关键词: 抗真菌蛋白, RNA结合蛋白Hfq, 孢子萌发, 附着胞形成, 菌丝形态结构

Abstract:

【Objective】The objective of this study is to separate and purify a kind of antifungal protein from Bacillus subtilis strain A(CGMCC No.1982), and study the antifungal mechanism using Bipolaris maydis as a indicator. 【Method】 The crude protein was subjected to precipitation with ammonium sulfate and then a DE52 anion exchange chromatography column was employed for further purification of the protein with antifungal properties. The active fraction was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The mechanism of antifungal protein on the conidium germination and appressorium formation, on the mycelium morphology and intracellular structure of B. maydis was studied. 【Result】 The analysis of the antifungal protein (FV) by MALDI-TOF-MS suggested that it might be a kind of RNA-binding protein Hfq gi|16078797, with a relative molecular mass of 8 508.5 Da and isoelectric point (pI) of 8.04. The similarity of amino acid sequence between the antifungal protein from the strain A and a kind of RNA-binding protein Hfq was 86.484%. The IC50 for inhibiting spore germination was 138.32 μg?mL-1, while the IC50 for inhibiting appressorium formation was 22.23 μg?mL-1. The mycelia treated by FV with a concentration of 34.58 μg?mL-1 exhibited misshapen and existing intumescentia vesicles in top or middle. Under scanning electron microscope, the surface of the deformed mycelia became rough and the swollen vesicle appearance exhibited sags and crests and had some chips. Under transmission electron microscope, the protoplasm of the treated mycelia cell became vacuolization and in some mycelia existing daughter mycelia. 【Conclusion】 The amino acid sequence between the antifungal protein(FV) and a kind of RNA-binded protein Hfq was of higher homology. This protein could inhibit mycelial growth by changing the morphological structure of mycelial cells and affect conidium germination and appressorium formation.

Key words: antifungal protein, RNA-binding protein Hfq, conidium germination, appressorium formation, mycelial morpha and structure