KIEBER J J,et al. Arabidopsis response regulators ARR3 and ARR4 play cytokinin-independent roles in the control of circadian period[J]. The Plant Cell,2006,18(1):55-69.
[6]孟文静.拟南芥B类ARRs调控茎端分生组织重塑与维持的分子机理研究[D].泰安:山东农业大学,2018.
[7]程潇.水稻细胞分裂素双元信号系统分析及反应调节因子的分子调控研究[D].合肥:安徽农业大学,2010.
[8] D’AGOSTINO I B,KIEBER J J. Phosphorelay signal transduction:the emerging family of plant response regulators[J].Trends in Biochemical Sciences,1999,24(11):452-456.
[9]田晓岩,杨翠萍,胡进,等.文心兰On ERS1全长基因克隆及表达分析[J].分子植物育种,2017,15(4):1265-1272.
[10]田晓岩,石乐松,潘英文,等.文心兰On ACO2基因的克隆及表达分析[J].分子植物育种,2015,13(7):1602-1610.
[11]李玲,赖钟雄,陈裕坤,等.文心兰On COBRA基因克隆及表达模式分析[J].热带作物学报,2014,35(8):1551-1558.
[12]杨光华,刘进平.文心兰ACC氧化酶基因On ACO1克隆与表达分析[J].热带作物学报,2014,35(4):693-699.
[13]张国付,蒋素华,崔波,等.文心兰GAPDH基因片段的克隆及序列分析[J].河南农业大学学报,2013,47(01):68-71.
[14]YANG C P,XIA Z Q,HU J,et al. Transcriptome analysis of Oncidium petals provides new insights into the initiation of petal senescence[J]. The Journal of Horticultural Science and Biotechnology,2018,93:1-12.
[15]YANG G-H,LIU J-P. Isolation of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey[J].Genetics and Molecular Research,2014,13(4):8480-8488.
[16]杨光华,刘进平.文心兰ACC氧化酶基因On ACO1克隆与表达分析[J].热带作物学报,2014,35(4):693-699.
[17]SHI L-S,LIU J-P. Molecular cloning and expression analysis of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower Ramsey[J]. Biochemical and Biophysical Research Communications,2016,469:203-209.
[18]石乐松,刘进平.文心兰RNA不同提取方法比较[J].生物技术,2012,22(6):42-45.
[19]SCOTTO-LAVINO E,Du G,Frohman M A. 5'end c DNA amplification using classic RACE[J]. Nature protocols,2006,1(6):2555.
[20]石乐松,孙慧,田晓岩,等.文心兰南茜种花发育进程与分期[J].热带生物学报,2016,7(4):440-443,449.
[21]VERMA V,SIVARAMAN J,SRIVASTAVA A K,et al. Destabilization of interaction between cytokinin signaling intermediates AHP 1 and ARR 4 modulates Arabidopsis development[J]. New Phytologist,2015,206(2):726-737.
[22]OSAKABE Y,MIYATA S,URAO T,et al. Overexpression of Arabidopsis response regulators,ARR4/ATRR1/IBC7 and ARR8/ATRR3,alters cytokinin responses differentially in the shoot and in callus formation[J]. Biochemical and biophysical research communications,2002,293(2):806-815.
[23]HOEBERICHTS F A,VAN DOORN W G,VORST O,et al. Sucrose prevents up-regulation of senescence-associated genes in carnation petals[J]. Journal of Experimental Botany,2007,58(11):2873-2885.
[24]CHANG H,JONES M L,BANOWETZ G M,et al. Overproduction of cytokinins in petunia flowers transformed with PSAG12-IPT delays corolla senescence and decreases sensitivity to ethylene[J]. Plant Physiology,2003,132(4):2174-2183.
[25]VAN STADEN J,DIMALLA G G. The effect of silver thiosulphate preservative on the physiology of cut carnations II. Influence on endogenous cytokinins[J]. Zeitschrift für Pflanzenphysiologie,1980,99(1):19-26.