二穗短柄草DREB1G基因表达模式分析和在拟南芥中的异源过量表达
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摘要
CBF/DREB基因是一类植物特异性转录因子,通过调控下游抗逆相关基因的表达,在植物逆境胁迫应答过程中发挥重要作用。为了研究新型的禾本科模式植物二穗短柄草DREB基因的功能,克隆BdDREB1G的cDNA序列,分析该基因在各种非生物胁迫条件下的表达,构建过表达载体,获得转基因拟南芥。结果表明:高温、低温和水杨酸胁迫处理显著诱导BdDREB1G的表达,暗示该基因响应高温和低温胁迫。初步观察结果说明,该与野生型拟南芥相比,转基因植株生长发育迟缓。这些为研究该基因响应非生物胁迫的功能奠定基础。
As transcription factors in plants,CBF/DREBs play key roles in responses to adversity stresses via regulating the expression of downstream genes.To elucidate the function of DREBgene in Brachypodium distachyon,agramineous plant,we cloned the cDNA sequence of BdDREB1G ,analyzed the expression pattern under different abiotic stress,constructed the over-expression vector and obtained the transgenic Arabidopsis.The results showed that heat,cold and salicylic acid notably induced the transcription of BdDREB1G ,suggesting that BdDREB1G was in response to high temperature and low temperature stress.Compared with wild type Arabidopsis,transgenic plant growth was retarded.These findings laid the foundation for studying BdDREB1G gene function in response to abiotic stress.
As transcription factors in plants,CBF/DREBs play key roles in responses to adversity stresses via regulating the expression of downstream genes.To elucidate the function of DREBgene in Brachypodium distachyon,agramineous plant,we cloned the cDNA sequence of BdDREB1G ,analyzed the expression pattern under different abiotic stress,constructed the over-expression vector and obtained the transgenic Arabidopsis.The results showed that heat,cold and salicylic acid notably induced the transcription of BdDREB1G ,suggesting that BdDREB1G was in response to high temperature and low temperature stress.Compared with wild type Arabidopsis,transgenic plant growth was retarded.These findings laid the foundation for studying BdDREB1G gene function in response to abiotic stress.
引文
[1]YAMAGUCHI-SHINOZAKI K,SHINOZAKI K.Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses[J].Annual Review of Plant Biology,2006,57(57):781-803.
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[16]张彦妮,焦孟月,刘奕佳.表达二穗短柄草CBF2基因拟南芥抗旱性分析[J].草业科学,2016,33(8):1518-1525.ZHANG Y N,JIAO M Y,LIU Y J.Drought resistance of Arabidopsis thaliana expressing CBF2 gene in Brachypodiumdistachyon[J].Pratacultural Science,2016,33(8):1518-1525.
[17]李川.二穗短柄草抗寒冻机理研究及作为早熟禾亚科研究模式植物的综合评估[D].四川雅安:四川农业大学,2012.LI CH.Comparative analyses reveal potential uses of Brachypodiumdistachyon as a model for cold stress responses in pooideae[D].Ya’an Sichuan:Sichuan University,2012.
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[19]窦艳华,韩萌萌,孙其信,等.二穗短柄草MADS-BOX基因AGL6和FUL1的可变拼接和表达模式分析[J].农业生物技术学报,2015,23(4):459-468.DOU Y H,HAN M M,SUN Q X,et al.Alternative splicing and expression pattern analyses of two MADSBOXgenes AGL6and FUL1in brachypodium distachyon[J].Journal of Agricultural Biotechnology,2015,23(4):459-468.
[20]FERNANDEZ-CABALLERO C,ROSALES R,ROMEROI,et al.Unraveling the roles of CBF1,CBF4 and dehydrin1genes in the response of table grapes to high CO2levels and low temperature[J].Journal of Plant Physiology,2012,169(7):744-748.
[21]MBOUP M,FISCHER I,LAINER H,et al.Trans-species polymorphism and allele-specific expression in the CBFgene family of wild tomatoes[J].Molecular Biology and Evolution,2012,29(12):3641-3652.
[22]MEDINA J,BARGUES M,TEROL J,et al.The Arabidopsis CBF gene family is composed of three genes encoding AP2domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration[J].Plant Physiology,1999,119(2):463-470.
[23]GILMOUR S J,ZARKA D G,STOCKINGER E J,et al.Low temperature regulation of the Arabidopsis CBF family of AP2transcriptional activators as an early step in coldinduced COR gene expression[J].The Plant Journal,1998,16(4):433-442.
[24]冯勋伟,才宏伟.结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证[J].作物学报,2014,40(9):1572-1578.FENG X W,CAI H W.Cloning of zoysiagrass CBFgene and validation of cold tolerance in transgenic Arabidopsis[J].Acta Agronomica Sinica,2014,40(9):1572-1578.
[25]KRASENSKY J,JONAK C.Drought,salt,and temperature stress-induced metabolic rearrangements and regulatory networks[J].Journal of Experimental Botany,2012,63(4):1593.
[26]Q LIU M K,Y SAKUMA,H ABE,et al.Two transcription factors,DREB1and DREB2,with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406.
[27]李健湄,李逸豪,林莹莹,等.过表达AhAREB1影响GA代谢调控拟南芥的株型[J].植物生理学报,2014(7):925-930.LI J M,LI Y H,LIN Y Y,et al.Overexpression of AhA-REB1 affected plant type of Arabidopsis regulated by GAmetabolism[J].Journal of Plant Physiology,2014(7):925-930.
[28]CARRERA E,BOU J,GARC AMART NEZ J L,et al.Changes in GA 20-oxidase gene expression strongly affect stem length,tuber induction and tuber yield of potato plants[J].The Plant Journal,2000,22(3):247-256.
[29]MARUYAMA K,SAKUMA Y,KASUGA M,et al.Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3transcriptional factor using two microarray systems[J].The Plant Journal,2004,38(6):982-993.
[2]LATA C,PRASAD M.Role of DREBs in regulation of abiotic stress responses in plants[J].Journal of Experimental Botary,2011,62(14):4731.
[3]MIURA K,FURUMOTO T.Cold signaling and cold response in plants[J].International Journal of Molecular Sciences,2013,14(3):5312-5337.
[4]MIZOI J,SHINOZAKI K,YAMAGUCHISHINOZAKI K.AP2/ERF family transcription factors in plant abiotic stress responses[J].Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2012,1819(2):86-96.
[5]STOCKINGER E J,GILMOUR S J,THOMASHOW M F.Arabidopsis thaliana CBF1encodes an AP2domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Procceeding of the National Academy of Science of the United States of America,1997,94(3):1035-1040.
[6]SAKUMA Y,LIU Q,DUBOUZET J G,et al.DNA-binding specificity of the ERF/AP2domain 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]HAAKE V,COOK D,RIECHMANN J,et al.Transcription factor CBF4is a regulator of drought adaptation in Arabidopsis[J].Plant Physiology,2002,130(2):639-648.
[8]DUBOUZET J G,SAKUMA Y,ITO Y,et al.OsDREBgenes 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(4):751.
[9]SAZEGARI S,NIAZI A.Isolation and molecular characterization of wheat(Triticum aestivum)dehydration responsive element binding factor(DREB)isoforms[J].Australian Journal of Crop Science,2012,6(6):1037-1044.
[10]CHOI D W,RODRIGUEZ E M,CLOSE T J.Barley Cbf3gene identification,expression pattern,and map location[J].Plant Physiology,2002,129(4):1781-1787.
[11]AGARWAL P K,AGARWAL P,REDDY M K,et al.Role of DREBtranscription factors in abiotic and biotic stress tolerance in plants[J].Plant Cell Reports,2006,25(12):1263-1274.
[12]JIANG Q,HU Z,ZHANG H,et al.Overexpression of GmDREB1improves salt tolerance in transgenic wheat and leaf protein response to high salinity[J].The Crop Journal,2014(Z1):120-131.
[13]OPANOWICZ M,VAIN P,DRAPER J,et al.Brachypodium distachyon:making hay with a wild grass[J].Trends in Plant Science,2008,13(4):172-177.
[14]BEVAN M W,GARVIN D F,VOGEL J P.Brachypodium distachyon genomics for sustainable food and fuel production[J].Current Opinion in Biotechnology,2010,21(2):211-217.
[15]VOGEL J P,GARVIN D F,MOCKLER T C,et al.Genome sequencing and analysis of the model grass Brachypodiumdistachyon[J].Nature,2010,463(7282):763-768.
[16]张彦妮,焦孟月,刘奕佳.表达二穗短柄草CBF2基因拟南芥抗旱性分析[J].草业科学,2016,33(8):1518-1525.ZHANG Y N,JIAO M Y,LIU Y J.Drought resistance of Arabidopsis thaliana expressing CBF2 gene in Brachypodiumdistachyon[J].Pratacultural Science,2016,33(8):1518-1525.
[17]李川.二穗短柄草抗寒冻机理研究及作为早熟禾亚科研究模式植物的综合评估[D].四川雅安:四川农业大学,2012.LI CH.Comparative analyses reveal potential uses of Brachypodiumdistachyon as a model for cold stress responses in pooideae[D].Ya’an Sichuan:Sichuan University,2012.
[18]TELLORUIZ M K,NAITHANI S,STEIN J C,et al.Gramene 2018:unifying comparative genomics and pathway resources for plant research[J].Nucleic Acids Research,2018,46(D1):1181-1189.
[19]窦艳华,韩萌萌,孙其信,等.二穗短柄草MADS-BOX基因AGL6和FUL1的可变拼接和表达模式分析[J].农业生物技术学报,2015,23(4):459-468.DOU Y H,HAN M M,SUN Q X,et al.Alternative splicing and expression pattern analyses of two MADSBOXgenes AGL6and FUL1in brachypodium distachyon[J].Journal of Agricultural Biotechnology,2015,23(4):459-468.
[20]FERNANDEZ-CABALLERO C,ROSALES R,ROMEROI,et al.Unraveling the roles of CBF1,CBF4 and dehydrin1genes in the response of table grapes to high CO2levels and low temperature[J].Journal of Plant Physiology,2012,169(7):744-748.
[21]MBOUP M,FISCHER I,LAINER H,et al.Trans-species polymorphism and allele-specific expression in the CBFgene family of wild tomatoes[J].Molecular Biology and Evolution,2012,29(12):3641-3652.
[22]MEDINA J,BARGUES M,TEROL J,et al.The Arabidopsis CBF gene family is composed of three genes encoding AP2domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration[J].Plant Physiology,1999,119(2):463-470.
[23]GILMOUR S J,ZARKA D G,STOCKINGER E J,et al.Low temperature regulation of the Arabidopsis CBF family of AP2transcriptional activators as an early step in coldinduced COR gene expression[J].The Plant Journal,1998,16(4):433-442.
[24]冯勋伟,才宏伟.结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证[J].作物学报,2014,40(9):1572-1578.FENG X W,CAI H W.Cloning of zoysiagrass CBFgene and validation of cold tolerance in transgenic Arabidopsis[J].Acta Agronomica Sinica,2014,40(9):1572-1578.
[25]KRASENSKY J,JONAK C.Drought,salt,and temperature stress-induced metabolic rearrangements and regulatory networks[J].Journal of Experimental Botany,2012,63(4):1593.
[26]Q LIU M K,Y SAKUMA,H ABE,et al.Two transcription factors,DREB1and DREB2,with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406.
[27]李健湄,李逸豪,林莹莹,等.过表达AhAREB1影响GA代谢调控拟南芥的株型[J].植物生理学报,2014(7):925-930.LI J M,LI Y H,LIN Y Y,et al.Overexpression of AhA-REB1 affected plant type of Arabidopsis regulated by GAmetabolism[J].Journal of Plant Physiology,2014(7):925-930.
[28]CARRERA E,BOU J,GARC AMART NEZ J L,et al.Changes in GA 20-oxidase gene expression strongly affect stem length,tuber induction and tuber yield of potato plants[J].The Plant Journal,2000,22(3):247-256.
[29]MARUYAMA K,SAKUMA Y,KASUGA M,et al.Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3transcriptional factor using two microarray systems[J].The Plant Journal,2004,38(6):982-993.