邓恩桉EdDREB2基因的克隆和表达分析
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摘要
邓恩桉是成功引种至中国的耐寒桉,为了深入挖掘邓恩桉耐寒基因资源,本研究利用同源克隆技术从邓恩桉叶片低温诱导的c DNA中分离出一个长度为1 392 bp的DNA序列,包含一个长度为972 bp的开放阅读框,编码323个氨基酸的蛋白质。该蛋白质具有一个AP2结构域,分子量为34.765 76 kD。聚类分析表明,该基因与冈尼桉的DREB2类基因和蓝桉的DREB2基因聚成一支,与蓝桉DREB2基因遗传关系最近,因此命名为EdDREB2。qRT-PCR分析表明,EdDREB2基因受低温诱导1 h时的表达量为1.08倍,至12 h时表达最高,说明该基因可能参与低温胁迫的调控。EdDREB2基因的克隆、生物信息学分析和表达分析为其功能的深入研究提供帮助。
Eucalyptus dunnii has been introduced into China as cold-resistant eucalyptus successfully. In order to explore cold-tolerant gene resources of Eucalyptus dunnii deeply, a DNA sequence of 1 392 bp with an openreading frame of 972 bp which coded 323 amino acids was isolated from the cold-induced cDNA of Eucalyptus dunnii leaves by homologous cloning technology. Molecular weight of this protein with a AP2 domain was 34.765 76 kD.Cluster analysis indicated that this gene was clustered with the DREB2 gene of Eucalyptus gunnii and DREB2 gene of Eucalyptus globulus, and had the closest genetic relationship with the DREB2 of Eucalyputs globulus, so it was named as EdDREB2. Quantit ative qPCR detection showed that the expression quantity of EdDREB2 was 1.08 times when induced by low temperature for 1 h, and was the highest when induced by low temperature for 12 h.This results indicated that this gene might participate in regulation of low temperature stress. The cloning,bioinformatic analysis and expression analysis of EdDREB2 gene would provide a solid fundamental for further functional study.
Eucalyptus dunnii has been introduced into China as cold-resistant eucalyptus successfully. In order to explore cold-tolerant gene resources of Eucalyptus dunnii deeply, a DNA sequence of 1 392 bp with an openreading frame of 972 bp which coded 323 amino acids was isolated from the cold-induced cDNA of Eucalyptus dunnii leaves by homologous cloning technology. Molecular weight of this protein with a AP2 domain was 34.765 76 kD.Cluster analysis indicated that this gene was clustered with the DREB2 gene of Eucalyptus gunnii and DREB2 gene of Eucalyptus globulus, and had the closest genetic relationship with the DREB2 of Eucalyputs globulus, so it was named as EdDREB2. Quantit ative qPCR detection showed that the expression quantity of EdDREB2 was 1.08 times when induced by low temperature for 1 h, and was the highest when induced by low temperature for 12 h.This results indicated that this gene might participate in regulation of low temperature stress. The cloning,bioinformatic analysis and expression analysis of EdDREB2 gene would provide a solid fundamental for further functional study.
引文
Agarwal P.,Agarwal P.K.,Nair S.,Sopory S.K.,and Reddy M.K.,2007,Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity,Mol.Genet.Genomics,277(2):189-198
Chen J.H.,Xia X.L.,and Yin W.L.,2009,Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica,Biochem.Biophys.Res.Commun.,378(3):483-487
Chen J.Q.,Meng X.P.,Zhang Y.,Xia M.,and Wang X.P.,2008,Over-expression of OsDREB genes lead to enhanced drought tolerance in rice,Biotechnol.Lett.,30(12):2191-2198
Chinnusamy V.,Zhu J.,and Zhu J.K.,2007,Cold stress regulation of gene expression in plants,Trends Plant Sci.,12(10):444-451
Chinnusamy V.,Zhu J.K.,and Sunkar R.,2010,Gene regulation during cold stress acclimation in plants,Plant Stress Tolerance,639:39-55
Deng Z.Y.,Chen J.B.,and Zhou W.,2015,Advances of research on the cold hardiness of Eucalyptus dunnii in China,Anshu Keji(Eucalypt Science&Technology),32(4):40-45(邓紫宇,陈健波,周维,2015,中国邓恩桉抗寒性研究进展,桉树科技,32(4):40-45)
Dubouzet J.G.,Sakuma Y.,Ito Y.,Kasuga M.,Dubouzet E.G.,Miura S.,Seki M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2003,OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-saltand cold-responsive gene expression,Plant J.,33(4):751-763
Egawa C.,Kobayashi F.,Ishibashi M.,Nakamura T.,Nakamura C.,and Takumi S.,2006,Differential regulation of transcript accumulation and alternative splicing of a DREB2 homology under abiotic stress conditions in common wheat,Genes Genet.Syst.,81(2):77-91
Lata C.,and Prasad M.,2011,Role of DREBs in regulation of abiotic stress responses in plants,J.Exp.Bot.,62(14):4731-4748
Liu L.Q.,Zhu K.,Yang Y.F.,Wu J.,Chen F.D.,and Yu D.Y.,2008,Molecular cloning,expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum(Dendranthema vestitum),J.Plant Res.,121(2):215-226
Liu Q.,Kasuga M.,Sakuma Y.,Abe H.,Miura S.,Yamaguchi-Shinozaki K.,and Shinozaki K.,1998,Two transcription factors,DREB1 and DREB2,with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis,Plant Cell,10(8):1391-1406
Nakano T.,Suzuki K.,Fujimura T.,and Shinshi H.,2006,Genome-wide analysis of the ERF gene family in Arabidopsis and rice,Plant Physiol.,140(2):411-432
Navarro M.,Ayax C.,Martinez Y.,Laur J.,El Kayal W.,Marque C.,and Teulieres C.,2010,Two EguCBF1 genes overexpressed in Eucalyptus display a different impact on stress tolerance and plant development,Plant Biotechnol.J.,9(1):50-63
Navarro M.,Marque G.,Ayax C.,Keller G.,Borges J.P.,Marque C.,and Teulieres C.,2009,Complementary regulation of four Eucalyptus CBF genes under various cold conditions,J.Exp.Bot.,60(9):2713-2724
Qin F.,Kakimoto M.,Sakuma Y.,Maruyama K.,Osakabe Y.,Tran L.S.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2007,Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L.,Plant J.,50(1):54-69
Sakuma Y.,Liu Q.,Dubouzet J.G.,Abe H.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2002,DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression,Biochem.Biophys.Res.Commun.,290(3):998-1009
Shi Y.T.,Ding Y.L.,and Yang S.H.,2018,Molecular regulation of CBF signaling in cold acclimation,Trends Plant Sci.,23(7):623-637
Wang P.L.,Jiang F.X.,Cai L.,Chen X.M.,Tan Z.H.,Chen B.W.,and Wu Y.M.,2013,Construction of genetic transformation system for Eucalyptus grandis×E.urophylla'GLGU9',Linye Keji Kaifa(China Forestry Science and Technology),27(3):76-80(王鹏良,姜福星,蔡玲,陈晓明,覃子海,陈博雯,吴幼媚,2013,广林巨尾桉9号遗传转化体系的建立,林业科技开发,27(3):76-80)
Wang Y.H.,Wu Y.M.,and Cai L.,2008,Effects of several factors on adventitious bud induction of Eucalyptus dunnii,Xibu Linye Kexue(Journal of West China Forestry Science),37(1):24-28(王以红,吴幼媚,蔡玲,2008,几种因子对邓恩桉组培中不定芽培养影响的研究,西部林业科学,37(1):24-28)
Chen J.H.,Xia X.L.,and Yin W.L.,2009,Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica,Biochem.Biophys.Res.Commun.,378(3):483-487
Chen J.Q.,Meng X.P.,Zhang Y.,Xia M.,and Wang X.P.,2008,Over-expression of OsDREB genes lead to enhanced drought tolerance in rice,Biotechnol.Lett.,30(12):2191-2198
Chinnusamy V.,Zhu J.,and Zhu J.K.,2007,Cold stress regulation of gene expression in plants,Trends Plant Sci.,12(10):444-451
Chinnusamy V.,Zhu J.K.,and Sunkar R.,2010,Gene regulation during cold stress acclimation in plants,Plant Stress Tolerance,639:39-55
Deng Z.Y.,Chen J.B.,and Zhou W.,2015,Advances of research on the cold hardiness of Eucalyptus dunnii in China,Anshu Keji(Eucalypt Science&Technology),32(4):40-45(邓紫宇,陈健波,周维,2015,中国邓恩桉抗寒性研究进展,桉树科技,32(4):40-45)
Dubouzet J.G.,Sakuma Y.,Ito Y.,Kasuga M.,Dubouzet E.G.,Miura S.,Seki M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2003,OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-saltand cold-responsive gene expression,Plant J.,33(4):751-763
Egawa C.,Kobayashi F.,Ishibashi M.,Nakamura T.,Nakamura C.,and Takumi S.,2006,Differential regulation of transcript accumulation and alternative splicing of a DREB2 homology under abiotic stress conditions in common wheat,Genes Genet.Syst.,81(2):77-91
Lata C.,and Prasad M.,2011,Role of DREBs in regulation of abiotic stress responses in plants,J.Exp.Bot.,62(14):4731-4748
Liu L.Q.,Zhu K.,Yang Y.F.,Wu J.,Chen F.D.,and Yu D.Y.,2008,Molecular cloning,expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum(Dendranthema vestitum),J.Plant Res.,121(2):215-226
Liu Q.,Kasuga M.,Sakuma Y.,Abe H.,Miura S.,Yamaguchi-Shinozaki K.,and Shinozaki K.,1998,Two transcription factors,DREB1 and DREB2,with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis,Plant Cell,10(8):1391-1406
Nakano T.,Suzuki K.,Fujimura T.,and Shinshi H.,2006,Genome-wide analysis of the ERF gene family in Arabidopsis and rice,Plant Physiol.,140(2):411-432
Navarro M.,Ayax C.,Martinez Y.,Laur J.,El Kayal W.,Marque C.,and Teulieres C.,2010,Two EguCBF1 genes overexpressed in Eucalyptus display a different impact on stress tolerance and plant development,Plant Biotechnol.J.,9(1):50-63
Navarro M.,Marque G.,Ayax C.,Keller G.,Borges J.P.,Marque C.,and Teulieres C.,2009,Complementary regulation of four Eucalyptus CBF genes under various cold conditions,J.Exp.Bot.,60(9):2713-2724
Qin F.,Kakimoto M.,Sakuma Y.,Maruyama K.,Osakabe Y.,Tran L.S.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2007,Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L.,Plant J.,50(1):54-69
Sakuma Y.,Liu Q.,Dubouzet J.G.,Abe H.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2002,DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression,Biochem.Biophys.Res.Commun.,290(3):998-1009
Shi Y.T.,Ding Y.L.,and Yang S.H.,2018,Molecular regulation of CBF signaling in cold acclimation,Trends Plant Sci.,23(7):623-637
Wang P.L.,Jiang F.X.,Cai L.,Chen X.M.,Tan Z.H.,Chen B.W.,and Wu Y.M.,2013,Construction of genetic transformation system for Eucalyptus grandis×E.urophylla'GLGU9',Linye Keji Kaifa(China Forestry Science and Technology),27(3):76-80(王鹏良,姜福星,蔡玲,陈晓明,覃子海,陈博雯,吴幼媚,2013,广林巨尾桉9号遗传转化体系的建立,林业科技开发,27(3):76-80)
Wang Y.H.,Wu Y.M.,and Cai L.,2008,Effects of several factors on adventitious bud induction of Eucalyptus dunnii,Xibu Linye Kexue(Journal of West China Forestry Science),37(1):24-28(王以红,吴幼媚,蔡玲,2008,几种因子对邓恩桉组培中不定芽培养影响的研究,西部林业科学,37(1):24-28)
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