日本结缕草ZjERF1的克隆、转录激活活性、亚细胞定位及表达分析
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摘要
ERF转录因子是植物体内最大的一类转录因子之一,在调控植物生长发育和响应环境胁迫等方面发挥着重要的作用。研究利用RACE技术,从日本结缕草中克隆到ZjERF1基因(GenBank登录号为MH294481),开放阅读框为630 bp,编码209个氨基酸残基,含有1个高度保守的AP2结构域,属于典型的ERF转录因子。利用染色体步移技术,成功获得ATG上游1581 bp的启动子序列,生物信息学分析表明其中含有多个响应MeJA等激素和非生物胁迫的作用元件。为分析其转录激活特性,构建了pGBKT7-ZjERF1载体,转化Y2HGold酵母感受态细胞,结果表明其具有较强的转录激活活性。为探析其亚细胞定位情况,成功构建35S::ZjERF1:YFP载体,本生烟草瞬时表达结果证明ZjERF1定位于细胞核。为进一步研究ZjERF1的表达特征,利用实时荧光定量的方法进行了验证,结果表明:ZjERF1在日本结缕草茎中表达量最高,并且与叶片的衰老程度呈正相关性;此外ZjERF1的表达可受200μmol·L~(-1) ET、10μmol·L~(-1) MeJA和300 mmol·L~(-1) NaCl处理的诱导,但受到20%PEG4000的抑制。研究表明ZjERF1是一个可参与多种信号转导途径的优良转录因子,为进一步探索ZjERF1基因的功能及其调控机制奠定了基础。
The ERF transcription factors(TFs) are one of the largest groups in plants and play important roles in plant developmental progress and responses to abiotic stresses. In this study, one novel ERF gene, ZjERF1(GenBank No. MH294481), was isolated from Zoysia japonica using the RACE method. The open reading frame of ZjERF1 is 630 bp in length, encoding 209 amino acids. One highly conserved AP2 domain was found in ZjERF1, indicating ZjERF1 was a typical ERF TF. By genome walking, a 1581 bp upstream sequence from the ATG of ZjERF1 was obtained. Bioinformatics analysis revealed that there were several cis-elements in response to MeJA and abiotic stresses in the promoter. To investigate the transcriptional activation character, a pGBKT7-ZjERF1 vector was constructed and then was transformed into Y2 HGold yeast cells; the results showed that ZjERF1 had strong transcriptional activity. To reveal the subcellular localization character, 35 S::ZjERF1:YFP was generated. The transient expression analysis in Nicotiana benthamiana demonstrated that ZjERF1 was localized in the nucleus. To further explore the expression characteristics, real-time quantitative PCR was carried out. The results showed that ZjERF1 was expressed most abundantly in the stem and its transcriptional abundance was positively correlated with leaf senescence. Moreover, its expression could be induced by 200 μmol·L~(-1) ET, 10 μmol·L~(-1) MeJA or 300 mmol·L~(-1) NaCl but was suppressed by 20% PEG4000. Taken together, these findings proves that ZjERF1 is a functional TF involved in various signaling pathways and paves the way for further study of the function of ZjERF1 and its regulatory mechanism in Z. japonica.
The ERF transcription factors(TFs) are one of the largest groups in plants and play important roles in plant developmental progress and responses to abiotic stresses. In this study, one novel ERF gene, ZjERF1(GenBank No. MH294481), was isolated from Zoysia japonica using the RACE method. The open reading frame of ZjERF1 is 630 bp in length, encoding 209 amino acids. One highly conserved AP2 domain was found in ZjERF1, indicating ZjERF1 was a typical ERF TF. By genome walking, a 1581 bp upstream sequence from the ATG of ZjERF1 was obtained. Bioinformatics analysis revealed that there were several cis-elements in response to MeJA and abiotic stresses in the promoter. To investigate the transcriptional activation character, a pGBKT7-ZjERF1 vector was constructed and then was transformed into Y2 HGold yeast cells; the results showed that ZjERF1 had strong transcriptional activity. To reveal the subcellular localization character, 35 S::ZjERF1:YFP was generated. The transient expression analysis in Nicotiana benthamiana demonstrated that ZjERF1 was localized in the nucleus. To further explore the expression characteristics, real-time quantitative PCR was carried out. The results showed that ZjERF1 was expressed most abundantly in the stem and its transcriptional abundance was positively correlated with leaf senescence. Moreover, its expression could be induced by 200 μmol·L~(-1) ET, 10 μmol·L~(-1) MeJA or 300 mmol·L~(-1) NaCl but was suppressed by 20% PEG4000. Taken together, these findings proves that ZjERF1 is a functional TF involved in various signaling pathways and paves the way for further study of the function of ZjERF1 and its regulatory mechanism in Z. japonica.
引文
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[9]Tan X,Fan Z,Shan W,et al.Association of BrERF72with methyl jasmonate-induced leaf senescence of Chinese flowering cabbage through activating JA biosynthesis-related genes.Horticulture Research,2018,5(1):22.
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[12]Xie X L,Xia X J,Kuang S,et al.A novel ethylene responsive factor CitERF13plays a role in photosynthesis regulation.Plant Science,2017,256:112-119.
[13]Li Y,Liu J X,Xiang Q B.Progress in zoysiagrases germplasm resourcs research.Acta Prataculturae Sinica,2002,11(2):7-14.李亚,刘建秀,向其伯.结缕草属种质资源研究进展.草业学报,2002,11(2):7-14.
[14]Dong D,Teng K,Yu A D,et al.Cloning,subcellular localization and expression analysis of a novel phytoene synthase gene,ZmPSY,in Zoysia matrella.Acta Prataculturae Sinica,2017,26(11):69-76.董笛,滕珂,于安东,等.沟叶结缕草八氢番茄红素基因ZmPSY的克隆、亚细胞定位及表达分析.草业学报,2017,26(11):69-76.
[15]Wei S,Du Z,Gao F,et al.Global transcriptome profiles of‘Meyer’Zoysiagrass in response to cold stress.PLoS One,2015,10(6):e0131153.
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[17]Sparkes I A,Runions J,Kearns A,et al.Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.Nature Protocols,2006,1(4):2019-2025.
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[20]Li S J.Research on regulation of the citric acid metabolism in citrus fruit by CitERF13and CitVHA-c4.Hangzhou:Zhejiang University,2016.李绍佳.CitERF13与CitVHA-c4协同调控柑橘果实柠檬酸代谢研究.杭州:浙江大学,2016.
[21]Zhao Y X,Pei L,Zhao-shi X U,et al.Analysis of specific binding and subcellular localization of wheat ERF transcription factor W17.Agricultural Sciences in China,2008,7(6):647-655.
[22]Huang W F,Huang P L,Do Y Y.Characterization of promoter activity of the ethylene receptor gene OgERS1fromOncidesain transgenic Arabidopsis.Biologia Plantarum,2016,60(2):261-268.
[23]Tan P H,Teng K,Li J,et al.Cloning and functional analysis of PpGA2ox gene promoter from Kentucky Bluegrass.Chinese Journal of Grassland,2018,40(1):1-8.檀鹏辉,滕珂,李俊,等.草地早熟禾PpGA2ox基因启动子的克隆及功能分析.中国草地学报,2018,40(1):1-8.
[24]Zhu Q,Zhang J,Gao X,et al.The Arabidopsis AP2/ERF transcription factor RAP2.6participates in ABA,salt and osmotic stress responses.Gene,2010,457(1/2):1-12.
[25]Iqbal N,Khan N A,Ferrante A,et al.Ethylene role in plant growth,development and senescence:Interaction with other phytohormones.Frontiers in Plant Science,2017,8(9):475.
[26]Liu J,Li J,Wang H,et al.Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments.Journal of Experimental Botany,2011,62(2):825.
[27]PréM,Atallah M,Champion A,et al.The AP2/ERF domain transcription factor ORA59integrates jasmonic acid and ethylene signals in plant defense.Plant Physiology,2008,147(3):1347-1357.
[28]Schippers J H M,Schmidt R,Wagstaff C,et al.Living to die and dying to live:The survival strategy behind leaf senescence.Plant Physiology,2015,169:914-930.
[29]Liu W,Wang Y,Gao C.The ethylene response factor(ERF)genes from Tamarix hispida respond to salt,drought and ABA treatment.Trees,2014,28(2):317-327.
[2]Riechmann J L,Heard J,Martin G,et al.Arabidopsis transcription factors:Genome-wide comparative analysis among eukaryotes.Science,2000,290(5499):2105-2110.
[3]Gutterson N,Reuber T L.Regulation of disease resistance pathways by AP2/ERF transcription factors.Current Opinion in Plant Biology,2004,7(4):465-471.
[4]Fu M,Kang H K,Son S H,et al.A subset of Arabidopsis RAV transcription factors modulates drought and salt stress responses independent of ABA.Plant&Cell Physiology,2014,55(11):1892-1904.
[5]Mizoi J,Shinozaki K,Yamaguchi-Shinozaki K.AP2/ERF family transcription factors in plant abiotic stress responses.Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2012,1819(2):86-96.
[6]Fujimoto S Y,Ohta M,Usui A,et al.Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC Box-mediated gene expression.Plant Cell,2000,12(3):393-404.
[7]Jung K H,Ronald P C.The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors.Plant Physiology,2010,152(3):1674-1692.
[8]Yin X R,Xie X L,Xia X J,et al.Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening.The Plant Journal,2016,86(5):403-412.
[9]Tan X,Fan Z,Shan W,et al.Association of BrERF72with methyl jasmonate-induced leaf senescence of Chinese flowering cabbage through activating JA biosynthesis-related genes.Horticulture Research,2018,5(1):22.
[10]Song C P,Agarwal M,Ohta M,et al.Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses.Plant Cell,2005,17(8):2384-2396.
[11]Liu D,Chen X,Liu J,et al.The rice ERF transcription factor OsERF922negatively regulates resistance to Magnaporthe oryzae and salt tolerance.Journal of Experimental Botany,2012,63(10):3899-3911.
[12]Xie X L,Xia X J,Kuang S,et al.A novel ethylene responsive factor CitERF13plays a role in photosynthesis regulation.Plant Science,2017,256:112-119.
[13]Li Y,Liu J X,Xiang Q B.Progress in zoysiagrases germplasm resourcs research.Acta Prataculturae Sinica,2002,11(2):7-14.李亚,刘建秀,向其伯.结缕草属种质资源研究进展.草业学报,2002,11(2):7-14.
[14]Dong D,Teng K,Yu A D,et al.Cloning,subcellular localization and expression analysis of a novel phytoene synthase gene,ZmPSY,in Zoysia matrella.Acta Prataculturae Sinica,2017,26(11):69-76.董笛,滕珂,于安东,等.沟叶结缕草八氢番茄红素基因ZmPSY的克隆、亚细胞定位及表达分析.草业学报,2017,26(11):69-76.
[15]Wei S,Du Z,Gao F,et al.Global transcriptome profiles of‘Meyer’Zoysiagrass in response to cold stress.PLoS One,2015,10(6):e0131153.
[16]Liu J,Chen X,Liang X,et al.Alternative splicing of rice WRKY62and WRKY76transcription factor genes in pathogen defense.Plant Physiology,2016,171(2):1427-1442.
[17]Sparkes I A,Runions J,Kearns A,et al.Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.Nature Protocols,2006,1(4):2019-2025.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25(4):402-408.
[19]Ohta M,Matsui K,Hiratsu K,et al.Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.Plant Cell,2001,13(8):1959-1968.
[20]Li S J.Research on regulation of the citric acid metabolism in citrus fruit by CitERF13and CitVHA-c4.Hangzhou:Zhejiang University,2016.李绍佳.CitERF13与CitVHA-c4协同调控柑橘果实柠檬酸代谢研究.杭州:浙江大学,2016.
[21]Zhao Y X,Pei L,Zhao-shi X U,et al.Analysis of specific binding and subcellular localization of wheat ERF transcription factor W17.Agricultural Sciences in China,2008,7(6):647-655.
[22]Huang W F,Huang P L,Do Y Y.Characterization of promoter activity of the ethylene receptor gene OgERS1fromOncidesain transgenic Arabidopsis.Biologia Plantarum,2016,60(2):261-268.
[23]Tan P H,Teng K,Li J,et al.Cloning and functional analysis of PpGA2ox gene promoter from Kentucky Bluegrass.Chinese Journal of Grassland,2018,40(1):1-8.檀鹏辉,滕珂,李俊,等.草地早熟禾PpGA2ox基因启动子的克隆及功能分析.中国草地学报,2018,40(1):1-8.
[24]Zhu Q,Zhang J,Gao X,et al.The Arabidopsis AP2/ERF transcription factor RAP2.6participates in ABA,salt and osmotic stress responses.Gene,2010,457(1/2):1-12.
[25]Iqbal N,Khan N A,Ferrante A,et al.Ethylene role in plant growth,development and senescence:Interaction with other phytohormones.Frontiers in Plant Science,2017,8(9):475.
[26]Liu J,Li J,Wang H,et al.Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments.Journal of Experimental Botany,2011,62(2):825.
[27]PréM,Atallah M,Champion A,et al.The AP2/ERF domain transcription factor ORA59integrates jasmonic acid and ethylene signals in plant defense.Plant Physiology,2008,147(3):1347-1357.
[28]Schippers J H M,Schmidt R,Wagstaff C,et al.Living to die and dying to live:The survival strategy behind leaf senescence.Plant Physiology,2015,169:914-930.
[29]Liu W,Wang Y,Gao C.The ethylene response factor(ERF)genes from Tamarix hispida respond to salt,drought and ABA treatment.Trees,2014,28(2):317-327.