College of Life Science, Hebei Agricultural University, 289 Lingyusi Road, 071001, Baoding, PR China.
Microb Cell Fact. 2020 Nov 23;19(1):214. doi: 10.1186/s12934-020-01475-1.
Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis.
An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NHASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Niaffinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10.
A novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.
小麦纹枯病是一种由立枯丝核菌引起的土传真菌病害，被认为是全球范围内对小麦最严重的威胁之一。枯草芽孢杆菌 Z-14 是从小麦根际土壤中分离得到的，被证实对立枯丝核菌具有很强的抗真菌活性。
从 Z-14 菌株的培养液上清液中通过硫酸铵沉淀、阴离子交换层析和反相色谱法分离得到一种抗菌蛋白，命名为 F2。F2 经 SDS-PAGE 电泳测定相对分子质量约为 8 kDa，Edman 降解法测定其 N 端氨基酸序列为 NHASGGTVGIYGANMRS，与枯草芽孢杆菌 FZB42 合成的 hypothetical protein RBAM_004680 (YP_001420098.1) 完全一致。重组 F2 蛋白（rF2）在毕赤酵母宿主中异源表达，经 Ni-NTA 亲和层析柱纯化，对立枯丝核菌表现出显著的抗真菌活性。纯化的 rF2 对不同真菌如尖孢镰刀菌、番茄枯萎病菌、玉蜀黍平脐蠕孢菌和层出镰刀菌等具有广谱抗真菌活性。rF2 热稳定性高，在 100°C 孵育 30min 后仍保持 91.5%的活性。同时，rF2 在蛋白酶 K 和胰蛋白酶处理以及 pH5-10 的较宽范围内保持活性。
从生防枯草芽孢杆菌 Z-14 发酵上清液中纯化出一种新型抗菌蛋白 F2，并在毕赤酵母中异源表达，以验证其对立枯丝核菌的抗真菌活性及 F2 基因编码的有效性。考虑到其显著的抗真菌活性和稳定的特性，蛋白 F2 为防治立枯丝核菌引起的真菌病害提供了一种替代化合物。
