中间锦鸡儿CiMYB60基因克隆及表达分析
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摘要
本研究以中间锦鸡儿作为实验材料,利用PCR技术分别对CiMYB60基因的cDNA与gDNA进行了克隆。测序结果表明:CiMYB60基因的开放阅读框为1 035 bp,编码345个氨基酸,其基因组DNA序列长度为1 485 bp,含有2个内含子和3个外显子。生物信息学分析显示:CiMYB60所编码蛋白的N端包含2个MYB结构域,因此认为其属于典型的R2R3-MYB类蛋白。克隆得到CiMYB60基因的ATG上游序列1 848 bp,分析显示该启动子中包含一些与光反应、组织特异性表达、激素和非生物胁迫相关的响应元件。对CiMYB60基因的表达模式进行分析发现在脱水、NaCl和UV-B处理下其表达量随胁迫处理时间的延长而降低。上述研究结果表明CiMYB60基因可能参与中间锦鸡儿对非生物胁迫等逆境胁迫的响应过程,为进一步研究该基因在非生物胁迫中的功能和表达调控提供了一定的参考。
In this study, C. in termedia was used as materials, and the cDNA and gDNA of CiMYB60 gene was cloned by PCR technique. Sequencing results indicate that CiMYB60 had an open reading frame of 1 035 bp,encoding 345 amino acids. The full-length gDNA of CiMYB60 was 1 485 bp, including two introns and three exons. Bioinformatics analysis showed that N-terminal of CiMYB60 contained two MYB domains, so it was regarded as a typical R2 R3-MYB protein. 1 848 bpATG upstream sequence of CiMYB60 was obtained. Analysis showed that the promoter contained some response elements related to light response, tissue specific expression,hormone and abiotic stress. The expression pattern of CiMYB60 was analyzed, which revealed the expression of it was decreased with the prolongation of dehydration, NaCl and UV-B stress. These results demonstrated that CiMYB60 might be involved in the response of of C. intermedia to abiotic stress, which could provide reference for further study on the function and expression regulation of the gene in abiotic stress.
In this study, C. in termedia was used as materials, and the cDNA and gDNA of CiMYB60 gene was cloned by PCR technique. Sequencing results indicate that CiMYB60 had an open reading frame of 1 035 bp,encoding 345 amino acids. The full-length gDNA of CiMYB60 was 1 485 bp, including two introns and three exons. Bioinformatics analysis showed that N-terminal of CiMYB60 contained two MYB domains, so it was regarded as a typical R2 R3-MYB protein. 1 848 bpATG upstream sequence of CiMYB60 was obtained. Analysis showed that the promoter contained some response elements related to light response, tissue specific expression,hormone and abiotic stress. The expression pattern of CiMYB60 was analyzed, which revealed the expression of it was decreased with the prolongation of dehydration, NaCl and UV-B stress. These results demonstrated that CiMYB60 might be involved in the response of of C. intermedia to abiotic stress, which could provide reference for further study on the function and expression regulation of the gene in abiotic stress.
引文
Abe H.,Urao T.,Ito T.,Seki M.,Shinozaki K.,and YamaguchiShinozaki K.,2002,Arabidopsis At MYC2(b HLH)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling,The Plant Cell,15(1):63-78
Century K.,Reuber T.L.,and Ratcliffe O.J.,2008,Regulating the regulators:the future prospects for transcription-factor-based agricultural biotechnology products,Plant Physiol.,147(1):20-29
Cominelli E.,Galbiati M.,Vavasseur A.,Conti L.,Sala T.,Vuylsteke M.,Leonhardt N.,Dellaporta S.L.,and Tonelli C.,2005,A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance,Curr Biol.,15(13):1196-1200
Du H.,Yang S.S.,Liang Z.,Feng B.R.,Liu L.,Huang Y.B.,and Tang Y.X.,2012,Genome-wide analysis of the MYB transcription factor superfamily in soybean,BMC Plant Biol.,12(1):1-22
Dubos C.,Stracke R.,Grotewold E.,Weisshaar B.,Martin C.and Lepiniec L.,2010,MYB transcription factors in Arabidopsis,Trends Plant Sci.,15(10):573-581
Galbiati M.,Matue J.T.,Francia P.,Rusconi F.,Caón P.,Medina C.,Conti L.,Cominelli E.,Tonelli C.,and Arce-Johnson P.2011,The grapevine guard cell-related Vv MYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress,BMC Plant Biol.,11(1):142
Han X.M.,2015,The cloning and analysis of three stress-related transcription factors from Caragana intermedia,Dissertation for Ph.D.,College of Life Sciences,Inner Mongolia Agricultural University,Supervisor:Li G.J.,pp.24-27(韩晓敏,2015中间锦鸡儿3个非生物胁迫相关转录因子的克隆与功能分析,博士学位论文,内蒙古农业大学生命科学学院,导师李国婧,pp.24-27)
Jung C.,Seo J.S.,Han S.W.,Koo Y.J.,Kim C.H.,Song S.I.Nahm B.H.,Choi Y.D.,and Cheong J.J.,2008,Overexpression of At MYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic,Arabidopsis Plant Biol.146(2):623-635
Liu L.,Du H.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.2008,The roles of MYB transcription factors on plan defense responses and its molecular mechanism,Yichuan(Hereditas),30(10):1265-1271(刘蕾,杜海,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,MYB转录因子在植物抗逆胁迫中的作用及其分子机理,遗传,30(10):1265-1271)
Liu L.Y.,Jia Z.Q.,Zhu Y.J.,Liu J.M.,Yang D.F.,and Wei D.X.2012,Water use strategy of different stand ages of Caragana intermedia in Alpine sandland,Ganhanqu Ziyuan Yu Huanjing(Journal of Arid Land Resources and Environment),26(5):119-125(刘丽颖,贾志清,朱雅娟,刘君梅,杨德福,魏登贤,2012,高寒沙地不同林龄中间锦鸡儿的水分利用策略,干旱区资源与环境,26(5):119-125)
Ogata K.,Morikawa S.,Nakamura H.,Hojo H.,Yoshimura S.Zhang R.,Aimoto S.,Ametani Y.,Hirata Z.,and Sarai A.1995,Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb,Nat.Struct.Biol.,2(4):309-320
Ogata K.,Kanei-Ishii C.,Sasaki M.,Hatanaka H.,Nagadoi A.,Enari M.,Nakamura H.,Nishimura Y.,Ishii S.,and Sarai A.,1996,The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and transactivation,Nat.Struct.Biol.,3(2):178-187
Ogata K.,Morikawa S.,Nakamura H.,Sekikawa A.,Inoue T.,Kanai H.,Sarai A.,Ishii S.,and Nishimura Y.,1994,Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices,Cell,79(4):639-648
Oh J.E.,Kwon Y.,Kim J.H.,Noh H.,Hong S.W.,and Lee H.J.,2011,A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress,Plant Mol.Biol.,77(1-2):91-103
Park J.S.,Kim J.B.,Cho K.J.,Cheon C.I.,Sung M.K.,Choung M.G.,and Roh K.H.,2008,Arabidopsis R2R3-MYBtranscription factor At MYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce(Lactuca sativa),Plant Cell Rep.,27(6):985-994
Riechamann J.L.,Heard J.,Martin G.,Reuber L.,Jiang C.,Keddie J.,Adam L.,Pineda O.,Ratcliffe O.J.,Samaha R.R.,Creelman R.,Pilgrim M.,Broun P.,Zhang J.Z.,Ghandehari D.,Sherman B.K.,and Yu G.,2000,Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes,Science,290(5499):2105-2110
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYBgene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456
Su L.T.,Wang Y.,Liu D.Q.,Li X.W.,Zhai Y.,Sun X.,Li X.Y.,Liu Y.J.,Li J.W.,and Wang Q.Y.,2015,The soybean gene,Gm MYBJ2,encodes a R2R3-type transcription factor involved in drought stress tolerance in Arabidopsis thaliana,Acta Physiol.Plant,37(7):1-12
Xiao D.C.,Zhang Z.J.,Xu Y.W.,Yang L.,Zhang F.X.,and Wang C.L.,2013,Cloning and functional analysis of Phyllostachys edulis MYB transcription factor Pe MYB2,Yichuan(Hereditas),35(10):1217-1225(肖冬长,张智俊,徐英武,杨丽,张凤雪,王超莉,2013,毛竹MYB转录因子Pe MYB2的克隆与功能分析,遗传,35(10):1217-1225)
Century K.,Reuber T.L.,and Ratcliffe O.J.,2008,Regulating the regulators:the future prospects for transcription-factor-based agricultural biotechnology products,Plant Physiol.,147(1):20-29
Cominelli E.,Galbiati M.,Vavasseur A.,Conti L.,Sala T.,Vuylsteke M.,Leonhardt N.,Dellaporta S.L.,and Tonelli C.,2005,A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance,Curr Biol.,15(13):1196-1200
Du H.,Yang S.S.,Liang Z.,Feng B.R.,Liu L.,Huang Y.B.,and Tang Y.X.,2012,Genome-wide analysis of the MYB transcription factor superfamily in soybean,BMC Plant Biol.,12(1):1-22
Dubos C.,Stracke R.,Grotewold E.,Weisshaar B.,Martin C.and Lepiniec L.,2010,MYB transcription factors in Arabidopsis,Trends Plant Sci.,15(10):573-581
Galbiati M.,Matue J.T.,Francia P.,Rusconi F.,Caón P.,Medina C.,Conti L.,Cominelli E.,Tonelli C.,and Arce-Johnson P.2011,The grapevine guard cell-related Vv MYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress,BMC Plant Biol.,11(1):142
Han X.M.,2015,The cloning and analysis of three stress-related transcription factors from Caragana intermedia,Dissertation for Ph.D.,College of Life Sciences,Inner Mongolia Agricultural University,Supervisor:Li G.J.,pp.24-27(韩晓敏,2015中间锦鸡儿3个非生物胁迫相关转录因子的克隆与功能分析,博士学位论文,内蒙古农业大学生命科学学院,导师李国婧,pp.24-27)
Jung C.,Seo J.S.,Han S.W.,Koo Y.J.,Kim C.H.,Song S.I.Nahm B.H.,Choi Y.D.,and Cheong J.J.,2008,Overexpression of At MYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic,Arabidopsis Plant Biol.146(2):623-635
Liu L.,Du H.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.2008,The roles of MYB transcription factors on plan defense responses and its molecular mechanism,Yichuan(Hereditas),30(10):1265-1271(刘蕾,杜海,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,MYB转录因子在植物抗逆胁迫中的作用及其分子机理,遗传,30(10):1265-1271)
Liu L.Y.,Jia Z.Q.,Zhu Y.J.,Liu J.M.,Yang D.F.,and Wei D.X.2012,Water use strategy of different stand ages of Caragana intermedia in Alpine sandland,Ganhanqu Ziyuan Yu Huanjing(Journal of Arid Land Resources and Environment),26(5):119-125(刘丽颖,贾志清,朱雅娟,刘君梅,杨德福,魏登贤,2012,高寒沙地不同林龄中间锦鸡儿的水分利用策略,干旱区资源与环境,26(5):119-125)
Ogata K.,Morikawa S.,Nakamura H.,Hojo H.,Yoshimura S.Zhang R.,Aimoto S.,Ametani Y.,Hirata Z.,and Sarai A.1995,Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb,Nat.Struct.Biol.,2(4):309-320
Ogata K.,Kanei-Ishii C.,Sasaki M.,Hatanaka H.,Nagadoi A.,Enari M.,Nakamura H.,Nishimura Y.,Ishii S.,and Sarai A.,1996,The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and transactivation,Nat.Struct.Biol.,3(2):178-187
Ogata K.,Morikawa S.,Nakamura H.,Sekikawa A.,Inoue T.,Kanai H.,Sarai A.,Ishii S.,and Nishimura Y.,1994,Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices,Cell,79(4):639-648
Oh J.E.,Kwon Y.,Kim J.H.,Noh H.,Hong S.W.,and Lee H.J.,2011,A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress,Plant Mol.Biol.,77(1-2):91-103
Park J.S.,Kim J.B.,Cho K.J.,Cheon C.I.,Sung M.K.,Choung M.G.,and Roh K.H.,2008,Arabidopsis R2R3-MYBtranscription factor At MYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce(Lactuca sativa),Plant Cell Rep.,27(6):985-994
Riechamann J.L.,Heard J.,Martin G.,Reuber L.,Jiang C.,Keddie J.,Adam L.,Pineda O.,Ratcliffe O.J.,Samaha R.R.,Creelman R.,Pilgrim M.,Broun P.,Zhang J.Z.,Ghandehari D.,Sherman B.K.,and Yu G.,2000,Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes,Science,290(5499):2105-2110
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYBgene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456
Su L.T.,Wang Y.,Liu D.Q.,Li X.W.,Zhai Y.,Sun X.,Li X.Y.,Liu Y.J.,Li J.W.,and Wang Q.Y.,2015,The soybean gene,Gm MYBJ2,encodes a R2R3-type transcription factor involved in drought stress tolerance in Arabidopsis thaliana,Acta Physiol.Plant,37(7):1-12
Xiao D.C.,Zhang Z.J.,Xu Y.W.,Yang L.,Zhang F.X.,and Wang C.L.,2013,Cloning and functional analysis of Phyllostachys edulis MYB transcription factor Pe MYB2,Yichuan(Hereditas),35(10):1217-1225(肖冬长,张智俊,徐英武,杨丽,张凤雪,王超莉,2013,毛竹MYB转录因子Pe MYB2的克隆与功能分析,遗传,35(10):1217-1225)