青稞HD-ZipⅣ 5基因克隆及表达探究
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摘要
同源异型域-亮氨酸拉链(homeodomain-leucine zipper, HD-Zip)蛋白是高等植物特有的转录因子,在植物的生长、发育和抗逆适应中发挥重要的调控作用。本研究通过RT-PCR技术,从青稞(肚里黄品种)中克隆了HD-ZipⅣ5基因的CDS序列,进行生物信息学分析,并以大麦(Morex)为对照,利用荧光定量PCR(qPCR)比较分析了HD-ZipⅣ5在青稞不同组织和不同胁迫条件下的表达模式。结果表明,青稞HD-ZipⅣ5基因的CDS序列(2 388 bp)编码了796个氨基酸,与大麦HD-ZipⅣ5基因序列相比存在8个SNP变异,生物信息学分析显示该蛋白分子质量为85 679.38 Da,等电点为6.08,具有亲水性。qPCR结果显示青稞各组织中HD-ZipⅣ5在叶片中表达量最高,与大麦的组织表达模式一致;冷、盐及干旱胁迫下,青稞幼苗叶片HD-ZipⅣ5受盐胁迫诱导强烈表达,根HD-ZipⅣ5对干旱胁迫显著响应,与大麦中表达模式存在差异。本研究为HD-ZipⅣ5可能在青稞的叶片生长发育及抗逆适应中的作用提供科学依据。
Homeodomain-Leucine Zipper(HD-Zip) protein is a transcription factor unique to higher plants and plays an important regulatory role in plant growth, development and stress tolerance. In this study, the CDS sequence of HD-ZipⅣ 5 gene was cloned from Tibet Hulless barley(Dulihuang variety) by RT-PCR, and bioinformatics analysis was performed. Using barley(Morex) as a control, quantitative PCR(qPCR) was used. The expression patterns of HD-ZipⅣ 5 in different tissues and different stress conditions were studied. The results showed that the CDS sequence of the HD-ZipⅣ 5 gene(2 388 bp) encoded 796 amino acids, and there were 8 SNPs variants compared with the HD-ZipⅣ 5 gene sequence of barley. Bioinformatics analysis showed that the protein molecule in Tibet Hulless barley the mass is 85 679.38 Da, and the isoelectric point is 6.08, which is hydrophilic. The results of qPCR showed that HD-ZipⅣ 5 had the highest leaf expression in Tibet Hulless barley, which was consistent with the tissue expression pattern of barley; Under cold, salt and drought stress, HD-ZipⅣ 5 in leaves of Tibet Hulless barley seedlings was strongly expressed by salt stress, and root HD-Zip Ⅳ 5 responded significantly to drought stress,which was different between the expression patterns in barley. This paper provides a theoretical basis for the role of HD-Zip Ⅳ 5 in leaf growth and stress tolerance in Tibet Hulless barley.
Homeodomain-Leucine Zipper(HD-Zip) protein is a transcription factor unique to higher plants and plays an important regulatory role in plant growth, development and stress tolerance. In this study, the CDS sequence of HD-ZipⅣ 5 gene was cloned from Tibet Hulless barley(Dulihuang variety) by RT-PCR, and bioinformatics analysis was performed. Using barley(Morex) as a control, quantitative PCR(qPCR) was used. The expression patterns of HD-ZipⅣ 5 in different tissues and different stress conditions were studied. The results showed that the CDS sequence of the HD-ZipⅣ 5 gene(2 388 bp) encoded 796 amino acids, and there were 8 SNPs variants compared with the HD-ZipⅣ 5 gene sequence of barley. Bioinformatics analysis showed that the protein molecule in Tibet Hulless barley the mass is 85 679.38 Da, and the isoelectric point is 6.08, which is hydrophilic. The results of qPCR showed that HD-ZipⅣ 5 had the highest leaf expression in Tibet Hulless barley, which was consistent with the tissue expression pattern of barley; Under cold, salt and drought stress, HD-ZipⅣ 5 in leaves of Tibet Hulless barley seedlings was strongly expressed by salt stress, and root HD-Zip Ⅳ 5 responded significantly to drought stress,which was different between the expression patterns in barley. This paper provides a theoretical basis for the role of HD-Zip Ⅳ 5 in leaf growth and stress tolerance in Tibet Hulless barley.
引文
Ahuja I.,de Vos R.C.H.,Bones A.M.,and Hall R.D.,2010,Plant molecular stress responses face climate change,Trends in Plant Science,15(12):664-674
Ariel F.D.,Manavella P.A.,Dezar C.A.,and Chan R.L.,2007,The true story of the HD-Zip family,Trends in Plant Science,12(9):419-426
Bürglin R.T.,and Affolter M.,2016,Homeodomain proteins:an update,Chromosoma,125(3):497-521
Cai J.,Li P.F.,Luo X.,Chang T.L.,Li J.X.,Zhao Y.W.,and Xu Y.,2018,Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan Hulless Barley,PLoS One,13(1):e0190559
Chen X.,Long H.,Gao P.,Deng G.B.,Pan Z.F.,Liang J.J.,Tang Y.W.,Tashi N.,and Yu M.Q.,2014,Transcriptome assembly and analysis of tibetan hulless barley(Hordeum vulgare L.var.nudum)developing grains,with emphasis on quality properties,PLoS One,9(5):e98144
Chen X.C.,and Xiong D.J.,2010,Research progress in plant stress resistance,Hubei Nongye Kexue(Hubei Agricultural Sciences),49(9):2253-2256(陈秀晨,熊冬金,2010,植物抗逆性研究进展,湖北农业科学,49(9):2253-2256)
Chew W.,Hrmova M.,and Lopato S.,2013,Role of homeodomain leucine zipper(HD-Zip)Ⅳtranscription factors in plant development and plant protection from deleterious environmental factors,International Journal of Molecular Sciences,14(4):8122-8147
Cristina M.D.,Sessa G.,Dolan L.,Linstead P.,Baima S.,Ruberti I.,and Morelli G.,1996,The Arabidopsis Athb-10(GLA-BRA2)is an HD-Zip protein required for regulation of root hair development,The Plant Journal,10(3):393-402
Elhiti M.,and Stasolla C.,2009,Structure and function of homodomain-leucine zipper(HD-Zip)proteins,Plant Signaling&Behavior,4(2):86-88
Fu R.,2013,Bioinformatics and function preliminary analysis of HD-ZipⅣgenes in cucumber,Thesis for M.S.,Shandong A-gricultural University,Supervisor:Ren Z.H.,pp.23-30(付娆,2013,黄瓜HD-ZipⅣ基因的生物信息学及功能初步分析,硕士学位论文,山东农业大学,导师:任仲海,pp.23-30)
Fu R.,Liu W.,Li Q.,Li J.,Wang L.,and Ren Z.H.,2013,Comprehensive analysis of the homeodomain-leucine zipperⅣtranscription factor family in Cucumis sativus,Genome,56(7):395-405
He Z.Z.,Xing J.J.,Chen L.,and Lan H.Y.,2013,Review on progress of stress tolerance mechanisms of plant seedlings,Shengwu Jishu Tongbao(Biotechnology Bulletin),1(2):1-7(贺转转,邢佳佳,陈玲,兰海燕,2013,植物幼苗抗逆机制研究进展,生物技术通报,1(2):1-7)
Johannesson H.,Wang Y.,and Engstr觟m P.,2001,DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro,Plant Mol.Biol.,45(1):63-73
Kovalchuk N.,Wu W.,Eini O.,Bazanova N.,Pallotta M.,Shirley N.,Singh R.,Ismagul A.,Eliby S.,Johnson A.,Langridge P.,and Lopato S.,2012,The scutellar vascular bundle-specific promoter of the wheat HD-ZipⅣtranscription factor shows similar spatial and temporal activity in transgenic wheat,barley and rice,Plant Biotechnol.J.,10(1):43-53
Li B.,Meng X.Z.,Shan L.B.,and Ping H.,2016,Transcriptional regulation of pattern-triggered immunity in plants,Cell Host&Microbe,19(5):641-650
Li Y.D.,Zhu Y.X.,Yao J.,Zhang S.L.,Wang L.,Guo C.L.,Van Nocker S.,and Wang X.P.,2017a,Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes,Scientific Reports,7(1):12638
Li Z.Q.,Zhang C.,Guo Y.R.,Niu W.L.,Wang Y.J.,and Xu Y.,2017b,Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes(Vitis vinifera L.),BMC Genomics,18(1):744
Lin Z.F.,Hong Y.G.,Yin M.G.,Li C.Y.,Zhang K.,and Grierson D.,2008,A tomato HD-Zip homeobox protein,LeHB-1,plays an important role in floral organogenesis and ripening,The Plant Journal,55(2):301-310
Ma J.,and Ptashne M.,1987,A new class of yeast transcriptional activators,Cell,51(1):113-119
Nakamura M.,Katsumata H.,Abe M.,Yabe N.,Komeda Y.,Yamamoto K.T.,and Takahashi T.,2006,Characterization of the class IV homeodomain-leucine zipper gene family in Arabidopsis,Plant Physiol.,141(4):1363-1375
Perotti M.F.,Ribone P.A.,and Chan R.L.,2017,Plant transcription factors from the homeodomain-leucine zipper family I.role in development and stress responses,IUBMB Life,69(5):280-289
Roodbarkelari F.,and Groot E.P.,2017,Regulatory function of homeodomain-leucine zipper(HD-Zip)family proteins during embryogenesis,New Phytologist,213(1):95-104
Ruan L.,Chen L.J.,Chen Y.H.,He J.L.,Zhang W.,Gao Z.L.,and Zhang Y.H.,2012,Expression of Arabidopsis HOMEO-DOMAIN GLABROUS 11 enhances tolerance to drought stress in transgenic sweet potato plants,J.Plant Biol.,55(2):151-158
Verslues P.E.,Agarwal M.,Katiyar-agarwal S.,Zhu J.H.,and Zhu J.K.,2006,Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status,The Plant Journal,45(4):523-539
Wang H.,Li G.B.,Zhang D.Y.,Lin J.,Sheng B.L.,Han J.L.,and Chang Y.H.,2013,Biological functions of HD-Zip transcrip tion factors,Yichuan(Hereditas),35(10):1179-1188(王宏,李刚波,张大勇,蔺经,盛宝龙,韩金龙,常有宏,2013,植物HD-Zip转录因子的生物学功能,遗传,35(10):1179-1188)
Wang R.,Lu L.L.,Liu T.,Zhang Z.Q.,Ma Z.B.,Li L.L.,Liu W.,Yang Z.R.,and Zhu W.,2017,Genome-wide identification and characterization of the HD-ZipⅣgene family in cotton(Gossypium spp.),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):2921-2936(王瑞,鲁丽丽,刘拓,章志强,马宗斌,李伶俐,刘伟,杨作仁,朱伟,2017,棉花中HD-ZipⅣ家族基因的全基因组鉴定及特征分析,分子植物育种,15(8):2921-2936)
Xiong H.Y.,Zhang C.,Duo J.C.,Zhou W.,and Duan R.J.,2018,Bioinformatic analysis of the gene HD-ZipⅣfamily in barley,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(2):352-361(熊辉岩,张超,多杰措,周武,段瑞君,2018,大麦HD-ZipⅣ基因家族的生物信息学分析,分子植物育种,16(2):352-361)
Yu H.,Chen X.,Hong Y.Y.,Wang Y.,Xu P.,Ke S.D.,Liu H.Y.,Zhu J.K.,Oliver D.J.,and Xianga C.B.,2008,Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density,The Plant Cell,20(4):1134-1151
Yu L.H.,Chen X.,Wang Z.,Wang S.M.,Wang Y.P.,Zhu Q.S.,Li S.G.,and Xiang C.B.,2013,Arabidopsis enhanced drought tolerance1/HOMEODOMAIN GLABROUS11 confers drought tolerance in transgenic rice without yield penalty,Plant Physiol.,162(3):1378-1391
Ariel F.D.,Manavella P.A.,Dezar C.A.,and Chan R.L.,2007,The true story of the HD-Zip family,Trends in Plant Science,12(9):419-426
Bürglin R.T.,and Affolter M.,2016,Homeodomain proteins:an update,Chromosoma,125(3):497-521
Cai J.,Li P.F.,Luo X.,Chang T.L.,Li J.X.,Zhao Y.W.,and Xu Y.,2018,Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan Hulless Barley,PLoS One,13(1):e0190559
Chen X.,Long H.,Gao P.,Deng G.B.,Pan Z.F.,Liang J.J.,Tang Y.W.,Tashi N.,and Yu M.Q.,2014,Transcriptome assembly and analysis of tibetan hulless barley(Hordeum vulgare L.var.nudum)developing grains,with emphasis on quality properties,PLoS One,9(5):e98144
Chen X.C.,and Xiong D.J.,2010,Research progress in plant stress resistance,Hubei Nongye Kexue(Hubei Agricultural Sciences),49(9):2253-2256(陈秀晨,熊冬金,2010,植物抗逆性研究进展,湖北农业科学,49(9):2253-2256)
Chew W.,Hrmova M.,and Lopato S.,2013,Role of homeodomain leucine zipper(HD-Zip)Ⅳtranscription factors in plant development and plant protection from deleterious environmental factors,International Journal of Molecular Sciences,14(4):8122-8147
Cristina M.D.,Sessa G.,Dolan L.,Linstead P.,Baima S.,Ruberti I.,and Morelli G.,1996,The Arabidopsis Athb-10(GLA-BRA2)is an HD-Zip protein required for regulation of root hair development,The Plant Journal,10(3):393-402
Elhiti M.,and Stasolla C.,2009,Structure and function of homodomain-leucine zipper(HD-Zip)proteins,Plant Signaling&Behavior,4(2):86-88
Fu R.,2013,Bioinformatics and function preliminary analysis of HD-ZipⅣgenes in cucumber,Thesis for M.S.,Shandong A-gricultural University,Supervisor:Ren Z.H.,pp.23-30(付娆,2013,黄瓜HD-ZipⅣ基因的生物信息学及功能初步分析,硕士学位论文,山东农业大学,导师:任仲海,pp.23-30)
Fu R.,Liu W.,Li Q.,Li J.,Wang L.,and Ren Z.H.,2013,Comprehensive analysis of the homeodomain-leucine zipperⅣtranscription factor family in Cucumis sativus,Genome,56(7):395-405
He Z.Z.,Xing J.J.,Chen L.,and Lan H.Y.,2013,Review on progress of stress tolerance mechanisms of plant seedlings,Shengwu Jishu Tongbao(Biotechnology Bulletin),1(2):1-7(贺转转,邢佳佳,陈玲,兰海燕,2013,植物幼苗抗逆机制研究进展,生物技术通报,1(2):1-7)
Johannesson H.,Wang Y.,and Engstr觟m P.,2001,DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro,Plant Mol.Biol.,45(1):63-73
Kovalchuk N.,Wu W.,Eini O.,Bazanova N.,Pallotta M.,Shirley N.,Singh R.,Ismagul A.,Eliby S.,Johnson A.,Langridge P.,and Lopato S.,2012,The scutellar vascular bundle-specific promoter of the wheat HD-ZipⅣtranscription factor shows similar spatial and temporal activity in transgenic wheat,barley and rice,Plant Biotechnol.J.,10(1):43-53
Li B.,Meng X.Z.,Shan L.B.,and Ping H.,2016,Transcriptional regulation of pattern-triggered immunity in plants,Cell Host&Microbe,19(5):641-650
Li Y.D.,Zhu Y.X.,Yao J.,Zhang S.L.,Wang L.,Guo C.L.,Van Nocker S.,and Wang X.P.,2017a,Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes,Scientific Reports,7(1):12638
Li Z.Q.,Zhang C.,Guo Y.R.,Niu W.L.,Wang Y.J.,and Xu Y.,2017b,Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes(Vitis vinifera L.),BMC Genomics,18(1):744
Lin Z.F.,Hong Y.G.,Yin M.G.,Li C.Y.,Zhang K.,and Grierson D.,2008,A tomato HD-Zip homeobox protein,LeHB-1,plays an important role in floral organogenesis and ripening,The Plant Journal,55(2):301-310
Ma J.,and Ptashne M.,1987,A new class of yeast transcriptional activators,Cell,51(1):113-119
Nakamura M.,Katsumata H.,Abe M.,Yabe N.,Komeda Y.,Yamamoto K.T.,and Takahashi T.,2006,Characterization of the class IV homeodomain-leucine zipper gene family in Arabidopsis,Plant Physiol.,141(4):1363-1375
Perotti M.F.,Ribone P.A.,and Chan R.L.,2017,Plant transcription factors from the homeodomain-leucine zipper family I.role in development and stress responses,IUBMB Life,69(5):280-289
Roodbarkelari F.,and Groot E.P.,2017,Regulatory function of homeodomain-leucine zipper(HD-Zip)family proteins during embryogenesis,New Phytologist,213(1):95-104
Ruan L.,Chen L.J.,Chen Y.H.,He J.L.,Zhang W.,Gao Z.L.,and Zhang Y.H.,2012,Expression of Arabidopsis HOMEO-DOMAIN GLABROUS 11 enhances tolerance to drought stress in transgenic sweet potato plants,J.Plant Biol.,55(2):151-158
Verslues P.E.,Agarwal M.,Katiyar-agarwal S.,Zhu J.H.,and Zhu J.K.,2006,Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status,The Plant Journal,45(4):523-539
Wang H.,Li G.B.,Zhang D.Y.,Lin J.,Sheng B.L.,Han J.L.,and Chang Y.H.,2013,Biological functions of HD-Zip transcrip tion factors,Yichuan(Hereditas),35(10):1179-1188(王宏,李刚波,张大勇,蔺经,盛宝龙,韩金龙,常有宏,2013,植物HD-Zip转录因子的生物学功能,遗传,35(10):1179-1188)
Wang R.,Lu L.L.,Liu T.,Zhang Z.Q.,Ma Z.B.,Li L.L.,Liu W.,Yang Z.R.,and Zhu W.,2017,Genome-wide identification and characterization of the HD-ZipⅣgene family in cotton(Gossypium spp.),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(8):2921-2936(王瑞,鲁丽丽,刘拓,章志强,马宗斌,李伶俐,刘伟,杨作仁,朱伟,2017,棉花中HD-ZipⅣ家族基因的全基因组鉴定及特征分析,分子植物育种,15(8):2921-2936)
Xiong H.Y.,Zhang C.,Duo J.C.,Zhou W.,and Duan R.J.,2018,Bioinformatic analysis of the gene HD-ZipⅣfamily in barley,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(2):352-361(熊辉岩,张超,多杰措,周武,段瑞君,2018,大麦HD-ZipⅣ基因家族的生物信息学分析,分子植物育种,16(2):352-361)
Yu H.,Chen X.,Hong Y.Y.,Wang Y.,Xu P.,Ke S.D.,Liu H.Y.,Zhu J.K.,Oliver D.J.,and Xianga C.B.,2008,Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density,The Plant Cell,20(4):1134-1151
Yu L.H.,Chen X.,Wang Z.,Wang S.M.,Wang Y.P.,Zhu Q.S.,Li S.G.,and Xiang C.B.,2013,Arabidopsis enhanced drought tolerance1/HOMEODOMAIN GLABROUS11 confers drought tolerance in transgenic rice without yield penalty,Plant Physiol.,162(3):1378-1391