| 摘要: |
| 【目的】从枯草芽孢杆菌E1R-j菌株的发酵液中分离纯化抗菌蛋白，分析确其对苹果树腐烂病菌菌丝的抑制作用，为更好地利用该菌株防治植物病害和研制生物农药奠定基础。【方法】采用盐酸沉淀法从E1R-j发酵液中提取粗蛋白，并筛选酸沉淀的最佳pH值，然后通过反相柱RESOURCETMRPC、凝胶柱Superdex-75、阴离子交换柱DEAE-Sepharose和脱盐柱层析技术，提取并分离纯化粗蛋白；在粗蛋白分离纯化过程中，以皿内对峙试验测定蛋白的抗菌活性；通过扫描电镜技术，观察抗菌蛋白对病原菌菌丝的抑制作用。【结果】以pH 4.0盐酸沉淀获得的粗蛋白活性最强，抑菌效果最佳。反相柱RESOURCETMRPC获得的活性峰收集物经凝胶柱Superdex-75、阴离子交换柱DEAE-Sepharose和脱盐柱层析后，得到1个对称的活性峰，其收集物经非变性聚丙烯酰胺凝胶电泳（Native-PAGE）检测为单一条带，变性聚丙烯酰胺凝胶电泳（SDS-PAGE）图谱中显示有2个分子质量相近的条带，表明该抗菌蛋白可能含有2个亚基，2个亚基的分子质量分别为55和60 ku，将该活性蛋白命名为EP-1。扫描电镜结果显示，抗菌蛋白EP-1可使苹果树腐烂病菌菌丝出现膨大、弯曲以及原生质外渗等现象。【结论】通过盐酸沉淀及柱层析技术，从枯草芽孢杆菌E1R-j发酵液中分离纯化出一种分子质量为115 ku的抗菌蛋白EP-1，其对苹果树腐烂病菌菌丝生长具有明显的抑制和破坏作用。 |
| 关键词: 枯草芽孢杆菌 抗菌蛋白 分离纯化 抑制作用 苹果树腐烂病菌 |
| DOI： |
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| 基金项目:公益性行业（农业）科研专项（201203034）；陕西省科技统筹创新工程计划项目（2011KTZB02-02-02） |
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| Isolation and purification of antifungal protein from Bacillus subtilis E1R-j against Valsa mali |
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NIU Huanjie,LI Hui,WANG Nana,et al
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| Abstract: |
| 【Objective】This study isolated and purified antifungal protein from the fermentation liquid of E1R-j and clarified the inhibitory effect on the hyphae of Valsa mali (03-8) to use the strain for prevention of plant disease and development of biological chemicals.【Method】The antifungal crude extract was separated from the fermentation filtrate by HCl precipitation and the optimal pH value was selected before the antifungal protein was purified on column chromatography including RESOURCETMRPC,Superdex-75,DEAE-Sepharose and desalting column.During the whole process,the antifungal activity was determined using the oxford plate assay system and the inhibitory effect against hyphae of 03-8 was studied using scanning electron microscope.【Result】When pH was 4.0,the antifungal activity of protein precipitated by HCl was the strongest.A symmetrical active peak was obtained through RESOURCETMRPC,Superdex-75,DEAE-Sepharose and desalting column.A single protein band showed in polyacrylamide gel electrophoresis (Native-PAGE) while two close bands showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),indicating that the antifungal protein named as EP-1 had two subunits with molecular weights of 55 and 60 ku.The scanning electron microscopy results showed that the hyphae of 03-8 treated with EP-1 were severely deformed with symptoms of mycelium swollen,hyphal bending and cytoplasm leak.【Conclusion】The antifungal protein EP-1 with a molecular of 115 ku was isolated and purified through HCl precipitation and column chromatography,and it showed inhibitory and destructive effect on hyphae of 03-8. |
| Key words: Bacillus subtilis antifungal protein isolation and purification inhibitory effect Valsa mali |
