利用VIGS技术研究NtbHLH93基因在烟草甾醇代谢中的功能
|
摘要
为进一步研究烟草NtbHLH93基因的功能,利用病毒诱导基因沉默(Virus induced gene silence,VIGS)技术降低烟草中NtbHLH93基因的表达。通过测定pTRV2-NtbHLH93烟株中的甾醇含量,发现NtbHLH93基因表达下调后,总甾醇、豆甾醇、胆固醇和β-谷甾醇的含量显著降低。转录组测序发现NtbHLH93基因下调后,共有259个基因差异表达。对这些差异表达基因进行KEGG表达通路分析,发现质体萜类代谢2-C-甲基-D-赤藓糖醇-4-磷酸(2-C-Methyl-D-Erythritol-4-Phosphate, MEP)途径中有两个基因(Niben101Scf00063g13014.1和Niben101Scf06249g03002.1)上调表达,这两个基因与MEP途径中的牻牛儿基牻牛儿基还原酶(Geranylgeranyl diphosphate synthases, GGPPS)基因序列高度相似,推测当甾醇合成代谢通路受阻后导致Niben101Scf00063g13014.1和Niben101Scf06249g03002.1基因上调表达。
To study the function of NtbHLH93, virus-induced gene silencing(VIGS) was used to downregulate NtbHLH93 expression in tobacco. The results showed that when NtbHLH93 expression was downregulated, the contents of total sterol, stigmasterol, cholesterol and β-sitosterol significantly decreased in pTRV2-NtbHLH93 tobacco seedlings. Transcriptome sequencing revealed that there were 259 differentially expressed genes in NtbHLH93 expression downregulated tobacco seedlings. KEGG pathway analysis indicated that two genes(Niben101 Scf00063 g13014.1 and Niben101 Scf06249 g03002.1) were upregulated in MEP(2-C-methyl-D-erythritol-4-phosphate) pathway, a terpenoid metabolism pathway in plastids. The sequences of the two genes were highly similar to geranylgeranyl diphosphate synthases(GGPPS) gene in MEP pathway, which suggested that the upregulation of Niben101 Scf00063 g13014.1 and Niben101 Scf06249 g03002.1 might relate to the inhibition of sterol synthesis.
To study the function of NtbHLH93, virus-induced gene silencing(VIGS) was used to downregulate NtbHLH93 expression in tobacco. The results showed that when NtbHLH93 expression was downregulated, the contents of total sterol, stigmasterol, cholesterol and β-sitosterol significantly decreased in pTRV2-NtbHLH93 tobacco seedlings. Transcriptome sequencing revealed that there were 259 differentially expressed genes in NtbHLH93 expression downregulated tobacco seedlings. KEGG pathway analysis indicated that two genes(Niben101 Scf00063 g13014.1 and Niben101 Scf06249 g03002.1) were upregulated in MEP(2-C-methyl-D-erythritol-4-phosphate) pathway, a terpenoid metabolism pathway in plastids. The sequences of the two genes were highly similar to geranylgeranyl diphosphate synthases(GGPPS) gene in MEP pathway, which suggested that the upregulation of Niben101 Scf00063 g13014.1 and Niben101 Scf06249 g03002.1 might relate to the inhibition of sterol synthesis.
引文
[1]Heim M A,Jakoby M,Werber M,et al.The basic helixloop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity[J].Molecular Biology and Evolution,2003,20(5):735-747.
[2]Pires N,Dolan L.Origin and diversification of basic-helix-loop-helix proteins in plants[J].Molecular Biology and Evolution,2010,27(4):862-874.
[3]Toledo-Ortiz G,Huq E,Quail P H.The Arabidopsis basic/helix-loop-helix transcription factor family[J].The Plant Cell,2003,15(8):1749-1770.
[4]Jones S.An overview of the basic helix-loop-helix proteins[J].Genome Biology,2004,5:226.
[5]Massari M E,Murre C.Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms[J].Molecular and Cellular Biology,2000,20(2):429-440.
[6]Robinson K A,Lopes J M.SURVEY ANDSUMMARY:Saccharomyces cerevisiae basic helix-loophelix proteins regulate diverse biological processes[J].Nucleic Acids Research,2000,28(7):1499-1505.
[7]Atchley W R,Terhalle W,Dress A.Positional dependence,cliques,and predictive motifs in the bHLHprotein domain[J].Journal of Molecular Evolution,1999,48(5):501-516.
[8]Li X X,Duan X P,Jiang H X,et al.Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis[J].Plant Physiology,2006,141(4):1167-1184.
[9]Sharma N.Role of b HLH93 in controlling flowering time in Arabidopsis thaliana[D].Austin:The University of Texas at Austin,2011.
[10]Feldman M J.Identifying regulatory components of phytosterol biosynthesis in the genomics era[D].Austin:University of Texas at Austin,2012.
[11]Stedman R L.Chemical composition of tobacco and tobacco smoke[J].Chemical Reviews,1968,68(2):153-207.
[12]Bindler G N,Piade J J,Schulthess D.Evaluation of selected steroids as chemical markers of past or presently occurring fungal infections on tobacco[J].Beitr?ge zur Tabakforschung International,1988,14(2):127-134.
[13]Breinh?lder P,Mosca L,Lindner W.Concept of sequential analysis of free and conjugated phytosterols in different plant matrices[J].Journal of Chromatography B,2002,777(1/2):67-82.
[14]Lagarda M J,García-Llatas G,FarréR.Analysis of phytosterols in foods[J].Journal of Pharmaceutical and Biomedical Analysis,2006,41(5):1486-1496.
[15]Johnstone R A W,Plimmer J R.The chemical constituents of tobacco and tobacco smoke[J].Chemical Reviews,1959,59(5):885-936.
[16]Badger G M,Kimber R W L,Spotswood T M.Mode of formation of 3,4-benzopyrene in human environment[J].Nature,1960,187(4738):663-665.
[17]Britt P F,BuchananⅢA C,Kidder M M,et al.Mechanistic investigation into the formation of polycyclic aromatic hydrocarbons from the pyrolysis of plant steroids[J].Fuel,2001,80(12):1727-1746.
[18]聂建群.卷烟烟气中稠环芳烃的分析、形成及其相关降害技术的初步研究[D].昆明:云南师范大学,2006.NIE Jianqun.Analysis and formation of polycyclic aromatic hydrocarbons in cigarette smoke and preliminary study on the relative harm-reducing techniques[D].Kunming:Yunnan Normal University,2006.
[19]武明珠,李锋,王燃,等.烟草转录因子bHLH93基因的克隆及表达分析[J].烟草科技,2015,48(3):1-7.WU Mingzhu,LI Feng,WANG Ran,et al.Cloning and expression of transcription factor gene bHLH93 from Nicotiana tabacum[J].Tobacco Science&Technology,2015,48(3):1-7.
[20]魏攀,史艳梅,李锋,等.烟草β-胡萝卜素羟化酶基因的转录表达模式及功能分析[J].烟草科技,2015,48(4):1-6,18.WEI Pan,SHI Yanmei,LI Feng,et al.Transcription expression pattern and function analysis ofβ-ohase gene in Nicotiana tabacum[J].Tobacco Science&Technology,2015,48(4):1-6,18.
[21]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.
[22]张松涛,陈红丽,崔红,等.植物MEP途径的代谢调控机制[J].西北植物学报,2012,32(7):1500-1504.ZHANG Songtao,CHEN Hongli,CUI Hong,et al.Multiple regulation mechanisms of MEP pathway in plant[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(7):1500-1504.
[2]Pires N,Dolan L.Origin and diversification of basic-helix-loop-helix proteins in plants[J].Molecular Biology and Evolution,2010,27(4):862-874.
[3]Toledo-Ortiz G,Huq E,Quail P H.The Arabidopsis basic/helix-loop-helix transcription factor family[J].The Plant Cell,2003,15(8):1749-1770.
[4]Jones S.An overview of the basic helix-loop-helix proteins[J].Genome Biology,2004,5:226.
[5]Massari M E,Murre C.Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms[J].Molecular and Cellular Biology,2000,20(2):429-440.
[6]Robinson K A,Lopes J M.SURVEY ANDSUMMARY:Saccharomyces cerevisiae basic helix-loophelix proteins regulate diverse biological processes[J].Nucleic Acids Research,2000,28(7):1499-1505.
[7]Atchley W R,Terhalle W,Dress A.Positional dependence,cliques,and predictive motifs in the bHLHprotein domain[J].Journal of Molecular Evolution,1999,48(5):501-516.
[8]Li X X,Duan X P,Jiang H X,et al.Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis[J].Plant Physiology,2006,141(4):1167-1184.
[9]Sharma N.Role of b HLH93 in controlling flowering time in Arabidopsis thaliana[D].Austin:The University of Texas at Austin,2011.
[10]Feldman M J.Identifying regulatory components of phytosterol biosynthesis in the genomics era[D].Austin:University of Texas at Austin,2012.
[11]Stedman R L.Chemical composition of tobacco and tobacco smoke[J].Chemical Reviews,1968,68(2):153-207.
[12]Bindler G N,Piade J J,Schulthess D.Evaluation of selected steroids as chemical markers of past or presently occurring fungal infections on tobacco[J].Beitr?ge zur Tabakforschung International,1988,14(2):127-134.
[13]Breinh?lder P,Mosca L,Lindner W.Concept of sequential analysis of free and conjugated phytosterols in different plant matrices[J].Journal of Chromatography B,2002,777(1/2):67-82.
[14]Lagarda M J,García-Llatas G,FarréR.Analysis of phytosterols in foods[J].Journal of Pharmaceutical and Biomedical Analysis,2006,41(5):1486-1496.
[15]Johnstone R A W,Plimmer J R.The chemical constituents of tobacco and tobacco smoke[J].Chemical Reviews,1959,59(5):885-936.
[16]Badger G M,Kimber R W L,Spotswood T M.Mode of formation of 3,4-benzopyrene in human environment[J].Nature,1960,187(4738):663-665.
[17]Britt P F,BuchananⅢA C,Kidder M M,et al.Mechanistic investigation into the formation of polycyclic aromatic hydrocarbons from the pyrolysis of plant steroids[J].Fuel,2001,80(12):1727-1746.
[18]聂建群.卷烟烟气中稠环芳烃的分析、形成及其相关降害技术的初步研究[D].昆明:云南师范大学,2006.NIE Jianqun.Analysis and formation of polycyclic aromatic hydrocarbons in cigarette smoke and preliminary study on the relative harm-reducing techniques[D].Kunming:Yunnan Normal University,2006.
[19]武明珠,李锋,王燃,等.烟草转录因子bHLH93基因的克隆及表达分析[J].烟草科技,2015,48(3):1-7.WU Mingzhu,LI Feng,WANG Ran,et al.Cloning and expression of transcription factor gene bHLH93 from Nicotiana tabacum[J].Tobacco Science&Technology,2015,48(3):1-7.
[20]魏攀,史艳梅,李锋,等.烟草β-胡萝卜素羟化酶基因的转录表达模式及功能分析[J].烟草科技,2015,48(4):1-6,18.WEI Pan,SHI Yanmei,LI Feng,et al.Transcription expression pattern and function analysis ofβ-ohase gene in Nicotiana tabacum[J].Tobacco Science&Technology,2015,48(4):1-6,18.
[21]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.
[22]张松涛,陈红丽,崔红,等.植物MEP途径的代谢调控机制[J].西北植物学报,2012,32(7):1500-1504.ZHANG Songtao,CHEN Hongli,CUI Hong,et al.Multiple regulation mechanisms of MEP pathway in plant[J].Acta Botanica Boreali-Occidentalia Sinica,2012,32(7):1500-1504.