玉米内生菌YY1菌株抗菌蛋白的稳定性和活性组分分析  

Stability and active component analysis of antifungal protein from maize endophyte strain YY1 of bacterium Bacillus subtilis

作　　者：钱欣雨 王会敏 候美玲 申珅[1,2] 郝志敏 董金皋[1,2,4] Qian Xinyu;Wang Huimin;Hou Meiling;Shen Shen;Hao Zhimin;Dong Jingao(National Key Laboratory of Crop Improvement and Regulation in North China,Hebei Agricultural University,Baoding 071001,Hebei Province,China;Hebei Key Laboratory of Plant Physiology and Molecular Pathology,School of Life Sciences,Hebei Agricultural University,Baoding 071001,Hebei Province,China;College of Life Sciences,Northwest A&F University,Yangling 712100,Shaanxi Province,China;College of Plant Protection,Hebei Agricultural University,Baoding 071001,Hebei Province,China)

机构地区：[1]河北农业大学,华北作物改良与调控国家重点实验室,保定071001 [2]河北农业大学生命科学学院,河北省植物生理与分子病理学重点实验室,保定071001 [3]西北农林科技大学生命科学学院,陕西杨凌712100 [4]河北农业大学植物保护学院,保定071001

出　　处：《植物保护学报》2023年第3期725-732,共8页Journal of Plant Protection

基　　金：河北省重点研发计划(20326503D);国家现代农业(玉米)产业技术体系(CARS-02-25);国家重点研发计划(2018YFD0800607)。

摘　　要：为探究枯草芽胞杆菌Bacillus subtilis菌株YY1抗菌蛋白的基本特性,采用硫酸铵分级沉淀法对蛋白粗提液进行提取,测定温度、酸碱性、蛋白酶和金属离子对抗菌蛋白稳定性的影响,利用阴离子交换柱层析和葡聚糖凝胶柱层析对其组分进行分离,通过MALDI-TOF质谱对其活性组分进行初步鉴定。结果表明,当硫酸铵饱和度为50%时,菌株YY1产生的粗蛋白对玉米大斑病菌Setosphaeria turcica的抑制效果最好,抑菌圈半径达2.20 cm。该粗蛋白具有较高的热稳定性,90℃处理120 min其抑菌活性未受到明显影响,121℃处理120 min其抑菌活性仍可保持原有活性的85.0%。该粗蛋白具有较好的耐碱性,pH 5~11条件下其抑菌活性稳定,pH 3~4条件下其抑菌活性显著下降。该粗蛋白对蛋白酶K和胰蛋白酶不敏感,两者处理后的抑菌活性分别为对照的97.5%和98.9%;不同浓度金属离子对其抑菌活性的影响存在差异,Cr^(3+)可使该粗蛋白完全失活,Zn^(2+)、Li^(+)、K^(+)、Na^(+)、Mg^(2+)和Fe^(2+)对其抑菌活性有促进作用。该抗菌蛋白的分子量约为30 kD,命名为YY1-AFP6,将其初步鉴定为烯酰辅酶A水合酶同源蛋白。In order to explore the basic characteristics of the antifungal protein of Bacillus subtilis strain YY1,the crude protein extract was precipitated in grades with the ammonium sulfate,and the effects of temperature,acid-base,protease and metal ions on the stability of the antifungal protein were deter-mined.The components of the crude protein extract were separated by the DEAE Sepharose Fast Flow column and SEPHADEX G-150 column chromatography,and its active components were preliminarily identified by MALDI-TOF-MS.The results showed that when the saturation of ammonium sulfate was 50%,the crude protein produced by strain YY1 had the best inhibition effect on Setosphaeria turcica,and the radius of inhibition circle was 2.20 cm.The crude protein has a high thermal stability,and its bacteriostatic activity was not significantly affected when treated at 90℃for 120 min;after 120 min treatment at 121℃,the antifungal activity could still maintain 85.0%of the original activity.The crude protein has a good alkali resistance,and the antifungal activity was stable under pH 5-11 conditions,and the antifungal activity was significantly reduced under pH 3-4 conditions.Crude protein was not sensitive to proteinase K and trypsin,and the bacteriostatic activities of the two were 97.5%and 98.9%of the control,respectively.There were differences in the effect of different concentrations of metal ions on their antifungal activity,Cr^(3+)could completely inactivate the crude protein,and Zn^(2+),Li^(+),K^(+),Na^(+),Mg^(2+)and Fe^(2+)had a promoting effect on the bacteriostatic activity of crude protein.The antimicrobial protein has a molecular weight of about 30 kD and was named as YY1-AFP6.It was preliminarily iden-tified as a homologous protein of enoyl-CoA hydratase.

关 键 词：玉米内生菌 枯草芽胞杆菌 抗菌蛋白 生物防治 玉米大斑病菌 

分 类 号：S476[农业科学—农业昆虫与害虫防治]