几种植物抗真菌蛋白（肽）的纯化和性质研究
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摘要
植物源抗真菌蛋白或抗真菌肽(AFPs)种类丰富,来源广泛,因其广谱,无毒的特征一直受到学者的关注。抗真菌蛋白的研究与农业,食品工业密切相关。本文选取了三种我国的特色作物种子为研究材料,从中纯化抗真菌蛋白或抗真菌肽,并分析其生物化学特性及抗真菌活性。首先以苦荞种子为实验材料,经提取,热处理,Resource S阳离子交换层析及Superdex Peptide分子筛层析,纯化得到一种抗真菌肽,Tricine-SDS-PAGE显示其表观分子质量约为8.0 kDa,表观纯度很高。表面增强激光解吸电离飞行时间质谱(SELDI-TOF)显示其实际分子质量为3.909 kDa。该抗菌肽对白腐菌(Panus conchatus),绿色木霉(Trichoderma reesei)和链格孢霉(Alternaria alternata)等均表现出显著的生长抑制活性。绿色木霉的形态学分析显示受到影响的菌丝生长停滞,分支加剧,基内菌丝端部膨大,原生质凝缩。
以莜麦(裸燕麦)种子为实验材料经过Resource S阳离子交换层析和superdex 75凝胶排阻层析,纯化得到两种抗真菌蛋白,分别命名为α-avechin和β-avechin,其中,β-avechin的含量约为α-avechin的三倍。SDS-PAGE显示两种蛋白的分子量均为35 kDa左右,推测是两种相似蛋白或是同一种蛋白的两种修饰形式应用固相pH梯度胶条测定β-avechin的等电点为8.9。两种蛋白均对白腐菌(Panus conchatus),绿色木霉(Trichoderma reesei)显示出显著的生长抑制作用并呈现剂量依赖性,其中,对于绿色木霉的最低抑制量为0.21μg。胶内酶解和串联质谱实验的匹配结果显示,该蛋白与莜麦种子中的几丁质酶具有显著的相似性。以胶体几丁质为底物测定了该蛋白的几丁质酶活性,结果显示该蛋白是一种几丁质酶,其比活力为3.59U／mg,同时测定了酶活力的最适pH和最适温度。
以薏苡种子为实验材料,经过Resource S阳离子交换层析和superdex 75凝胶排阻层析等步骤纯化得到一种28 kDa的薏苡抗真菌蛋白,该蛋白此前未见报道,对链格孢霉(Alternaria alternate),绿色木霉(Trichoderma reesei)和白腐菌(Panus conchatus)三株丝状真菌具有显著生长抑制活性,并呈现出剂量依赖的特征。
Antifungal peptides(AFPs), serve to protect organisms against fungal invasion, have been isolated from a variety of plants species.The experimental materials of present investigation was seeds of tartary buckwheat (Fagopyrum tataricum).The purification procedure involved extraction with aqueous buffer, heat treatment, cation exchange chromatography on Resource S column, and size exclusion chromatography (SEC) on Superdex Peptide.The apparent molecular weight of purified peptide was approximately 8.0 kDa by Tricine-SDS-PAGE,and actual molecular weight was 3.909 kDa by surface-enhanced laser desorption ionization-time of flight (SELDI-TOF). This peptide showed strong antifungal activity against Panus conchatus, Trichoderma reesei and Alternaria alternata. Light microscopic examination disclosed antifungal peptide induced swelling of hyphal tips, hyphal distortions and fragmentation in the Trichoderma reesei.
A chitinase with antifungal activity was purified from naked oat (Avena chinensis) seeds by extraction with an aqueous buffer, cation exchange and size exclusion chromatography. The molecular weight and isoelectric point of the purified chitinase were estimated at 35 kDa and 8.9, respectively. Peptide mass fingerprint analysis indicated the primary structure of the purified naked oat (Avena chinensis) chitinase is highly homologic to oat (Avena sativa) class I chitinase.When colloidal chitin is used as a substrate, with a specific activity 3.596 U/mg, which is consistent with reported chitinase activity, the purified chitinase is highly active.The chitinase activity is dependent on both pH and temperature, with an optimum pH of 7.0 and optimum temperature of 40℃.Measurement of in vitro antifungal activity demonstrated that the purified chitinase has potent, dose-dependent inhibitory activity against fungi Panus conchatus and Trichoderma reesei. All these results provided evidence that the chitinase purified from naked oat seeds is a class I chitinase with antifungal activity..To isolate and purify an antifungal proteins from seeds of adlay(Coix chinensis Tod.)and analyze its antifungal activity. Methods The procedure involved extraction with aqueous buffer, ion exchange chromatography on Resource S,and size exclusion chromatography (SEC) on Superdex 75.Results The molecule weight of antifungal protein is approximately 28kDa, which was determined by SDS-PAGE.It shows strong fungal growth inhibitory activity against Alternaria alternate, Trichoderma reesei and Panus conchatus, in a dose-dependent manner. Conclusion The method is simple,and the purify of protein is over 95%,it can be used for purification of some novel antifungal protein from plant seeds.
以莜麦(裸燕麦)种子为实验材料经过Resource S阳离子交换层析和superdex 75凝胶排阻层析,纯化得到两种抗真菌蛋白,分别命名为α-avechin和β-avechin,其中,β-avechin的含量约为α-avechin的三倍。SDS-PAGE显示两种蛋白的分子量均为35 kDa左右,推测是两种相似蛋白或是同一种蛋白的两种修饰形式应用固相pH梯度胶条测定β-avechin的等电点为8.9。两种蛋白均对白腐菌(Panus conchatus),绿色木霉(Trichoderma reesei)显示出显著的生长抑制作用并呈现剂量依赖性,其中,对于绿色木霉的最低抑制量为0.21μg。胶内酶解和串联质谱实验的匹配结果显示,该蛋白与莜麦种子中的几丁质酶具有显著的相似性。以胶体几丁质为底物测定了该蛋白的几丁质酶活性,结果显示该蛋白是一种几丁质酶,其比活力为3.59U／mg,同时测定了酶活力的最适pH和最适温度。
以薏苡种子为实验材料,经过Resource S阳离子交换层析和superdex 75凝胶排阻层析等步骤纯化得到一种28 kDa的薏苡抗真菌蛋白,该蛋白此前未见报道,对链格孢霉(Alternaria alternate),绿色木霉(Trichoderma reesei)和白腐菌(Panus conchatus)三株丝状真菌具有显著生长抑制活性,并呈现出剂量依赖的特征。
Antifungal peptides(AFPs), serve to protect organisms against fungal invasion, have been isolated from a variety of plants species.The experimental materials of present investigation was seeds of tartary buckwheat (Fagopyrum tataricum).The purification procedure involved extraction with aqueous buffer, heat treatment, cation exchange chromatography on Resource S column, and size exclusion chromatography (SEC) on Superdex Peptide.The apparent molecular weight of purified peptide was approximately 8.0 kDa by Tricine-SDS-PAGE,and actual molecular weight was 3.909 kDa by surface-enhanced laser desorption ionization-time of flight (SELDI-TOF). This peptide showed strong antifungal activity against Panus conchatus, Trichoderma reesei and Alternaria alternata. Light microscopic examination disclosed antifungal peptide induced swelling of hyphal tips, hyphal distortions and fragmentation in the Trichoderma reesei.
A chitinase with antifungal activity was purified from naked oat (Avena chinensis) seeds by extraction with an aqueous buffer, cation exchange and size exclusion chromatography. The molecular weight and isoelectric point of the purified chitinase were estimated at 35 kDa and 8.9, respectively. Peptide mass fingerprint analysis indicated the primary structure of the purified naked oat (Avena chinensis) chitinase is highly homologic to oat (Avena sativa) class I chitinase.When colloidal chitin is used as a substrate, with a specific activity 3.596 U/mg, which is consistent with reported chitinase activity, the purified chitinase is highly active.The chitinase activity is dependent on both pH and temperature, with an optimum pH of 7.0 and optimum temperature of 40℃.Measurement of in vitro antifungal activity demonstrated that the purified chitinase has potent, dose-dependent inhibitory activity against fungi Panus conchatus and Trichoderma reesei. All these results provided evidence that the chitinase purified from naked oat seeds is a class I chitinase with antifungal activity..To isolate and purify an antifungal proteins from seeds of adlay(Coix chinensis Tod.)and analyze its antifungal activity. Methods The procedure involved extraction with aqueous buffer, ion exchange chromatography on Resource S,and size exclusion chromatography (SEC) on Superdex 75.Results The molecule weight of antifungal protein is approximately 28kDa, which was determined by SDS-PAGE.It shows strong fungal growth inhibitory activity against Alternaria alternate, Trichoderma reesei and Panus conchatus, in a dose-dependent manner. Conclusion The method is simple,and the purify of protein is over 95%,it can be used for purification of some novel antifungal protein from plant seeds.
引文
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[5]Wang HX, Ng TB.Isolation of cicadin, a novel and potent antifungal peptide from dried juvenile cicadas. Peptides,2002,23,7-11.
[6]Vogelsang R, Barz W. Purification, characterization and differential hormonal regulation of a beta-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.). Planta,1993,189,60-69.
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[13]Boller T, Gehri A, Mauch F, et al.Chitinase in bean leaves:induction by ethylene, purification, properties, and possible function. Planta,1983,157,22-31.
[14]Izabela S.Santos, Maura Da Cunha, Olga L.T. Machado, et al.A chitinase from Adenanthera pavonina L.seeds:purification, characterisation and immunolocalisation. Plant Science,2004,167,1203-1210
[15]Ye XY, Wang HX, Ng TB.Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab).Biochem. Biophys. Res. Commun.,2000,269, 155-159.
[16]Huang X, Xie W, Gong Z. Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba. FEBS Lett.,2000,478,123-126.
[17]Lam SS,Wang H, Ng TB.Purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem. Biophys. Res. Commun.,1998,253,135-142.
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[19]Parkash A, Ng TB, Tso WW. Isolation and characterization of luffacylin, a ribosome inactivating peptide with anti-fungal activity from sponge gourd (Luffa cylindrica) seeds. Peptides.2002,23,1019-1024.
[20]Wang SY, Lin J, Ye MY, et al.Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities.Peptides,2006,27, 3129-3136.
[21]Oliveira AS,Migliolo L, Aquino RO, et al.Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity. J. Agric. Food Chem.,2007,55,7342-7349.
[22]Park Y, Choi BH, Kwak JS,et al.Kunitz-type serine protease inhibitor from potato (Solarium tuberosum L. cv. Jopung).J. Agric. Food Chem.,2005,53,6491-6496.
[23]Joshi BN, Sainani MN,Bastawade KB, et al.Cysteine protease inhibitor from pearl millet:a new class of antifungal protein. Biochem. Biophys. Res.Commun.,1998,246, 382-387.
[24]Ye XY, Ng TB, Rao PF. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. Biochem. Biophys. Res. Commun.,2001,289,91-96.
[25]Ye XY, Ng TB.Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun.,2000,273,1111-1115.
[26]Ye XY, Ng TB.Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities.Life Sci.,2002,70, 1129-1138.
[27]Kirubakaran SI, Begum SM, Ulaganathan K, et al.Characterization of a new antifungal lipid transfer protein from wheat. Plant Physiol.Biochem.,2008,46,918-927.
[28]Molina A, Segura A.Garcia-Olmedo F. Lipid transfer proteins (ns-LTPs) from barley and maize leaves are potent inhibitors of bacterialand fungal plant pathogens, FEBS Lett.. 1993,316,119-122.
[29]Nielsen KK, Nielsen JE, Madrid SM, et al.New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins. Plant Mol. Biol.,1996,31,539-552.
[30]Terras FRG, Goderis IJ, Van Leuven F, et al.In vitro antifungal activity of a radish (Raphanus sativus L.) seed protein homologous to nonspecific lipid transfer proteins. Plant Physiol.,1992,100,1055-1058.
[31]Wang SY, Wu JH, Ng TB, et al.A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides.2004,25,1235-1242.
[32]Lin P, Xia L, Ng TB.First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species. Peptides.2007,28,1514-1519.
[33]Wang HX, Ng TB.Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. Biochem. Biophys. Res.Commun., 2000,279,407-411.
[34]Ye XY, Wang HX, Ng TB.Sativin:a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon. Life Sci.,2000,67, 775-781.
[35]Sobott F, Watt SJ, Smith J, et al.Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination. J.Am.Soc. Mass Spectrom.,2009, 20,1652-1659.
[36]Schlumbaum A, Mauch F, Vogeli U, et al.Plant chitinases are potent inhibitors of fungal growth. Nature 1986,324,365-367. Leung EH, Ng TB.A relatively stable antifungal peptide from buckwheat seeds with antiproliferative activity toward cancer cells. J.Pept. Sci.,2007,13,762-767.
[37]刘颖,徐庆.抗真菌肽LP-1的分离纯化及特性分析.微生物学报,1999,39, 441-447.
[38]Peterson GL. A simplification of the protein assay method of Lowry et al.which is more generally applicable. Anal.Biochem.,1977,83,346-356.
[39]Laemmli UK, Favre M. Gel electrophoresis of proteins. J.Mol. Biol.,1973,80, 575-599.
[40]Gorg A, Weiss W,Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics.2004,4,3665-3685.
[41]Neuhoff V, Arold N, Taube D, et al.Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.Electrophoresis,1988,9,255-262.
[42]Burlingame AL, Boyd RK, Gaskell SJ.Mass Spectrometry. Anal. Chem.,1996,68, 599-652.
[43]Williams K, Lopresti M, Stone K. Internal protein sequencing of SDS-page-separated proteins:Optimization of an in gel digest protocol.In Techniques in Protein Chemistry VIII. Marshak, D.Academic Press:San Diego, CA.1997,8,79-90.
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[48]刘静,赵奎军,潘映红.利用色谱聚焦和离子交换色谱快速分离分析薏苡38ku抗真菌蛋白.东北农业大学学报,2008,39,23-28.
[2]Ng TB.Antifungal proteins and peptides of leguminous and non-leguminous origins. Peptides,2004,25,1215-22.
[3]张勇,吴建伟,顾莉娟.凝胶层析法分离家蝇幼虫血淋巴中抗真菌肽的研究.中国媒介生物学及控制杂志,2007,3,200-204.
[4]徐兴军,陈丽,吕建伟,等.中国林蛙和中华蟾蜍皮肤抗菌肽的分离纯化及其抗菌活性.四川动物,2009,28,164-167.
[5]Wang HX, Ng TB.Isolation of cicadin, a novel and potent antifungal peptide from dried juvenile cicadas. Peptides,2002,23,7-11.
[6]Vogelsang R, Barz W. Purification, characterization and differential hormonal regulation of a beta-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.). Planta,1993,189,60-69.
[7]Deshpande MV. Enzymatic degradation of chitin and its biological applications. J. Sci. Ind. Res.,1986,45,273-281.
[8]Kasprzewska A. Plant chitinases-regulation and function. Cell.Mol.Biol.Lett.,2003, 8,809-824.
[9]Taira T, Ohnuma T, Yamagami T, et al. Antifungal activity of rye (Secale cereale) seed chitinases:the different binding manner of class I and class II chitinases to the fungal cell walls.Biosci.Biotechnol.Biochem.,2002,66,970-977.
[10]Wang S,Wu J, Rao PF, et al.A chitinase with antifungal activity from the mung bean. Protein Expr. Purif.,2005,40,230-236.
[11]Krishnaveni S, Liang GH, Muthukrishnan S,et al.Purification and partial characterization of chitinases from sorghum seeds.Plant Science,1999,144,1-7.
[12]Cohen-Kupiec R, Chet I.The molecular biology of chitin digestion. Curr. Opin. Biotechnol.,1998,9,270-277.
[13]Boller T, Gehri A, Mauch F, et al.Chitinase in bean leaves:induction by ethylene, purification, properties, and possible function. Planta,1983,157,22-31.
[14]Izabela S.Santos, Maura Da Cunha, Olga L.T. Machado, et al.A chitinase from Adenanthera pavonina L.seeds:purification, characterisation and immunolocalisation. Plant Science,2004,167,1203-1210
[15]Ye XY, Wang HX, Ng TB.Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab).Biochem. Biophys. Res. Commun.,2000,269, 155-159.
[16]Huang X, Xie W, Gong Z. Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba. FEBS Lett.,2000,478,123-126.
[17]Lam SS,Wang H, Ng TB.Purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem. Biophys. Res. Commun.,1998,253,135-142.
[18]Ng TB, Parkash A. Hispin, a novel ribosome inactivating protein with antifungal activity from hairy melon seeds.Protein Expr. Purif.,2002,26,211-217.
[19]Parkash A, Ng TB, Tso WW. Isolation and characterization of luffacylin, a ribosome inactivating peptide with anti-fungal activity from sponge gourd (Luffa cylindrica) seeds. Peptides.2002,23,1019-1024.
[20]Wang SY, Lin J, Ye MY, et al.Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities.Peptides,2006,27, 3129-3136.
[21]Oliveira AS,Migliolo L, Aquino RO, et al.Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity. J. Agric. Food Chem.,2007,55,7342-7349.
[22]Park Y, Choi BH, Kwak JS,et al.Kunitz-type serine protease inhibitor from potato (Solarium tuberosum L. cv. Jopung).J. Agric. Food Chem.,2005,53,6491-6496.
[23]Joshi BN, Sainani MN,Bastawade KB, et al.Cysteine protease inhibitor from pearl millet:a new class of antifungal protein. Biochem. Biophys. Res.Commun.,1998,246, 382-387.
[24]Ye XY, Ng TB, Rao PF. A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. Biochem. Biophys. Res. Commun.,2001,289,91-96.
[25]Ye XY, Ng TB.Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun.,2000,273,1111-1115.
[26]Ye XY, Ng TB.Isolation of a new cyclophilin-like protein from chickpeas with mitogenic, antifungal and anti-HIV-1 reverse transcriptase activities.Life Sci.,2002,70, 1129-1138.
[27]Kirubakaran SI, Begum SM, Ulaganathan K, et al.Characterization of a new antifungal lipid transfer protein from wheat. Plant Physiol.Biochem.,2008,46,918-927.
[28]Molina A, Segura A.Garcia-Olmedo F. Lipid transfer proteins (ns-LTPs) from barley and maize leaves are potent inhibitors of bacterialand fungal plant pathogens, FEBS Lett.. 1993,316,119-122.
[29]Nielsen KK, Nielsen JE, Madrid SM, et al.New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins. Plant Mol. Biol.,1996,31,539-552.
[30]Terras FRG, Goderis IJ, Van Leuven F, et al.In vitro antifungal activity of a radish (Raphanus sativus L.) seed protein homologous to nonspecific lipid transfer proteins. Plant Physiol.,1992,100,1055-1058.
[31]Wang SY, Wu JH, Ng TB, et al.A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean. Peptides.2004,25,1235-1242.
[32]Lin P, Xia L, Ng TB.First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species. Peptides.2007,28,1514-1519.
[33]Wang HX, Ng TB.Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. Biochem. Biophys. Res.Commun., 2000,279,407-411.
[34]Ye XY, Wang HX, Ng TB.Sativin:a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon. Life Sci.,2000,67, 775-781.
[35]Sobott F, Watt SJ, Smith J, et al.Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination. J.Am.Soc. Mass Spectrom.,2009, 20,1652-1659.
[36]Schlumbaum A, Mauch F, Vogeli U, et al.Plant chitinases are potent inhibitors of fungal growth. Nature 1986,324,365-367. Leung EH, Ng TB.A relatively stable antifungal peptide from buckwheat seeds with antiproliferative activity toward cancer cells. J.Pept. Sci.,2007,13,762-767.
[37]刘颖,徐庆.抗真菌肽LP-1的分离纯化及特性分析.微生物学报,1999,39, 441-447.
[38]Peterson GL. A simplification of the protein assay method of Lowry et al.which is more generally applicable. Anal.Biochem.,1977,83,346-356.
[39]Laemmli UK, Favre M. Gel electrophoresis of proteins. J.Mol. Biol.,1973,80, 575-599.
[40]Gorg A, Weiss W,Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics.2004,4,3665-3685.
[41]Neuhoff V, Arold N, Taube D, et al.Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.Electrophoresis,1988,9,255-262.
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