梅花PmERF4基因的克隆及其表达分析
|
摘要
为阐明梅花(Prunus mume)ERF基因的功能,采用RT-PCR技术从梅花品种‘雪梅’中克隆获得1个PmERF4基因。生物信息学分析表明该基因cDNA全长序列为842 bp,完整开放阅读框(ORF)为690 bp,编码229个氨基酸。氨基酸序列比对发现,PmERF4蛋白包含1个AP2/ERF保守结构域。系统进化分析结果显示,梅花PmERF4蛋白与李亚科李属植物的ERF蛋白高度同源。利用荧光定量PCR(qRT-PCR)检测PmERF4基因在非生物胁迫和激素处理下的表达,结果表明:PmERF4基因受低温、氧化胁迫的诱导,但响应程度不同,它能够持续且强烈地响应低温胁迫,对甘露醇、MeJA、SA处理不敏感,而高盐和ABA则抑制其表达;PmERF4基因可能在梅花低温胁迫应答中发挥重要作用。
ERF(ethylene response factor) transcription factor plays a critical role in response to environmental stress and hormonal signal transduction in plants.To elucidate the function of ERF gene in Prunus mume,the PmERF4 gene was cloned from a Prunus mume ‘Xuemei' by RT-PCR.The full-length cDNA sequence of PmERF4 was 842 bp with an open reading frame(ORF) of 690 bp,encoding 229 amino acids.The deduced amino acid sequence alignment of PmERF4 contained a conserved AP2/ERF domain.The result of phylogenetic analysis showed the PmERF4 was highly homologous to other proteins in Prunus genus.qRT-PCR was conducted to examine the expression patterns of PmERF4 under abiotic stress and hormone treatment.The results showed that PmERF4 was induced by cold and H_2O_2 stress.The induction levels of the gene were different between cold and H_2O_2 stress.A continuous and strong response of PmERF4 was triggered by cold.PmERF4 was insensitive to mannitol,MeJA and SA treatment and its expression was inhibited by NaCl and ABA.It is indicated that PmERF4 from Prunus mume may be involved in response to cold and H_2O_2 stress.
ERF(ethylene response factor) transcription factor plays a critical role in response to environmental stress and hormonal signal transduction in plants.To elucidate the function of ERF gene in Prunus mume,the PmERF4 gene was cloned from a Prunus mume ‘Xuemei' by RT-PCR.The full-length cDNA sequence of PmERF4 was 842 bp with an open reading frame(ORF) of 690 bp,encoding 229 amino acids.The deduced amino acid sequence alignment of PmERF4 contained a conserved AP2/ERF domain.The result of phylogenetic analysis showed the PmERF4 was highly homologous to other proteins in Prunus genus.qRT-PCR was conducted to examine the expression patterns of PmERF4 under abiotic stress and hormone treatment.The results showed that PmERF4 was induced by cold and H_2O_2 stress.The induction levels of the gene were different between cold and H_2O_2 stress.A continuous and strong response of PmERF4 was triggered by cold.PmERF4 was insensitive to mannitol,MeJA and SA treatment and its expression was inhibited by NaCl and ABA.It is indicated that PmERF4 from Prunus mume may be involved in response to cold and H_2O_2 stress.
引文
[1] 陈俊愉.中国梅花品种图志[M].北京:中国林业出版社,2010:1-8.
[2] PENG T,GUO C,YANG J,et al.Overexpression of a Mei (Prunus mume) CBF gene confers tolerance to freezing and oxidative stress in Arabidopsis[J].Plant cell tissue and organ culture,2016,126(3):373-385.
[3] 张杰,张启翔,杨炜茹.梅花CBF转录因子的克隆及表达[J].西北植物学报,2012,32(8):1505-1510.
[4] OHME-TAKAGI M,SHINSHI H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].The plant cell,1995,7:173-182.
[5] NAKANO T,SUZUKI K,FUJIMURA T,et al.Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant physiology,2006,140(2):411-432.
[6] AKUMA Y,LIU Q,DUBOUZET J G,et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration- and cold-inducible gene expression[J].Biochemical and biophysical research communications,2002,290(3):998-1009.
[7] YAMAMOTO S,SUZUKI K,SHINSHI H.Elicitor-responsive,ethylene-independent activation of GCC box-mediated transcription that is regulated by both protein phosphorylation and dephosphorylation in cultured tobacco cells[J].The plant journal,1999,20(5):571-579.
[8] GU Y Q,YANG C,THARA V K,et al.Pti4 is induced by ethylene and salicylic acid,and its product is phosphorylated by the Pto kinase[J].The plant cell,2000,12:771-785.
[9] KLAY I,GOUIA S,LIU M,et al.Ethylene response factors (ERF) are differentially regulated by different abiotic stress types in tomato plants[J].Plant science:an international journal of experimental plant biology,2018,274:137-145.
[10] STOCKINGER E J,GILMOUR S J,THOMASHOW M F.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeatDRE,a cis-acting DNA regulatory element that stimulates transcription[J].Proceedings of the national academy of sciences of the United States of America,1997,94:1035-1040.
[11] LIU Q,KASUGA M,SAKUMA Y,et al.Two transcription factors,DREB1 and DREB2,with an EREBP_AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene[J].The plant cell,1998,10:1391-1406.
[12] DUBOUZET J G,SAKUMA Y,ITO Y,et al.OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt- and cold-responsive gene expression[J].The plant journal,2003,33:751-763.
[13] ZHANG Q,CHEN W,SUN L,et al.The genome of Prunus mume[J].Nature communications,2012,3:1318.
[14] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J].Methods,2001,25(4):402-408.
[15] JOFUKU K D,BOER B G W,VAN-MONTAGU M,et al.Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J].The plant cell,1994,6:1211-1225.
[16] ZHAI Y,SHAO S,SHA W,et al.Overexpression of soybean GmERF9 enhances the tolerance to drought and cold in the transgenic tobacco[J].Plant cell tissue and organ culture,2016,128(3):607-618.
[17] SUN X,ZHAO T,GAN S,et al.Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ethylene response factor 057[J].Scientific reports,2016,6:24066.
[18] MA Y,ZHANG L,ZHANG J,et al.Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco[J].Scientia horticulturae,2014,174:65-76.
[19] LESTARI R,RIO M,MARTIN F,et al.Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation[J].Plant biotechnology journal,2018,16(1):322-336.
[20] ZHUO C,LIANG L,ZHAO Y,et al.A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover,antioxidant protection,and proline accumulation[J].Plant cell and environment,2018,41(9):2021-2032.
[21] LI M Y,XU Z S,HUANG Y,et al.Genome-wide analysis of AP2_ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress[J].Molecular genetics genomics,2015,290:2049-2061.
[22] 侯晓婉,胡伟,徐碧玉,等.巴西蕉2个ERF转录因子的克隆及功能研究[J].果树学报,2017,34(7):806-816.
[23] CHEN N,YANG Q,SU M,et al.Cloning of six ERF family transcription factor genes from peanut and analysis of their expression during abiotic stress[J].Plant molecular biology reporter,2012,30:1415-1425.
[24] SINGH K B,FOLEY R C,ONATE-SáNCHEZ L.Transcription factors in plant defense and stress responses[J].Current opinion in plant biology,2002,5(5):430-436.
[25] DU D,HAO R,CHENG T,et al.Genome-wide analysis of the AP2/ERF gene family in Prunus mume[J].Plant molecular biology reporter,2012,31(3):741-750.
[2] PENG T,GUO C,YANG J,et al.Overexpression of a Mei (Prunus mume) CBF gene confers tolerance to freezing and oxidative stress in Arabidopsis[J].Plant cell tissue and organ culture,2016,126(3):373-385.
[3] 张杰,张启翔,杨炜茹.梅花CBF转录因子的克隆及表达[J].西北植物学报,2012,32(8):1505-1510.
[4] OHME-TAKAGI M,SHINSHI H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].The plant cell,1995,7:173-182.
[5] NAKANO T,SUZUKI K,FUJIMURA T,et al.Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant physiology,2006,140(2):411-432.
[6] AKUMA Y,LIU Q,DUBOUZET J G,et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration- and cold-inducible gene expression[J].Biochemical and biophysical research communications,2002,290(3):998-1009.
[7] YAMAMOTO S,SUZUKI K,SHINSHI H.Elicitor-responsive,ethylene-independent activation of GCC box-mediated transcription that is regulated by both protein phosphorylation and dephosphorylation in cultured tobacco cells[J].The plant journal,1999,20(5):571-579.
[8] GU Y Q,YANG C,THARA V K,et al.Pti4 is induced by ethylene and salicylic acid,and its product is phosphorylated by the Pto kinase[J].The plant cell,2000,12:771-785.
[9] KLAY I,GOUIA S,LIU M,et al.Ethylene response factors (ERF) are differentially regulated by different abiotic stress types in tomato plants[J].Plant science:an international journal of experimental plant biology,2018,274:137-145.
[10] STOCKINGER E J,GILMOUR S J,THOMASHOW M F.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeatDRE,a cis-acting DNA regulatory element that stimulates transcription[J].Proceedings of the national academy of sciences of the United States of America,1997,94:1035-1040.
[11] LIU Q,KASUGA M,SAKUMA Y,et al.Two transcription factors,DREB1 and DREB2,with an EREBP_AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene[J].The plant cell,1998,10:1391-1406.
[12] DUBOUZET J G,SAKUMA Y,ITO Y,et al.OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt- and cold-responsive gene expression[J].The plant journal,2003,33:751-763.
[13] ZHANG Q,CHEN W,SUN L,et al.The genome of Prunus mume[J].Nature communications,2012,3:1318.
[14] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J].Methods,2001,25(4):402-408.
[15] JOFUKU K D,BOER B G W,VAN-MONTAGU M,et al.Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J].The plant cell,1994,6:1211-1225.
[16] ZHAI Y,SHAO S,SHA W,et al.Overexpression of soybean GmERF9 enhances the tolerance to drought and cold in the transgenic tobacco[J].Plant cell tissue and organ culture,2016,128(3):607-618.
[17] SUN X,ZHAO T,GAN S,et al.Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ethylene response factor 057[J].Scientific reports,2016,6:24066.
[18] MA Y,ZHANG L,ZHANG J,et al.Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco[J].Scientia horticulturae,2014,174:65-76.
[19] LESTARI R,RIO M,MARTIN F,et al.Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation[J].Plant biotechnology journal,2018,16(1):322-336.
[20] ZHUO C,LIANG L,ZHAO Y,et al.A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover,antioxidant protection,and proline accumulation[J].Plant cell and environment,2018,41(9):2021-2032.
[21] LI M Y,XU Z S,HUANG Y,et al.Genome-wide analysis of AP2_ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress[J].Molecular genetics genomics,2015,290:2049-2061.
[22] 侯晓婉,胡伟,徐碧玉,等.巴西蕉2个ERF转录因子的克隆及功能研究[J].果树学报,2017,34(7):806-816.
[23] CHEN N,YANG Q,SU M,et al.Cloning of six ERF family transcription factor genes from peanut and analysis of their expression during abiotic stress[J].Plant molecular biology reporter,2012,30:1415-1425.
[24] SINGH K B,FOLEY R C,ONATE-SáNCHEZ L.Transcription factors in plant defense and stress responses[J].Current opinion in plant biology,2002,5(5):430-436.
[25] DU D,HAO R,CHENG T,et al.Genome-wide analysis of the AP2/ERF gene family in Prunus mume[J].Plant molecular biology reporter,2012,31(3):741-750.