烟草转录因子bHLH93基因的克隆及表达分析
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摘要
碱性/螺旋-环-螺旋(basic/helix-loop-helix,b HLH)转录因子是一类广泛存在于真核生物体内的重要转录因子,能够参与调控植物体多种生理途径。为了明确b HLH转录因子在烟草中的功能和调控机制,通过同源克隆技术从烟草中克隆到一个b HLH转录因子基因(Ntb HLH93),并结合生物信息学和基因表达模式分析进一步预测其生物学功能。结果表明:Ntb HLH93 c DNA序列长1 254 bp,包含一个长度为1 047 bp的开放阅读框,编码349个氨基酸。Ntb HLH93属于疏水性蛋白,其二级结构中的主要结构元件是α-螺旋和无规则卷曲,三级结构中具有b HLH家族典型的螺旋-环-螺旋结构特征。Ntb HLH93基因表达具有明显的组织特异性,主要在烟草不同生育期侧根和盛花期子房组织中高表达,这与植物甾醇集中在生长分裂旺盛组织中合成的特征相吻合。
Basic/helix-loop-helix(b HLH) is an important transcription factor commonly existed in eukaryotes, it participates in the regulation of many physiological pathways. In order to clarify its function and regulation mechanism in tobacco, a b HLH gene, Ntb HLH93, was cloned from Nicotiana tabacum by homologous cloning technology, and its biological function was predicted by bioinformatics combining with gene expression pattern analysis. The results showed that the c DNA sequence of Ntb HLH93 was 1254 bp in length with an open reading frame(ORF)of 1047 bp and encoding 349 amino acids. Ntb HLH93 was a hydrophobic protein, the main elements in its secondary structure were α-helix and random coil, and its tertiary structure featured helix-loop-helix structure typical of b HLH family. The gene expression of Ntb HLH93 was obviously tissue-specific, highly expressed in lateral roots at different growing stages and in the ovary at flowering stage, which was consistent with the feature that the synthesis of plantsterol concentrated in meristematic tissues.
Basic/helix-loop-helix(b HLH) is an important transcription factor commonly existed in eukaryotes, it participates in the regulation of many physiological pathways. In order to clarify its function and regulation mechanism in tobacco, a b HLH gene, Ntb HLH93, was cloned from Nicotiana tabacum by homologous cloning technology, and its biological function was predicted by bioinformatics combining with gene expression pattern analysis. The results showed that the c DNA sequence of Ntb HLH93 was 1254 bp in length with an open reading frame(ORF)of 1047 bp and encoding 349 amino acids. Ntb HLH93 was a hydrophobic protein, the main elements in its secondary structure were α-helix and random coil, and its tertiary structure featured helix-loop-helix structure typical of b HLH family. The gene expression of Ntb HLH93 was obviously tissue-specific, highly expressed in lateral roots at different growing stages and in the ovary at flowering stage, which was consistent with the feature that the synthesis of plantsterol concentrated in meristematic tissues.
引文
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[2]Jones S.An overview of the basic helix-loop-helix proteins[J].Genome Biol,2004,5(6):226.
[3]Espenshade P J,Hughes A L.Regulation of sterol synthesis in eukaryotes[J].Annu Rev Genet,2007,41:401-427.
[4]Robinson K A,Lopes J M.Survey and summary:Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes[J].Nucl Acid Res,2000,28(7):1499-1505.
[5]Menand B,Yi K,Jouannic S,et al.An ancient mechanism controls the development of cells with a rooting function in land plants[J].Science,2007,316(5830):1477-1480.
[6]Bruex A,Kainkaryam R M,Wieckowski Y,et al.A gene regulatory network for root epidermis cell differentiation in Arabidopsis[J/OL].PLo S Genet,2012,8(1):e1002446[2014-07-28].http://www.plosgene tics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002446.
[7]Heim M A,Jakoby M,Werber M,et al.The basic helix-loop-helix transcription factor family in plants:A genome-wide study of protein structure and functional diversity[J].Mol Biol Evol,2003,20(5):735-747.
[8]Toledo-Ortiz G,Huq E,Quail P H.The Arabidopsis basic/helix-loop-helix transcription factor family[J].Plant Cell,2003,15(8):1749-1770.
[9]Pires N,Dolan L.Origin and diversification of basic-helix-loop-helix proteins in plants[J].Mol Biol Evol,2010,27(4):862-874.
[10]Jiang Y,Yang B,Deyholos M K.Functional characterization of the Arabidopsis b HLH92transcription factor in abiotic stress[J].Mol Genet Genom,2009,282(5):503-516.
[11]王勇,陈克平,姚勤.b HLH转录因子家族研究进展[J].遗传,2008,30(7):821-830.
[12]Yuan Y,Wu H,Wang N,et al.FIT interacts with Atb HLH38 and Atb HLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis[J].Cell Res,2008,18(3):385-397.
[13]Li X,Duan X,Jiang H,et al.Genome-wide analysis of basic/helix-loop-helix transcripttion factor family in rice and Arabidopsis[J].Plant Physiol,2006,141(4):1167-1184.
[14]Sharma N.Role of b HLH93 in controlling flowering time in Arabidopsis thaliana[D].Austin:University of Texas at Austin,2011.
[15]Feldman M J.Identifying regulatory components of phytosterol biosynthesis in the genomics era[D].Austin:University of Texas at Austin,2012.
[16]覃碧.巴西橡胶树Hb UBC22基因克隆与表达分析[J].中国农学通报,2013,29(19):1-8.
[17]Wu M,Wang F,Zhang C,et al.Heme oxygenase-1is involved in nitric oxide-and c GMP-inducedα-Amy2/54 gene expression in GA-treated wheat aleurone layers[J].Plant Mol Biol,2013,81(1/2):27-40.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod[J].Methods,2001,25(4):402-408.
[19]Atchley W R,Terhalle W,Dress A.Positional dependence,cliques,and predictive motifs in the b HLH protein domain[J].J Mol Evol,1999,48(5):501-516.
[20]Ferré-D,AmaréA R,Prendergast G C,et al.Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain[J].Nature,1993,363(6424):38-44.
[21]O'Connor T R,Dyreson C,Wyrick J J.Athena:A resource for rapid visualization and systematic analysis of Arabidopsis promoter sequences[J].Bioinformatics,2005,21(24):4411-4413.
[22]Vance D E,Van Den Bosch H.Cholesterol in the year 2000[J].Biochim Biophys Acta,2000,1529(1/3):1-8.
[23]Gould R G,Taylor C B,Hagerman J S,et al.Cholesterol metabolism.I.Effect of dietary cholesterol on the synthesis of cholesterol in dog tissue in vitro[J].J Biol Chem,1953,201(2):519-528.
[24]Devarenne T P,Ghosh A,Chappell J.Regulation of squalene synthase,a key enzyme of sterol biosynthesis,in tobacco[J].Plant Physiol,2002,129(3):1095-1106.
[25]Cunillera N,Boronat A,Ferrer A.Spatial and temporal patterns of GUS expression directed by 5'regions of the Arabidopsis thaliana farnesyl diphosphate synthase genes FPS1 and FPS2[J].Plant Mol Biol,2000,44(6):747-758.