枯草芽孢杆菌Loq18抗菌蛋白的分离纯化及其对灰葡萄孢的抑制活性

Purification of a Novel Antifungal Protein from Bacillus subtilis Strain Loq18 and Its Inhibition of Botrytis cinerea

从枯草芽孢杆菌(Bacillus subtilis)Loq18的代谢产物中纯化一种抗菌蛋白,并进行理化性质及其对灰霉病病原菌灰葡萄孢(Botrytis cinerea)的抑制作用研究.采用柱层析分离纯化抗菌蛋白,应用基质辅助激光解吸电离串联飞行时间质谱(MALDI/TOF/TOF/MS)测定分子质量,并对其理化特性进行分析.通过显微观察法研究其对灰葡萄孢生长的影响,依据对灰葡萄孢生长抑制率与蛋白质量浓度的线性关系建立毒力方程,计算IC50值.通过体内接种灰葡萄孢,研究抗菌蛋白对采后草莓和葡萄腐烂率的影响,探究其对灰霉病的抑制活性.经测定抗菌蛋白LP18的分子质量为8 568.5 Da,含有Leu、Glu、Asp等14种氨基酸,不含脂基和糖基;具有明显抑制灰葡萄孢生长的能力,其抑制灰葡萄孢生长的毒力方程为y=0.214 0x+13.517,r2=0.996 4,IC50值为170μg/m L;经抗菌蛋白处理的灰葡萄孢,在显微镜下可观察到菌丝出现原生质囊泡化、变形、断裂.体内抑制实验表明当对照组果实的腐烂率达到100%时,处理组草莓和葡萄果实的腐烂率分别为60.33%±6.30%和45.33%±6.30%.抗菌蛋白LP18对灰葡萄孢有明显的抑制作用,可造成灰葡萄孢的菌丝出现原生质囊泡化、断裂、畸形,对采后草莓和葡萄的灰霉病有较明显的抑制效果,可延长其保质期.

An antifungal protein is isolated from a culture of Bacillus subtilis strain Loq18 to research its characterization and inhibition of Botrytis cinerea. Column chromatography is used for the antifungal protein separation and protein molecular mass is measared by MALDI / TOF / TOF / MS to analyse the physical and chemical properties. Observed its inhibition of B. cinerea from optical microscope,and the relationship between protein concentration and inhibition is studied to determine the antifungal effects of grey mould disease of strawberries and grapes under in vivo condition. The molecular mass of the antifungal protein LP18 is 8 568. 5 Da containing 14 kinds of amino acids,such as Leu,Glu and Asp,without glycoprotein and lipid. Treaded B. cinerea with LP18,its protoplasm of hyphae becomes vacuolization,deformed and fractured. The equation is y = 0. 214 0 ＋ 13. 517 x,r^2= 0. 996 4（ y： inhibition rate of hyphae growth,x：protein concentration）. The 50% inhibitory concentrations（ IC50） of its antifungal activity is 170 μg/m L. Treaded strawberry and grape with LP18,their decay rates were 60. 33% ± 6. 30% and 45. 33% ± 6. 30%,respectively instead of 100% without any treatment. The protein LP18 showes inhibition activity on hyphal growth of B. cinerea. The protoplasm of hyphae becomes vacuolization,deformed and fractured. It can decrease the postharvest decay of strawberry and grape,which leads to an extension of the shelf life of the product.