民勤、临泽沙丘芦苇特有内含物比较分析及其对蛋白质氧化损伤的保护作用研究
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摘要
研究发现,分布于甘肃省民勤县、临泽县的沙丘芦苇(phragmites communis Trin.)中含有一种多氨基芳香族化合物(polyamine aromatic compound,简称PAAC),分子量为856Dalton,该化合物在其它三种生态型芦苇均未发现。为进一步研究其分子结构,实验通过柱层析手段,TLC和质谱分析技术对两地沙丘芦苇中的PAAC进行提取纯化和分析比对。结果显示两地沙丘芦苇的特有内含物是同一物质,并通过元素分析,红外光谱和H-NMR分析,确定该化合物分子式为C_(42)O_(11)H_(64_N_8,不饱和度15。
植物在逆境中很容易累积大量自由基而损伤生物分子。蛋白质在细胞内外广泛分布,极易受到自由基氧化。研究发现PAAC与芦苇抗逆性有关,并能与蛋白质结合。为证实PAAC对蛋白质的保护作用,本试验以水生芦苇(Phragmites communis Trin.)叶片为材料,提取可溶性蛋白,研究了PAAC对外源·OH引起的水生芦苇可溶性蛋白氧化损伤的影响;并用临氮二菲法测定了PAAC直接清除·OH的能力。
研究结果表明,在0～25μmol/L范围内,随着PAAC浓度的增加,由·OH引起的蛋白质羰基含量明显减少,PAAC浓度达到25μmol/L后,蛋白质羰基减少逐渐减缓,当PAAC浓度达到40μmol/L,蛋白质羰基含量基本维持不变,说明在0～25μmol/L范围内,PAAC处理能有效减轻外源·OH引起的水芦可溶性蛋白氧化损伤程度。用临氮二菲法测定PAAC直接清除·OH能力的实验也表明PAAC在低浓度时能有效清除·OH,0.45mmol/L时清除率达到55.2%。
通过实验可以证实生长于两地的沙丘芦苇所含有的该小分子物质是否同一物质,并对与该化合物的结构信息进一步了解;验证PAAC对蛋白质保护作用以及直接清除自由基的能力,这对揭示它作为相容性物质在芦苇抗逆中的作用及作用机理具有重要的理论价值和潜在的实用价值。
Preliminary studies suggest the compound, only exists in the dune reed coming from Minqin and Linze of Gansu province. Our work demonstrate it is a special polyamine aromatic compound(PACC) associated with anti-stress and not exist in other three ecotype reeds(swamp reed, light salt meadow reed and heavy salt meadow reed). We used three different techniques(TLC,H-NMR,IR) to analyse the structure of PACC from the reed of Minqin and Linze, and the results show they are identical, molecular weight 856Dalton. In addition, element analysis, ultra-red spectrum analysis and H-NMR analysis all demonstrate the molecular forum of this compound is C_(42)O_(11)H_(64)N_8 and the unsaturation is 15.
To further investigate its antioxidant function in plant, experiments were design to identify its protection on protein against reactive oxygen species damage in vitro. Study was processed as followed procedures: first, damage model was constructed by addition of exogenous hydroxyl radical(·OH) on the salt-reed protein solution; second, different doses of PAAC were feeded to the damage system, respectively; third, the protein carbonyl of the solutions was estimated. In addition, a supplementary test was carried out to examine the direct scavenging capacity of PAAC based on the method of orthophenanthroline.
Our data suggest PAAC can decrease the protein damage when treated with hydrol radical(·OH), and this protection effect is related significantly with the concentration of PAAC. With PAAC concentration increasing from 0 to 25μmol/L, the carbonyl amount declined rapidly, but declined gradually in the range of 25 to 40μmol/L. This protection function of low concentration of PAAC was also confirmed by hydrol radical(·OH) removal experiment, the scavenging rate is 55.2% when PAAC concentration is 0.45mmol/L.
植物在逆境中很容易累积大量自由基而损伤生物分子。蛋白质在细胞内外广泛分布,极易受到自由基氧化。研究发现PAAC与芦苇抗逆性有关,并能与蛋白质结合。为证实PAAC对蛋白质的保护作用,本试验以水生芦苇(Phragmites communis Trin.)叶片为材料,提取可溶性蛋白,研究了PAAC对外源·OH引起的水生芦苇可溶性蛋白氧化损伤的影响;并用临氮二菲法测定了PAAC直接清除·OH的能力。
研究结果表明,在0～25μmol/L范围内,随着PAAC浓度的增加,由·OH引起的蛋白质羰基含量明显减少,PAAC浓度达到25μmol/L后,蛋白质羰基减少逐渐减缓,当PAAC浓度达到40μmol/L,蛋白质羰基含量基本维持不变,说明在0～25μmol/L范围内,PAAC处理能有效减轻外源·OH引起的水芦可溶性蛋白氧化损伤程度。用临氮二菲法测定PAAC直接清除·OH能力的实验也表明PAAC在低浓度时能有效清除·OH,0.45mmol/L时清除率达到55.2%。
通过实验可以证实生长于两地的沙丘芦苇所含有的该小分子物质是否同一物质,并对与该化合物的结构信息进一步了解;验证PAAC对蛋白质保护作用以及直接清除自由基的能力,这对揭示它作为相容性物质在芦苇抗逆中的作用及作用机理具有重要的理论价值和潜在的实用价值。
Preliminary studies suggest the compound, only exists in the dune reed coming from Minqin and Linze of Gansu province. Our work demonstrate it is a special polyamine aromatic compound(PACC) associated with anti-stress and not exist in other three ecotype reeds(swamp reed, light salt meadow reed and heavy salt meadow reed). We used three different techniques(TLC,H-NMR,IR) to analyse the structure of PACC from the reed of Minqin and Linze, and the results show they are identical, molecular weight 856Dalton. In addition, element analysis, ultra-red spectrum analysis and H-NMR analysis all demonstrate the molecular forum of this compound is C_(42)O_(11)H_(64)N_8 and the unsaturation is 15.
To further investigate its antioxidant function in plant, experiments were design to identify its protection on protein against reactive oxygen species damage in vitro. Study was processed as followed procedures: first, damage model was constructed by addition of exogenous hydroxyl radical(·OH) on the salt-reed protein solution; second, different doses of PAAC were feeded to the damage system, respectively; third, the protein carbonyl of the solutions was estimated. In addition, a supplementary test was carried out to examine the direct scavenging capacity of PAAC based on the method of orthophenanthroline.
Our data suggest PAAC can decrease the protein damage when treated with hydrol radical(·OH), and this protection effect is related significantly with the concentration of PAAC. With PAAC concentration increasing from 0 to 25μmol/L, the carbonyl amount declined rapidly, but declined gradually in the range of 25 to 40μmol/L. This protection function of low concentration of PAAC was also confirmed by hydrol radical(·OH) removal experiment, the scavenging rate is 55.2% when PAAC concentration is 0.45mmol/L.
引文
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[2]Ana R,Fernando V.Water stress responses of two Mediterranean tree species influenced by native soil microorganisms and inoculation with a plant growth promoting rhizobacterium[J].Tree Physiol,2008,28:1693-1701.
[3]Ashraf M,Foolad MR.Roles of glycine betaine and proline in improving plant abiotic stress resistance[J].Environ Exp Botl,2006,59:206-216.
[4]Beal MF.Oxidative damage in neuro degeneratived is eases[J].The Neuro scientist,1997,3(l):21- 27.
[5]Bhattacharya RC,Maheswari M,Dineshkumar V.Transformation of Brassica oleracea var.capitata with bacterial betA gene enhances tolerance to salt stress[J].Scientia Horticulturae,2004(100):215-227.
[6]Bohnert HJ,Jensen RG.Strategies for engineering water-stress tolerance in plants[J].Trends Biotechnol 1996,14:89-97.
[7]Braam J,Sistrunk ML,Polisensky,DH.Plant responses to environmental stress:regulation and functions of the Arabidopsis TCH genes[J].Planta,1997,203:S35-S41.
[8]Chaturvedi V,Bartiss A,Wong B.Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol defective mutant from high-salt and oxidative stress[J].J Bacterid 1997,179:157-162.
[9]Cheng YF,Pu TL,Zhang CL,et al.PcTGD,a highly expressed gene in stem,is related to water tress in reed (Phragmites communis Trin.) [J].Chi Sci Bull,2001,46 (10):850-854.
[10]Chun PS,Agarwal M,Ohta M,et al.Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in ABA and drought stress responses[J].Plant Cell,2005,17:2384-2396.
[11]Creelman RA,Mul JE et al.Biosynthesis and action of jasmonates in plants[J].Ann Rev Plant Physiol Plant Mol Biol,1997,48:355-361.
[12]Deshnium P,Gombos Z,Nishiyama Y,et al.The action of glycine betaine in enhancement of tolerance of syechococcus sp.Strain PCC 7942 to low temperature[J].J Bacterid,1997,179:339-344.
[13]Emily S.Protein oxidative damage[J].methods in enzymology,2000,319:428-436.
[14]European Pharmacopoeia.EP5.0,2005.
[15]Frank J,Pompella A,Biesalski HK.Histo chemical visualization of oxidant stress[J].Free Radical Biology & Medicine,2000,29(l 1):1096-1105.
[16]Garcia AB,Almeida EJ,Iyer S,,et al.Effects of osmoprotectants upon NaCl stress in rice[J].Plant Physiol,1997,115;159-169.
[17]Graciela LS,Andrea CP,Walter A.Fructose-containing oligosaccharides:novel compatible solutes in Anabaena cells exposed to salt stress[J].Plant Science,2004,167:1003-1008.
[18]Hare PD,Cress WA,Staden JV.Dissecting the role of osmolyte accumulation during stress[J].Plant Cell and Environ.,1998,21:535-553.
[19]Hare PD,Cress WA.Metabolic implications of stress-induced proline accumulation inplants[J].Plant Growth Regulation,1997,21:79-102.
[20]Holmberg N,Bulow L.Improving stress tolerance in plants by gene transfer[M].Trends in Plant Science,1998,3:61-66.
[21]Kishor PB,Sangam S,Amrutha RN,et al.Regulation of proline biosynthesis,degradation,uptake and transport in higher plants:Its implicationsin plant growth and abiotic stress tolerance[J].Current Science,2005 88(3):424-438.
[22]Kiyosue T,Yoshiba Y,Yamaguchi SK,et al.A nuclear gene encoding mitochondrial proline dehydrogenase,an enzyme involved in proline metabolism,is upregulated by proline but downregulated by dehydration in Arabidopsis[J].Plant Cell,1996,8:1323-1335.
[23]Kumar YR,Babur JM,Sarma MS,et al.Structural identification and characterization of impurities in moxifloxacin[J].Pharm.Biomed.Anal.2004,34:1125-29.
[24]Lee MS,Kems EH.LC/MS applications in drug development[M].Mass Spec.Rev,1999,18:187-279.
[25]Leonidas L,Robert H P,Elaine K P,et al.Oxidative damage to proteins,lipids,and DNA in cortical brain regions from patients with dementia with levy bodies[J].Journal of Neuro chemistry,1998,71(1):302-312.
[26]McNeil SD,Nuccio ML,and Hanson AD.Betaines and related osmoprotectants:targets for metabolic engineering of stress resistance[J].Plant Physiol.,1999,120:945-949.
[27]Mittler R.Oxidative stress,antioxidants and stress tolerance[J].Trends in Plant science,2002,7:405-410.
[28]Nicolle S,Katrin M,Thomas Z,et al.Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts[J].Free Radical Biology &Medicine,2000,28(5):701-708.
[29]Nuccio ML,Rhodes D,McNeil SD,et al.Metabolic engineering of plants for osmotic stress resistance[J].Current Opinion in Plant Biology 1999,2:128-134.
[30]Pei SF,Wang DH,Sun CR,Pan YJ.Characterization of a novel trace-level impurity in lisinopril using multi-stage mass spectrometry[J].Eur J Mass Spectrom,2006,12:121-127.
[31]Ren DT,Zhang CHL.Analyses of soluble prtein,total and free amino acids in leaves of different ecotypes of Phragmites Communis growing in Hexi Corridor[J].Acta Bot Sin,1992,34:698-704.
[32]Ricardo A,Paolo V,Juan RL.Mycorrhizal and non-mycorrhizal Lactuca sativa plants exhibit contrasting responses to exogenous ABA during drought stress and recovery[J].Journal of Experimental Botany,2008,59,8:2029-2041.
[33]Savoure A,Hua X-J,Bertauche N,et al.Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana[J].Molecular and General Genetics,1997,254:104-109.
[34]Shena B,Jensen RG,Bohnert HJ.Increased resistance to oxidative stress in transgenicplants by targeting mannitol biosynthesis to chloroplasts[J].Plant Physiology,1997,113:1177-1183.
[35]Sheveleva E,Chmara W,Bohnert HJ.Increased salt and drought tolerance by D-ononitol production in transgenic Nicotiana tabacum[J].Plant Physiol,1997,115:1211-1219.
[36]Shinde V,Trivedi A,Upadhayay P R,Gupta NL,Kanase DG,Chikate R.Identification of a new impurity in lisinopril[J].J.Pharm Biomed.Anal,2007,43:381-386.
[37]Singh K,Foley RC,Onate SL.Transcrption factors in plant defense and stress response[J].Curr Opin Plant Biol,2002,5(5):430-436.
[38]Street TO,Bolen DW,Rose GD.A molecular mechanism for osmolyte-induced protein stability[J].Proc Natl Acad Sci,USA,2006,103(38):13997-14002.
[39]Takamiya K,Tsuchiya T,Ohta H.Degradation pathway of chlorophyll:What has gene cloning revealed[J].Trends Plant Sci,2000,5,10:426-431.
[40]Xiong LM,Ishitani M,Zhu JK.Interaction of osmotic stress,temperature and abscisic acid in theregulation of gene expression in Arabidopsis[J].Plant Physiol,1999,119:205-211.
[41]Yasuko N,Barbara SB,Earl RS.Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen,carbon,orphosphate starvation[J].PNAS,2007,47:18456-18460.
[42]Zafar IA,Barry H,Peter J.No evidence for increased oxidative damage to lipids,proteins,or DNA in huntington's disease[J].J Neurochem,2000,75(2):840-846.
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