枯草芽孢杆菌（Bacillus subtilis RN-61）抗菌蛋白的纯化及鉴定
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摘要
研究开发生防菌对经济林病害进行生物防治,是目前病害防治的研究方向,具有重要的理论意义和实际应用价值。本文针对目前经济林病害发生严重,使用化学农药不符合对经济林产品无公害和可持续发展要求这一难题,较系统的研究了应用于经济林病害防治的高效广谱拮抗细菌。本研究为获得应用于经济林病害防治的高效广谱拮抗细菌菌株,从土样中分离到103株细菌。采用平板对峙培养法,发酵产物活性测定法结合筛选拮抗菌株,根据16S rRNA基因分析以及生理生化反应鉴定筛选出的菌种。结果表明：菌株RN-61和RN-88对棉花红腐病(Fusarium moniliforme)、辣椒炭疽病(Colletotrichum gloesporioides)、茄子黄萎病(Verticillium dahliae)、马铃薯十腐病(Fusarium solani)、玉米青枯病(Ralstonia solanacearum)、辣椒根腐病(Fusarium vasinfectum)、黄瓜霜霉病(Pseudoperonospora cubensis)、李干腐病(Polvporus hirsutus)、草莓灰霉病(Botrytis cinerea)和桃褐腐病(Monilinia fructicola)10种病原真菌有明显抑制作用；菌株RN-61和RN-88的发酵液对桃褐腐病的抑制率最大在80%以上；对棉花红腐病、辣椒炭疽病的抑制率最小,在25%以上。经鉴定：菌株RN-61与枯草芽孢杆菌,相似度为99%,菌株RN-88与荧光假单胞杆菌的相似度也为99%,分别将其命名为Bacillus subtilis RN-61和Pseudomonas fluorescens RN-88。
通过单因素试验,在LB培养基的基础上做了改良,获得最适宜Bacillus subtilisRN-61抑菌物质产生的发酵配方：酵母提取物为碳源,大豆蛋白胨为氮源,其为培养基的优化结果。在对培养条件进行优化后,获得培养时间为24h,转速为180rpm,温度为37℃,pH为6.5,接菌量为10%为最佳培养条件。
对该菌的代谢产物进行环境稳定性测试分析,发现产生的抑菌物质粗提物不耐强酸,在偏酸、中性和强碱的条件下活性变化不大,紫外线对其活性也没有显著影响。温度对该抑菌物质的活性影响不强。
本试验利用柱层析技术从Bacillus subtilisRN-61发酵液中分离和纯化了枯草芽孢杆菌RN-61抗菌蛋白质。利用80%饱和度的硫酸铵提取的Bacillus subtilisRN-61粗蛋白质经DEAE-Cellulose柱层析后获得10个蛋白质吸收峰,其中第二个峰收集液具有抑菌活性,再将流分2收集液经层析柱Sephadex G-25gel层析后获得3个吸收峰,其中第二个峰收集液具有抑菌活性,将该活性蛋白进行通过SDS-PAGE进行观察,发现该蛋白质的分子量约为32.0kDa。
针对枯草芽孢杆菌RN-61的抗菌蛋白质的特性,应用现代串联质谱仪(Q-TOF)技术对抗菌蛋白质进行了鉴定。经过电喷雾四极杆飞行时间串联质谱分析后(Q-TOF)后获得了6个肽序列标签,氨基酸序列分别为INHNIAALNTLNR; LSSGLR; AGDDAAGLAISEK; NSQDGISLIQTAEGALTETHAILQRTEFNGK; LGAVQNRLEHTINNLS; ASGENLTAAESRIRDVDMAK; EMSEFTKNNI LSQASQAMLAQANQQPQNVLQLLR.利用肽序列标签进行鉴定,通过MASCOT数据库检索发现该抗菌蛋白质与枯草芽孢杆菌的鞭毛蛋白有显著相关性,相似度达到了44%。查新发现鞭毛蛋白的抗菌活性尚未见报道。
为了证实该蛋白的抑菌作用,用编码该蛋白的基因用Bacillus subtilisRN-61基因组DNA做模板,对其进行克隆。并将克隆的FLA-61基因插入表达载体pET28a(+),构建重组表达质粒pET28a-FLA61,将其转化到大肠杆菌E.coli中在IPTG诱导下表达该蛋白。将该菌的代谢产物对桃褐腐病原菌进行生物测定,结果表明具有较高的抑菌活性。96小时后,其对病原真菌的抑制率达到了36.72%。鉴于其较好的抑菌活性,我们将对其抑菌机制进行进一步研究。
An antifungal bacterial strain was isolated from soil samples collected from different agricultural sites in Beijing, China. An novel antifungal protein was also isolated and identified from the bacteria strain. In this study, Fusarium moniliforme、Colletotrichum gloesporioides、 Verticillium dahliae、Fusarium solani、Ralstonia solanacearum、Fusarium vasinfectum、 Pseudoperonospora cubensis、Polvporus hirsutus、Botrytis cinerea、Monilinia fructicola was used as indicators to screen antifungal bacteria. The bacteria were identified, based on their biochemical characteristics, which showed the highest antifungal activity, were selected for further identification by partial sequence analysis of their16S rRNA genes. Because of the effective antifungal activity of the strain RN-61and RN-88, it was investigated.The strain RN-61shared99%similarity with that of Bacillus subtilis subsp. subtilis str, named Bacillus subtilis RN-61, which was exhibited the hightest antifungal activity.The medium and culture conditions of the strain RN-61were optimized by single factor experiments.In the investigation of the culture condition, the major ingredients being investigated included carbon sources and nitrogen sources. The optimal antifungal activity production condition were determined as follows:initial pH6.5,culture temperature at37℃, culture time24h, rotation rate180rpm, soybean peptone10.0g/L, yeast extract5g/L.The antifungal metabolites from the bacteria B.subtilis RN-61were further purified by consecutive chromatographic fractionations including ion exchange chromatography and gel filtration chromatography. The purified fractions were tested for their antifugal effect.the result showed that the fraction B had the best effect with the inhibition of40.94%after96h culture.The purified fraction B was shown to have a relative low molecular weight of32kd by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE).Antifungal protein was ideniified with Q-TOF after digested with trypsin. six Peptides were get.six peptides sequenee was INHNIAAL NTLNR；LSSGLR; AGDDAAGLAISEK; NSQDGI SLIQTAEGAL TETHAILQRTEFNGKK: LGAVQNRLEHTINNLS: ASGENLTAAE SRIRDVDMAK EMSEFTKNNI LSQASQAMLA QANQQPQNVLQLLR.These peptides were used to search in MASCOT database:we found the protein was observed related with this antifungal Protein.The protein was flagllin. LC/ESI-MS analysis confirmed the fraction from the metabolites of B.subtilis RN-61which has the strongly inhibition to the indicator that formed as a result of a novel protein belonged to the flagellin family. A flagellin gene (FLA-RN61) was amplified from the B.subtilis RN-61genome by using specifically designed PCR primers. The sequence analysis showed the new cloned gene had a degree of91%identity to a known FLA gene in a new different B.subtilis. The sequence was also expressed in E.coli and showed high antifungal effect. We report here the isolation of a new antifungal bacteria from soils in Beijing China that may be very well adapted to the environmental conditions of this area. We also first report the complete cloning of the new antifungal gene which is belongs to the flagellin family. Due to their high antifungal activity, the bacteria and the novel protein isolated from this work merit further study as a potential biological agents for the organic food production in agriculture.
通过单因素试验,在LB培养基的基础上做了改良,获得最适宜Bacillus subtilisRN-61抑菌物质产生的发酵配方：酵母提取物为碳源,大豆蛋白胨为氮源,其为培养基的优化结果。在对培养条件进行优化后,获得培养时间为24h,转速为180rpm,温度为37℃,pH为6.5,接菌量为10%为最佳培养条件。
对该菌的代谢产物进行环境稳定性测试分析,发现产生的抑菌物质粗提物不耐强酸,在偏酸、中性和强碱的条件下活性变化不大,紫外线对其活性也没有显著影响。温度对该抑菌物质的活性影响不强。
本试验利用柱层析技术从Bacillus subtilisRN-61发酵液中分离和纯化了枯草芽孢杆菌RN-61抗菌蛋白质。利用80%饱和度的硫酸铵提取的Bacillus subtilisRN-61粗蛋白质经DEAE-Cellulose柱层析后获得10个蛋白质吸收峰,其中第二个峰收集液具有抑菌活性,再将流分2收集液经层析柱Sephadex G-25gel层析后获得3个吸收峰,其中第二个峰收集液具有抑菌活性,将该活性蛋白进行通过SDS-PAGE进行观察,发现该蛋白质的分子量约为32.0kDa。
针对枯草芽孢杆菌RN-61的抗菌蛋白质的特性,应用现代串联质谱仪(Q-TOF)技术对抗菌蛋白质进行了鉴定。经过电喷雾四极杆飞行时间串联质谱分析后(Q-TOF)后获得了6个肽序列标签,氨基酸序列分别为INHNIAALNTLNR; LSSGLR; AGDDAAGLAISEK; NSQDGISLIQTAEGALTETHAILQRTEFNGK; LGAVQNRLEHTINNLS; ASGENLTAAESRIRDVDMAK; EMSEFTKNNI LSQASQAMLAQANQQPQNVLQLLR.利用肽序列标签进行鉴定,通过MASCOT数据库检索发现该抗菌蛋白质与枯草芽孢杆菌的鞭毛蛋白有显著相关性,相似度达到了44%。查新发现鞭毛蛋白的抗菌活性尚未见报道。
为了证实该蛋白的抑菌作用,用编码该蛋白的基因用Bacillus subtilisRN-61基因组DNA做模板,对其进行克隆。并将克隆的FLA-61基因插入表达载体pET28a(+),构建重组表达质粒pET28a-FLA61,将其转化到大肠杆菌E.coli中在IPTG诱导下表达该蛋白。将该菌的代谢产物对桃褐腐病原菌进行生物测定,结果表明具有较高的抑菌活性。96小时后,其对病原真菌的抑制率达到了36.72%。鉴于其较好的抑菌活性,我们将对其抑菌机制进行进一步研究。
An antifungal bacterial strain was isolated from soil samples collected from different agricultural sites in Beijing, China. An novel antifungal protein was also isolated and identified from the bacteria strain. In this study, Fusarium moniliforme、Colletotrichum gloesporioides、 Verticillium dahliae、Fusarium solani、Ralstonia solanacearum、Fusarium vasinfectum、 Pseudoperonospora cubensis、Polvporus hirsutus、Botrytis cinerea、Monilinia fructicola was used as indicators to screen antifungal bacteria. The bacteria were identified, based on their biochemical characteristics, which showed the highest antifungal activity, were selected for further identification by partial sequence analysis of their16S rRNA genes. Because of the effective antifungal activity of the strain RN-61and RN-88, it was investigated.The strain RN-61shared99%similarity with that of Bacillus subtilis subsp. subtilis str, named Bacillus subtilis RN-61, which was exhibited the hightest antifungal activity.The medium and culture conditions of the strain RN-61were optimized by single factor experiments.In the investigation of the culture condition, the major ingredients being investigated included carbon sources and nitrogen sources. The optimal antifungal activity production condition were determined as follows:initial pH6.5,culture temperature at37℃, culture time24h, rotation rate180rpm, soybean peptone10.0g/L, yeast extract5g/L.The antifungal metabolites from the bacteria B.subtilis RN-61were further purified by consecutive chromatographic fractionations including ion exchange chromatography and gel filtration chromatography. The purified fractions were tested for their antifugal effect.the result showed that the fraction B had the best effect with the inhibition of40.94%after96h culture.The purified fraction B was shown to have a relative low molecular weight of32kd by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE).Antifungal protein was ideniified with Q-TOF after digested with trypsin. six Peptides were get.six peptides sequenee was INHNIAAL NTLNR；LSSGLR; AGDDAAGLAISEK; NSQDGI SLIQTAEGAL TETHAILQRTEFNGKK: LGAVQNRLEHTINNLS: ASGENLTAAE SRIRDVDMAK EMSEFTKNNI LSQASQAMLA QANQQPQNVLQLLR.These peptides were used to search in MASCOT database:we found the protein was observed related with this antifungal Protein.The protein was flagllin. LC/ESI-MS analysis confirmed the fraction from the metabolites of B.subtilis RN-61which has the strongly inhibition to the indicator that formed as a result of a novel protein belonged to the flagellin family. A flagellin gene (FLA-RN61) was amplified from the B.subtilis RN-61genome by using specifically designed PCR primers. The sequence analysis showed the new cloned gene had a degree of91%identity to a known FLA gene in a new different B.subtilis. The sequence was also expressed in E.coli and showed high antifungal effect. We report here the isolation of a new antifungal bacteria from soils in Beijing China that may be very well adapted to the environmental conditions of this area. We also first report the complete cloning of the new antifungal gene which is belongs to the flagellin family. Due to their high antifungal activity, the bacteria and the novel protein isolated from this work merit further study as a potential biological agents for the organic food production in agriculture.
引文
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