Wang N N, Yan X, Gao X N, Niu H J, Kang Z S, Huang L L
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
World J Microbiol Biotechnol. 2016 Apr;32(4):63. doi: 10.1007/s11274-016-2024-5. Epub 2016 Feb 29.
In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC-ESI-MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu(2+), Zn(2+), Mg(2+), or K(+). However, the antifungal activity was negatively affected by Proteinase K treatment.
为了鉴定枯草芽孢杆菌E1R-J产生的拮抗物质作为防治苹果腐烂病的生物防治剂候选物，采用了盐酸沉淀、反相色谱、凝胶过滤和离子交换色谱法。纯化后的组分EP-2在非变性聚丙烯酰胺凝胶电泳（native-PAGE）和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中均显示出单一条带。从native-PAGE中洗脱的组分EP-2对苹果黑腐皮壳菌03-8显示出明显的抑菌圈。这些结果证明，EP-2是枯草芽孢杆菌E1R-J在发酵液中产生的最重要的抗真菌物质之一。SDS-PAGE和纳米液相色谱-电喷雾串联质谱（Nano-LC-ESI-MS/MS）分析结果表明，EP-2可能是一种抗真菌肽（trA0A086WXP9），相对分子质量为12.44 kDa，等电点为9.94。扫描电子显微镜（SEM）和透射电子显微镜（TEM）下的拮抗机制研究表明，EP-2似乎通过引起菌丝肿胀、扭曲、异常和原生质体外渗来抑制苹果黑腐皮壳菌03-8。抑菌谱结果表明，抗真菌蛋白EP-2对16种植物病原真菌有显著抑制作用。稳定性测试结果表明，蛋白EP-2在高达100℃的温度下保持30分钟、在pH值为1.0至8.0的范围内、或与5 mM的铜离子（Cu(2+)）、锌离子（Zn(2+)）、镁离子（Mg(2+)）或钾离子（K(+)）孵育时，其抗真菌活性均保持稳定。然而，蛋白酶K处理会对其抗真菌活性产生负面影响。
