【作者】 刘永锋；

【导师】 周明国； 陈志谊；

【作者基本信息】 南京农业大学 ， 农药学， 2006， 博士

【摘要】 枯草芽孢杆菌Bs-916是我们自行分离的一株拮抗菌，目前用于防治水稻纹枯病和稻曲病，该菌株表现出广谱的抑菌活性。本文研究了枯草芽孢杆菌Bs-916及分泌液对水稻纹枯病菌（Rhizoctonia solani）、蚕豆枯萎病菌（Fusanum oxysporum Schlecht．f．fabae Yu et Fang）、水稻恶苗病菌（Fusarium monitiforme Sheld．）、西瓜枯萎病菌（Fusanum oxysporum（Schl．）f．sp．nieveum（E．F．Smith）Snyderet Hansen）、水稻稻瘟病菌（Pyricularia grisea）、水稻稻曲病菌（Ustilaginoidea virens）、水稻白叶枯病菌（Xanthomonas oryzae pv．oryzae（Ishiyama）Zoo）和水稻细菌性条斑病菌（Xanthomonas oryzae pv．oryzicola Dowson）的抑菌活性，发现枯草芽孢杆菌Bs-916及其分泌物对上述病原菌均具有较强的抑菌活性，具有广谱性的特点。同时，采用丙酮沉淀、PEG沉淀、等电点沉淀和超微浓缩，发现沉淀物和截留物都有抻菌活性，提示枯草芽孢杆菌胞外存在蛋白质类抗菌物质。用硫酸铵分级沉淀枯草芽孢杆菌Bs-916胞外抗菌蛋白质，共获得8个蛋白质粗提物，其中硫酸铵饱和度为40％～50％和50％～60％提取的粗蛋白质具有较强的抑菌活性，说明该抗菌蛋白质在硫酸铵饱和度为40-60％时就可完全沉淀。研究结果表明：硫酸铵饱和度40％～50％和50％～60％提取的粗蛋白质浓度分别为17.261μg／ml和18.899μg／ml，上述两者粗提物对水稻纹枯病菌和水稻恶苗病菌有较强的抑菌活性，对马铃薯晚疫病菌没有抑菌活性；当温度高于40℃时抑菌活性均降低，但在120℃时仍有部分抑菌活性；对pH值适应范围较宽，在pH值为13.6时抑菌活性均丧失；经蛋白酶K、蛋白酶E、胃蛋白酶、胰蛋白酶和木瓜蛋白酶处理后，抑菌活性均下降。SDS-PAGE电泳结果表明枯草芽孢杆菌Bs-916能分泌多种蛋白质。采用了层析技术分离和纯化了枯草芽孢杆菌Bs-916胞外的抗菌蛋白质。利用DEAE Sepharose Fast Flow柱层析，后获得3个蛋白质吸收峰，其中B峰具有抑菌活性，将B峰收集液过疏水层析柱Phenyl Sepharose 6 F.F.后获得2个吸收峰，其中第E峰具有抑菌活性，再将第E峰收集液过羟基磷石灰石柱层析后得到了2个蛋白质吸收峰，其中第G峰具有抑菌活性，经SDS-PAGE检测后为单一的蛋白质条带（暂定名：Bacisubin），该蛋白质的分子量约为41.9kDa。研究了胞外抗菌蛋白质（bacisubin）的抑菌活性。发现抗菌蛋白质（Bacisubin）对水稻纹枯病菌（Rhizoctonia solani）、水稻稻瘟病菌（Magnaporthe grisease）、油菜菌核病菌（Sclerotinia sclerotiorum）、甘蓝黑斑病菌（Ahernaria oleracea）、白菜黑斑病菌病菌（A．brassicae）、灰霉病菌（Botrytis cinerea）、辣椒炭疽病菌（C．capsici）和水稻恶苗病菌（F．moniliforme）均有抑菌活性。尤其对Alternaria属的病原菌具有较高的毒力。结果显示抗菌蛋白质Bacisubin具有广谱、高毒力的特点。抗菌蛋白质（Bacisubin）对水稻纹枯病菌、灰霉病菌、甘蓝黑斑病菌和白菜黑斑病菌的EC₅₀分别为4.011μM、2.740μM、0.087μM和0.055μM。枯草芽孢杆菌Bs-916胞外抗菌蛋白质（Bacisubin）的抑菌机制是造成病原菌菌丝营养吸收困难，致使菌丝顶端膨大，分支增加，胞壁加厚、断裂，从而抑制了病原菌的生长。同时该抗菌蛋白质具有凝集素活性和核糖核酸酶活性。枯草芽孢杆菌Bs-916胞外抗菌蛋白质（Bacisubin）无蛋白酶活性和蛋白酶抑制活性。利用生物质谱技术鉴定了枯草芽孢杆菌胞外抗菌蛋白质。胞外抗菌蛋白质（Bacisubin）经过胰蛋白酶消解后，经基质协助激光解吸附离子化-飞行时间质谱（MALDI-TOF-MS）获得22个质荷比峰（M／Z），Matrix Science数据库检索未发现显著相关的蛋白质。再经电喷雾四极杆飞行时间串联质谱分析后（Q-TOF2）后获得五个肽的氨基酸序列，氨基酸序列分别为VTIVDEKGR，FSDSDVMNR，VYIADSTNFK，ELPISENLASVNMR，EAEWAYMITGK。经鉴定，该蛋白质和一种枯草芽孢杆菌的草酸盐脱羧酶和地衣芽孢杆菌的草酸盐脱羧酶具有显著的相关性，关于草酸盐脱羧酶的抑菌活性和其他生物活性未见报道。更多还原

【Abstract】 Bacillus subtilis Strain Bs-916 was isolated from rice field soil. B-916 was used incontrolling rice shealth blight and rice false smut in field, while showed a high controleffect. The Bs-916 and its extract showed a high antifungal （antibacterial） activityagainst hizoctonia solani, Fusarium oxysporum Schlecht. f. fabae Yu et Fang, Fusariummoniliforme Sheld., Fusarium oxysporum （Schl.） f.sp.nieveum （E.F.Smith）SnyderetHansen, Pyricularia grisea, Ustilaginoidea virens, Xanthomonas oryzae pv. oryzae（Ishiyama） Zoo, Xanthomonas oryzae pv. oryzicola Dowson. Bs-916 and its extractshowed a broad-spectrum antifungal activity. The extract of Bacillus subtilis strain B-916was deposited by acetone, PEG6000, ammonium sulfate and ultra-4 centrifugal filterdevices exhibited a high antifungal activity （detected toward Rhizoctonia solani）. Theantifungal activity of the extract treated with protease K was lower than that withoutprotease K treated. The results implied that the extract of Bs-916 secrete an antifungalprotein.The filtrated cultured liquid of Bacillus subtilis strain Bs-916 was precipitated ingrades with the ammonium sulfate to draw the antifungal proteins, and eight kinds crudeproteins were drawn, we studied character of two kinds crude proteins drawn withammonium sulfate at 40%～50% and 50%～60% saturation respectively, which showeda strong antifungal activity. The concentration of crude proteins drawn with ammoniumsulfate at 40%～50% and 50%～60% saturation were 17.261μg/ml and 18.899μg/mlrespectively. Both of them showed high antifungal activity against R solani, F.moniliforme while no antifungal activity against P. infestans. Crude proteins drawn withammonium sulfate at 40%～50% and 50%～60% saturation showed a stable and highantifungal activity against R. solani during 0～40℃, decreased antifungal activity whenthe temperature was more than 40℃, kept only a little antifungal activity at 120℃. Crudeproteins drawn with ammonium sulfate at 40%～50% and 50%～60% saturation showed a stable and high antifungal activity in broad range of pH value, however, lost fullantifungal activity when pH value was 13.6, kept partial antifungal activity after treatedwith protease K, protease E, Pepsin, Papatin and Trypsin. Results of SDS-PAGE showedthat Bacillus subtilis Bs-916 secreted abundance of proteins.Bacillus subtilis strain Bs-916 secreted the antifungal protein, but litter is knownabout the antifungal protein. In this study, the crude proteins were purified by AmershamAKTA Explore system. The crude proteins were eluted through the DEAE SepharoseFast Flow column and three peaks were found; only the second fraction （fraction B） hadantifungal activity. After the fraction B were pooled and eluted in the Phenyl Sepharose 6F.F. column, two peaks were obtained, only the second fraction （Fraction E） hadantifungal activity. When the Fraction E were pooled and eluted in the hydroxyapatitecolumns, there were also two peaks and the last peak （Fraction G） exhibited antifungalactivity, which showed only one protein band in the SDS-PAGE, designated as Bacisubinwith 41.9 kDa molecular weight compared with standard proteins.Bacisubin was measured antifungal activity toward Rhizoctonia solani, Fusariummoniliforme, Magnaporthe grisease, Sclerotinia sclerotiorum, Alternaria oleracea, A.brassicae, C. capsici and Botrytis cinerea. The results exhibited that the antifungalprotein had a broad-spectrum of antifungal activity and high virulence. Especially, theantifungal protein exhibited a stronger antifungal to pathology fungi, such as genericAlternaria. EC₅₀ value of antifungal protein to Rhizoctonia solani, Botrytis cinerea,Alternaria oleracea and A. brassicae were 4.01、2.74、0.08 and 0.055μM, respectively.The mechanism to antifungal activity of Bacisubin caused mycelia alimentation obstacle.Mycelia apex appeared distortion, tumescence, rupture and cell wall thickened. HoweverBacisubin did not showed protease activity, protease inhibitory activity, but exhibited acertain ribonuclease activity and hemagglutinating activity,.Antifungal protein bacisubin was identified with MALDI-TOF-MS and Q-TOFafter digested with trypsin. When Bacisubin （digested with trypsin） was identified withMALDI-TOF-MS, 22 peaks of absorbance were found and identification in MatrixScience Database, where were no observably protein related. Then mixed peptides wereidentified with Q-TOF-MS, five peptide tags （PT） were get. Five PT sequence wasVTIVDEKGR, FSFSDSDVHNR, VYIADSTNFK, ELPISENLASVNMR, and EAEWAYMITGK respectively. These PTs were used to search in Matrix Science Database; we found two proteins were observably related with this antifungal protein. The two proteinswas oxalte decarboxylase. One was an oxalte decarboxylase from Bacillus subtilis strainB-168, another was an oxalte decarboxylase from the Bacillus licheniformis ATCC14580.However, antifungal activity of oxalte decarboxylase was not reported elsewhere.更多还原