摘要
铜伴侣蛋白是结合铜的小蛋白质,并将结合的铜运送到依赖铜的目标蛋白上。铜结合蛋白不仅可以保护细胞免受铜的毒害,而且可以消除超氧化阴离子的毒性。在植物中,已经鉴定出许多铜分子伴侣家族的成员。根据功能的不同,可以分为三种类型：第一种类型是拟南芥CCH基因,与酵母的ATX1同源,参与了铜的运输和活性氧的清除；该基因在RNA水平和蛋白质水平被研究最透彻的植物铜分子伴侣基因。第二种类型的铜分子伴侣为AtCOX17基因,最近从拟南芥中被分离出来,在功能上与酵母的COX1l7基因同源,也能将铜运输到细胞色素氧化酶分子上,用来修复ROS诱导的电子传递链的中断。第三种类型是从拟南芥基因组中所分离鉴定出的CCS基因,其功能是将铜送至Cu/Zn超氧化物歧化酶。
铜锌超氧化物歧化酶是一类抵抗外界氧化胁迫的保护类蛋白,先前我们已将其提交至NCBI,登录号为GQ472846。序列分析表明从第2位氨基酸残基至36位氨基酸残基为其高度保守序列。荧光定量PCR分析表明,在3%NaHCO3铜锌超氧化物歧化酶基因在叶、茎与地下茎中皆有表达,揭示铜锌超氧化物歧化酶基因在盐胁迫下具有不同的表达模式。
目前我们已将铜伴侣蛋白基因从西伯利亚蓼中克隆出来,将其与我们先期得到的Cu/Zn超氧化物歧化酶基因共转化至烟草中。结果证明,共转化烟草具有更强的耐NaCl的能力。
Copper (Cu) chaperones constitute a family of small Cu+ binding proteins required for Cu homeostasis in cytoplasm. The ATX1 family of Cu chaperones was involved in antioxidative activity.The partial sequence of a PsATX-1 gene was obtained from a random clone in polygonum sibicum SSH library and PsATX-1 was cloned by using rapid amplification of cDNA end (RACE) technology. The gene accession nucleotidesequence number in GenBank was EU620702.Sequence analysis indicated that the protein, such as most of the plant ATX1,including anamino-terminal (N-terminal) signature sequence of "MXCXXC",which possess to the heavy metal binding site,but in lack of carboxy-terminal domain (CTD)signature sequence. Quantitative analysis of PsATX-1 gene expression was carried by using Real-time PCR method.The result showed that PsATX-1 gene express in leaves,stem and underground stem.PsATX-1 was influence by 3% NaHCO3, the different express modes can be find in leaves,stem and underground stem.
In aerobic organisms,protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes,such as Cu/Zn superoxide dismutase.In this work,a cDNA clone encoding a Cu/Zn superoxide dismutase gene, named PsSOD-1,has been identified from Polygonum sibiricum Laxm by the rapid amplification of cDNA ends method(RACE).The Gene Accession nucleotide sequence Number in GenBank was GQ472846.Sequence analysis indicated that the protein, such as most of the plant SOD,including the conserve domain named'Cu-Zn_Superoxide_Dismutase'from the second amino acids to the one hundred thirty-sixth amino acids.Expression analysis by real-time quantitative PCR revealed that PsSOD-1 gene expression in leaves,stem and underground stem.PsSOD-1 gene expression can be induced by 3% NaHCO3,the different expressing of PsSOD-1 mRNA levels showed that the gene different express modes in leaves,stem and underground stem under the Salt Stress.
In salt-stress condition, the content of superoxide dismutase(SOD) and proline of transgenic plants were generally higher than those of the control, while relative ROS and MDA of transgenic plants were generally lower than those of the control.These results showed that the co-transformed PsATX-1 and PsSOD-1 gene contributed to improved salt resistance of the transgenic tobaccos.
引文
[1]张建锋,李吉跃,宋玉民,等.植物耐盐机理与耐盐植物选育研究进展[J].世界林业研究2003.16(2)：16-22
[2]Wang W X.,Vinocur B,Altman A. Plant responses to drought, salinity and extreme temperatures towards genetic engineering for stress tolerance. Planta, 2003,218:1～14
[3]Sairam RK, Arunaa Tysgi.Physiology and molecular biology of salinity stress tolerance in plants.Current Science,2004,86(3):407～421
[4]Marschner H. Mineral Nutrition of Higher Plants. London:Academic Press; 1995.
[5]Maksymiec W. Effect of copper on cellular processes in higher plants. Photosynthetica 1997 34:321-342.
[6]Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB.A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.Science 1999,283: 996-998
[7]Halliwell B.,Gutteridge JMC.Radicals in Biology and Medicine. Oxford, UK: Clarendon Press; 1989.
[8]Vulpe CD, Packman S.Cellular copper transport. Annu Rev Nutr.1995,15: 293～322.
[9]Koch K, Pena M, Thiele D.Copper-binding motifs in catalysis, transport, detoxification and signaling. Chem Biol.1997,4:549～560.
[10]Taraai KT, Gralla EB, Ellerby LM, Valentine JS, Thiele DJ.Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase. Proc Natl Acad Sci USA.1993,90:8013～8017.
[11]Culotta VC, Joh HD, Slekar KH, Strain J. A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J Biol Chem. 1995,270:29991-29997.
[12]Lin S,Culotta VC. The Atxl gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Sci USA.1995,92:3784-3788.
[13]Lin SJ, Pufahl RA, Dancis A, O'Halloran TV, Culotta VC. A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport. J Biol Chem.1997,272:9215～9220.
[14]Pufahl R, Singer C, Peariso K, Lin SJ, Schmidt J, Fahrni C, Cizewski Culotta V, Penner-Hahn J, O'Halloran T. Metal ion chaperone function of the soluble Cuo receptor Atrl.Science.1997,278:853～856.
[15]Hung IH, Casareno RLB,Labesseq Mathews FS, Gitlin JD.HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense. J Biol Chem.1998,273:1749～1754.
[16]Edward Himelblau,Helena Mira, Su-Ju Lin,Valeria Cizewski Culotta,Lola Peiiarrubia, and Richard M. Amasino.Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis.Plant Physiol.1998 August 1,117(4):1227～1234.
[17]Klomp LWJ, Lin SJ, Yaun DS,Klausner RD,Culotta VC, Gitlin JD.Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis.J Biol Chem.1997,272:9221～9226.
[18]Clerum DM, Shtanko A, Tzagoloff A. Characterization of COX17, a ycast gene involved in copper metabolism and assembly of cvtochrome oxidase. J Biol Chem. 1996,271:14504～14509.
[19]Amaravadi R, Glerum DM, Tzagoloff A. Isolation of a cDNA encoding the human homolog of COX17,a yeast gene essential for mitochondria copper recruitment. Hum Genet.1997,99:329～333.
[20]Teresa Balandin and Carmen Castresana. AtCOX17, an Arabidopsis Homolog of the Yeast Copper Chaperone COX17.Plant Physiol.2002,129(4):1852～1857.
[21]Zhou J, Goldsbrough PB.Structure, organization and expression of the metallothionein gene family in Arabidopsis. Mol Gen Genet.1994,248:318～328
[22]Ogra Y, Suzuki KT. Nuclear trafficking of metallothionein:possible mechanisms and current knowledge. Cell Mol Biol.2000,46:357～365
[23]Ye B, Maret W, Valle BL. Zinc metallothioneinried into liver mitochondria modulates respiration. Proc Natl Acad Sci USA.2001,98:2317～2322.
[24]Harrison MD, Jones CE, Dameron CT. Copper chaperones:function, structure and copper-binding properties. J Biol Inorg Chem.1999,4:145～153
[25]Gamonet F, Lauquin GJM.The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection. Eur J Biochem.1998,251:716～723
[26]Culotta VC, Klomp LW, Strain J, Casareno R, Krems B, Gitlin JD. The copper chaperone for superoxide dismutase. J Biol Chem.1997,272:23469～23472
[27]Zhu H, Shipp E, Sanchez RJ, Liba A, Stine JE, Hart J, Gralla EB, Nersissian AM, Valentine JS.Cobalt(2+) binding to human and tomato copper chaperone for superoxide dismutase:implications for the metal ion transfer mechanism. Biochemistry.2000,39:5413～5421
[28]Wintz H, Vulpe C.Plant copper chaperones. Biochem Soc Trans.2002,30: 732～735
[29]Bowler C, van Camp W, van Montage M, Inze D. Superoxide dismutase in plants. Crit Rev Plant Sci.1994,13:199～218.
[30]McCord JM, Fridovich. Superoxide dismutase:an enzymatic function for erythrocuprein (hemocuprein).Biol Chem.1969,244:6049～6055
[31]Himelblau E, Mira H, Lin SJ, Culotta VC, PenarrubiaL. Amasino RM.Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis. Plant Physiol.1998,117:1227～1234
[32]Mira H, Martinez-Garcia F, Penarrubia L. Evidence for the plant-specific intercellular transport of the Arabidopsis copper chaperone CCH. Plant.2001,25:521～528.
[33]Mira H,Vilar M,Pbres-Paya E,Penarrubia L. Functional and conformational properties of the exclusive C-domain from the Arabidopsis copper chaperone (CCI).Biochem.2001,357:545～549.
[34]Balandin T, Castresana C. AtCOX17, an Arabidopsis homolog of the yeast copper chaperone COX17.Plant Physiol.2002,129:1852～1857
[35]Schmidt PJ, Rae TD,Pufahl RA, Hamma T, Strain J, O'Halloran TV, Culotta VC. Multiple protein domains contribute to the action of the copper chaperone foe superoxide dismutase. J Biol Chem.1999,274:23719～23725
[36]Steven NeiIl, Radhika Desikan and John, Hancock. Hydrogen peroxide signaling, Current Opinion in Plant Biology,2002(5):388～395
[37]赵会杰,林学梧,刘国顺.干旱胁迫对香料烟叶生理特性的影响.中国烟草,1993,(1)：1-3
[38]OgawaK KS,Adada K. Intra-and-extra-cellular localization of "cytosolic" Cu /Zn-superox idase dismutase in spinach.2004,3,207～219
[39]Carlioz A, Touati D, Isolation of superoxide dismutase mutants in Escherichia coli: Is superoxide dismutase necessary for aerobic life. EMBO,1986,5:623～630
[40]Van Loon APGM, Pesold-Hurt B,Schatz G,A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc Natl Acad Sci USA,1986,83:3820～3824
[41]Lin C.T., Lin M.T., Chen YT.,Shaw J.R, Subunit interaction enhances enzyme activity and stability of sweet potatocy to cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. Plant Mol Biol,1995, 28(2):303～311
[42]Ryan C, Scandalios J.C.,Molecular evolution and structure-function relation ships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutase. Archives of Biochemistry and Biophysics,2002,399(1):19～36
[43]Bannisster J.V, Bannister W.H.,Rotilio G,Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem,1987,22(2): 111～180
[44]Richardson J.,Thomas K.A.,Rubin B.H.,Richardson D.C.,Crystal structure of bovine Cu, Zn superoxide dismutase at 3 A resolution:chain tracing and metal ligands. Proc Natl Acad Sci USA,1975,72(4):1349～1353
[45]Bowler C.,Van Camp W.,Montage M.,Inze D.,Superoxide dismutase in plants. Critical Reviews in Plant Science,1994,13:199～218
[46]C.,Sollten L.,Vandenbranden S.,et al.Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO, 1991,10:1723～1732
[47]Bowler C.,lliotte T.,Deloose M., et al. The induction of manganese superoxide in response to stress in Nicotiana plunbaginifolia. EMBO,1989,8:31～38
[48]Tanaka Y, ibino.T.,ayashi Y, et al.,Salt of transgenic rice over expressing yeast mitochondrial Mn SOD in chloroplasts, Plant Science,1999,148:131～138
[49]Kazoo Tsugane, Kyoko kobayashi et al. A Recessive a Arabidopsis Mutant That under Salt Stress Shows Enhanced Active Oxygen Detoxification, The Plant Cell, 1999,11:1195～1206
[50]胡道芬.植物细胞工程育种.科学中国人,1995,：21～24
[51]邓丽琴,祝朋芳,陈长青.试论常规育种与分子育种的研究应用.杂粮作物.2004,24(5)：280～281
[52]Shinichi Itoh, Kiyoshi Ozumi, Ha Won Kim, Osamu Nakagawa, Ronald D. McKinney, Rodney J. Folz, Igor N. Zelko, Masuko Ushio-Fukai, Tohru Fukai. Novel mechanism for regulation of extracellular SOD transcription and activity by copper:Role of antioxidant-1Free Radical Biology & Medicine (2009) 46 95～104