月季真核生物起始因子5A（eIF5A）基因的功能研究
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摘要
真核起始因子5A(eIF5A)是目前发现的唯一一种含有特殊氨基酸hypusine(吡咯赖氨酸)的高度保守性蛋白因子。已有的研究表明其参与翻译的延伸、mRNA的转运及降解、细胞的生长发育以及程序性死亡等多种代谢途径。本研究通过对月季(Rosa Chinensis) eIF5A基因(RceIF5A)进行洋葱表皮亚细胞定位,结果显示RceIF5A主要定位于细胞质中。用RT-PCR方法研究该基因在多种非生物胁迫下的表达模式,初步推断该基因与高温、氧化和渗透胁迫有关。于是将该基因转入真核模式生物毕氏酵母(Pichia pastoris)SMD1168中,通过各种胁迫处理及生长势测定分析证明该基因的表达能够提高宿主菌的高温和氧化胁迫抗性。进一步将该基因转入模式植物拟南芥中,通过对不同条件下植株的表型及生理指标分析,证明过表达该基因能够提高阳性植株的高温、氧化及渗透胁迫的抗性。
Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein known to contain the unusual amino acid hypusine. It is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies suggest that eIF5A is mainly involved in translation elongation, mRNA turnover and decay, cell proliferation, and programmed cell death. However, the precise cellular function of eIF5A remains largely unknown, especially in plants. In this work, we characterized the RceIF5A from Rosa chinensis. Subcellular localization of RceIF5A showed that it localized predominantly to the cytoplasm of onion epidermal cells. RceIF5A expression is up-regulated in Rosa chinensis under high temperature, oxidative and osmotic stress conditions. Recombinant RceIF5A was overexpressed in Pichia pastoris strain SMD1168 to study its possible function under stress conditions. The recombinant SMD1168 cells that accumulated RceIF5A showed improved viability under thermal and oxidative stress conditions compared to control cultures. We then produced transgenic Arabidopsis that constitutively enhanced or suppressed expression of RceIF5A. The RceIF5A over-expression plants exhibited increased resistance to heat, oxidative and osmotic stresses, while the suppressed expression plant (three AteIF5A isoforms in Arabidopsis were down-regulated) showed more susceptibility to these stresses.
Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein known to contain the unusual amino acid hypusine. It is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies suggest that eIF5A is mainly involved in translation elongation, mRNA turnover and decay, cell proliferation, and programmed cell death. However, the precise cellular function of eIF5A remains largely unknown, especially in plants. In this work, we characterized the RceIF5A from Rosa chinensis. Subcellular localization of RceIF5A showed that it localized predominantly to the cytoplasm of onion epidermal cells. RceIF5A expression is up-regulated in Rosa chinensis under high temperature, oxidative and osmotic stress conditions. Recombinant RceIF5A was overexpressed in Pichia pastoris strain SMD1168 to study its possible function under stress conditions. The recombinant SMD1168 cells that accumulated RceIF5A showed improved viability under thermal and oxidative stress conditions compared to control cultures. We then produced transgenic Arabidopsis that constitutively enhanced or suppressed expression of RceIF5A. The RceIF5A over-expression plants exhibited increased resistance to heat, oxidative and osmotic stresses, while the suppressed expression plant (three AteIF5A isoforms in Arabidopsis were down-regulated) showed more susceptibility to these stresses.
引文
[1]. Kemper, W.M., K.W. Berry, and W.C. Merrick. Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta [J]. J Biol Chem,1976.251(18):p.5551-7.
[2]. Park, M.H., E.C. Wolff, and J.E. Folk. Hypusine:its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation [J]. Biofactors,1993.4(2):p.95-104.
[3]. Gordon, E.D., et al. Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes [J]. J Biol Chem,1987.262(34):p. 16585-9.
[4]. Park, M.H. The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities [J]. J Biol Chem,1989.264(31):p.18531-5.
[5]. Hanauske-Abel, H.M., et al. Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation [J]. Biochim Biophys Acta, 1994.1221(2):p.115-24.
[6]. Saini, P., et al. Hypusine-containing protein eIF5A promotes translation elongation [J]. Nature,2009.459(7243):p.118-21.
[7]. Jao, D.L. and K.Y. Chen. Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex [J]. J Cell Biochem,2006.97(3):p. 583-98.
[8]. Zanelli, C.F., et al. eIF5A binds to translational machinery components and affects translation in yeast [J]. Biochem Biophys Res Commun,2006.348(4): p.1358-66.
[9]. Frigieri, M.C., et al. Synthetic lethality between eIF5A and Yptl reveals a connection between translation and the secretory pathway in yeast [J]. Mol Genet Genomics,2008.280(3):p.211-21.
[10]. Cano, V.S., et al. Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification [J]. Febs J,2008.275(1):p.44-58.
[11]. Elfgang, C., et al. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals [J]. Proc Natl Acad Sci U S A, 1999.96(11):p.6229-34.
[12]. Park, M.H., Y.A. Joe, and K.R. Kang. Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae [J]. J Biol Chem,1998.273(3):p.1677-83.
[13]. Park, M.H. The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A) [J]. J Biochem (Tokyo),2006.139(2):p.161-9.
[14]. Kang, H.A. and J.W. Hershey. Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae [J]. J Biol Chem,1994.269(6):p.3934-40.
[15]. Wolff, E.C., et al. Posttranslational synthesis of hypusine:evolutionary progression and specificity of the hypusine modification [J]. Amino Acids, 2007.
[16]. Nishimura, K., et al. Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin [J]. Biochem J,2002.363(Pt 3):p.761-8.
[17]. Tome, M.E. and E.W. Gerner. Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs [J]. Biochem J,1996.320 (Pt 1):p. 55-60.
[18]. Chen, Z.P., et al. Effects of inhibitors of deoxyhypusine synthase on the differentiation of mouse neuroblastoma and erythroleukemia cells [J]. Cancer Lett,1996.105(2):p.233-9.
[19]. Zanelli, C.F. and S.R. Valentini. Pkc1 acts through Zdsl and Gicl to suppress growth and cell polarity defects of a yeast eIF5A mutant [J]. Genetics,2005. 171(4):p.1571-81.
[20]. 靳宝锋,何昆,胡美茹等.真核起始因子5A与细胞周期G12S调控的研究[J].中国实验血液学杂志,2003.
[21]. Chen, G, et al. Proteomic analysis of eIF-5A in lung adenocarcinomas [J]. Proteomics,2003.3(4):p.496-504.
[22]. Li, A.L., et al. A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis [J]. J Biol Chem,2004.279(47):p.49251-8.
[23]. Rosorius, O., et al. Nuclear pore localization and nucleocytoplasmic transport of eIF-5A:evidence for direct interaction with the export receptor CRM1[J]. J Cell Sci,1999.112 (Pt 14):p.2369-80.
[24].何昆,周涛,靳宝锋等.eIF-5A的亚细胞定位研究[J].军事医学科学院院刊,2003.
[25]. Hopkins, M.T., et al. eIF5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis thaliana [J]. Plant Physiol, 2008.148(1):p.479-489.
[26]. Gao, Y. and Y. Chen. Advances in the study of the effects of danggui buxue decoction and its modified recipes on immunity and hematopoietic function [J]. Zhong Yao Cai,2000.23(3):p.177-80.
[27]. Shi, X.P., K.C. Yin, and L. Waxman. Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein [J]. Biol Signals,1997.6(3):p. 143-9.
[28]. Valentini, S.R., et al. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling [J]. Genetics,2002.160(2):p.393-405.
[29]. Lee, S.B., et al. The effect of hypusine modification on the intracellular localization of eIF5A [J]. Biochem Biophys Res Commun,2009.383(4):p. 497-502.
[30]. Chamot, D. and C. Kuhlemeier. Differential expression of genes encoding the hypusine-containing translation initiation factor, eIF-5A, in tobacco [J]. Nucleic Acids Res,1992.20(4):p.665-9.
[31]. Requejo, R. and M. Tena. Maize response to acute arsenic toxicity as revealed by proteome analysis of plant shoots [J]. Proteomics,2006.6 Suppl 1:p. S156-62.
[32]. Dresselhaus, T., S. Cordts, and H. Lorz. A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize [J]. Plant Mol Biol,1999.39(5):p.1063-71.
[33]. Wang, T.W., et al. Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato [J]. J Biol Chem,2001.276(20):p.17541-9.
[34]. Chou, W.C., et al. Expression of genes encoding the rice translation initiation factor, eIF5A, is involved in developmental and environmental responses [J]. Physiol Plant,2004.121(1):p.50-57.
[35]. Mehta, A.M., et al. Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A [J]. Plant Physiol,1994.106(4):p.1413-1419.
[36]. Wang, T.W., et al. Pleiotropic effects of suppressing deoxyhypusine synthase expression in Arabidopsis thaliana [J]. Plant Mol Biol,2003.52(6):p. 1223-35.
[37]. Duguay, J., et al. Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects [J]. J Plant Physiol,2007.164(4):p.408-20.
[38]. Thompson, J.E., et al. Regulation of senescence by eukaryotic translation initiation factor 5A:implications for plant growth and development [J]. Trends Plant Sci,2004.9(4):p.174-9.
[39]. Liu, Z., et al. Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana [J]. J Exp Bot,2008.59(4):p.939-50.
[40]. Feng, H., et al. Functional Characterization of the Arabidopsis Eukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in Plant Growth and Development by Regulating Cell Division, Cell Growth, and Cell Death [J]. Plant Physiol,2007.144(3):p.1531-45.
[41].姜蕊,胡永红,蒋昌华.热胁迫下月季叶片蛋白双向电泳分析[J].中国生物工程杂志,2006.26(4)：p.91-94.
[42].石金磊,徐健遥,蒋昌华等.月季eIF5A基因的表达增强宿主大肠杆菌对高温和低温的耐性[J].中国生物工程杂志,2008.28(1)：p.18-24.
[43]. Jao, D.L. and K. Yu Chen. Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging [J]. J Cell Biochem,2002.86(3):p.590-600.
[44]. Yang, X., et al. Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem Ⅱ in tobacco plants [J]. Planta,2007. 225(3):p.719-33.
[45]. Tang, W., T.M. Charles, and R.J. Newton. Overexpression of the pepper transcription factor CaPFl in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth [J]. Plant Mol Biol,2005.59(4):p.603-17.
[46]. Bates, L.S., R.P. Waldren, and I.D. Teare. Rapid determination of free proline for water-stress studies [J]. plant and soil,1973.39(0032-079X):p.205-207.
[2]. Park, M.H., E.C. Wolff, and J.E. Folk. Hypusine:its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation [J]. Biofactors,1993.4(2):p.95-104.
[3]. Gordon, E.D., et al. Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes [J]. J Biol Chem,1987.262(34):p. 16585-9.
[4]. Park, M.H. The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities [J]. J Biol Chem,1989.264(31):p.18531-5.
[5]. Hanauske-Abel, H.M., et al. Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation [J]. Biochim Biophys Acta, 1994.1221(2):p.115-24.
[6]. Saini, P., et al. Hypusine-containing protein eIF5A promotes translation elongation [J]. Nature,2009.459(7243):p.118-21.
[7]. Jao, D.L. and K.Y. Chen. Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex [J]. J Cell Biochem,2006.97(3):p. 583-98.
[8]. Zanelli, C.F., et al. eIF5A binds to translational machinery components and affects translation in yeast [J]. Biochem Biophys Res Commun,2006.348(4): p.1358-66.
[9]. Frigieri, M.C., et al. Synthetic lethality between eIF5A and Yptl reveals a connection between translation and the secretory pathway in yeast [J]. Mol Genet Genomics,2008.280(3):p.211-21.
[10]. Cano, V.S., et al. Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification [J]. Febs J,2008.275(1):p.44-58.
[11]. Elfgang, C., et al. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals [J]. Proc Natl Acad Sci U S A, 1999.96(11):p.6229-34.
[12]. Park, M.H., Y.A. Joe, and K.R. Kang. Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae [J]. J Biol Chem,1998.273(3):p.1677-83.
[13]. Park, M.H. The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A) [J]. J Biochem (Tokyo),2006.139(2):p.161-9.
[14]. Kang, H.A. and J.W. Hershey. Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae [J]. J Biol Chem,1994.269(6):p.3934-40.
[15]. Wolff, E.C., et al. Posttranslational synthesis of hypusine:evolutionary progression and specificity of the hypusine modification [J]. Amino Acids, 2007.
[16]. Nishimura, K., et al. Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin [J]. Biochem J,2002.363(Pt 3):p.761-8.
[17]. Tome, M.E. and E.W. Gerner. Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs [J]. Biochem J,1996.320 (Pt 1):p. 55-60.
[18]. Chen, Z.P., et al. Effects of inhibitors of deoxyhypusine synthase on the differentiation of mouse neuroblastoma and erythroleukemia cells [J]. Cancer Lett,1996.105(2):p.233-9.
[19]. Zanelli, C.F. and S.R. Valentini. Pkc1 acts through Zdsl and Gicl to suppress growth and cell polarity defects of a yeast eIF5A mutant [J]. Genetics,2005. 171(4):p.1571-81.
[20]. 靳宝锋,何昆,胡美茹等.真核起始因子5A与细胞周期G12S调控的研究[J].中国实验血液学杂志,2003.
[21]. Chen, G, et al. Proteomic analysis of eIF-5A in lung adenocarcinomas [J]. Proteomics,2003.3(4):p.496-504.
[22]. Li, A.L., et al. A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis [J]. J Biol Chem,2004.279(47):p.49251-8.
[23]. Rosorius, O., et al. Nuclear pore localization and nucleocytoplasmic transport of eIF-5A:evidence for direct interaction with the export receptor CRM1[J]. J Cell Sci,1999.112 (Pt 14):p.2369-80.
[24].何昆,周涛,靳宝锋等.eIF-5A的亚细胞定位研究[J].军事医学科学院院刊,2003.
[25]. Hopkins, M.T., et al. eIF5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis thaliana [J]. Plant Physiol, 2008.148(1):p.479-489.
[26]. Gao, Y. and Y. Chen. Advances in the study of the effects of danggui buxue decoction and its modified recipes on immunity and hematopoietic function [J]. Zhong Yao Cai,2000.23(3):p.177-80.
[27]. Shi, X.P., K.C. Yin, and L. Waxman. Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein [J]. Biol Signals,1997.6(3):p. 143-9.
[28]. Valentini, S.R., et al. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling [J]. Genetics,2002.160(2):p.393-405.
[29]. Lee, S.B., et al. The effect of hypusine modification on the intracellular localization of eIF5A [J]. Biochem Biophys Res Commun,2009.383(4):p. 497-502.
[30]. Chamot, D. and C. Kuhlemeier. Differential expression of genes encoding the hypusine-containing translation initiation factor, eIF-5A, in tobacco [J]. Nucleic Acids Res,1992.20(4):p.665-9.
[31]. Requejo, R. and M. Tena. Maize response to acute arsenic toxicity as revealed by proteome analysis of plant shoots [J]. Proteomics,2006.6 Suppl 1:p. S156-62.
[32]. Dresselhaus, T., S. Cordts, and H. Lorz. A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize [J]. Plant Mol Biol,1999.39(5):p.1063-71.
[33]. Wang, T.W., et al. Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato [J]. J Biol Chem,2001.276(20):p.17541-9.
[34]. Chou, W.C., et al. Expression of genes encoding the rice translation initiation factor, eIF5A, is involved in developmental and environmental responses [J]. Physiol Plant,2004.121(1):p.50-57.
[35]. Mehta, A.M., et al. Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A [J]. Plant Physiol,1994.106(4):p.1413-1419.
[36]. Wang, T.W., et al. Pleiotropic effects of suppressing deoxyhypusine synthase expression in Arabidopsis thaliana [J]. Plant Mol Biol,2003.52(6):p. 1223-35.
[37]. Duguay, J., et al. Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects [J]. J Plant Physiol,2007.164(4):p.408-20.
[38]. Thompson, J.E., et al. Regulation of senescence by eukaryotic translation initiation factor 5A:implications for plant growth and development [J]. Trends Plant Sci,2004.9(4):p.174-9.
[39]. Liu, Z., et al. Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana [J]. J Exp Bot,2008.59(4):p.939-50.
[40]. Feng, H., et al. Functional Characterization of the Arabidopsis Eukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in Plant Growth and Development by Regulating Cell Division, Cell Growth, and Cell Death [J]. Plant Physiol,2007.144(3):p.1531-45.
[41].姜蕊,胡永红,蒋昌华.热胁迫下月季叶片蛋白双向电泳分析[J].中国生物工程杂志,2006.26(4)：p.91-94.
[42].石金磊,徐健遥,蒋昌华等.月季eIF5A基因的表达增强宿主大肠杆菌对高温和低温的耐性[J].中国生物工程杂志,2008.28(1)：p.18-24.
[43]. Jao, D.L. and K. Yu Chen. Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging [J]. J Cell Biochem,2002.86(3):p.590-600.
[44]. Yang, X., et al. Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem Ⅱ in tobacco plants [J]. Planta,2007. 225(3):p.719-33.
[45]. Tang, W., T.M. Charles, and R.J. Newton. Overexpression of the pepper transcription factor CaPFl in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth [J]. Plant Mol Biol,2005.59(4):p.603-17.
[46]. Bates, L.S., R.P. Waldren, and I.D. Teare. Rapid determination of free proline for water-stress studies [J]. plant and soil,1973.39(0032-079X):p.205-207.