拮抗香蕉枯萎病菌的解淀粉芽孢杆菌LX1菌株的鉴定及其抗菌蛋白基因的克隆

摘要

收集海南不同盐碱地的土样进行拮抗香蕉枯萎病菌的微生物分离,对具有较强抑菌作用的菌株进行形态特征、生理生化和分子鉴定,并克隆其抗菌蛋白基因.结果分离到1株具有较强抑制香蕉枯萎病菌能力的细菌LXl,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).抑菌实验发现LX1菌株的发酵上清液中粗蛋白有一定的抑菌作用,经过Sephacryl S-200HR柱层析、DEAE Sepharose Fast Flow离子交换层析分离纯化了其中抑菌作用最强的抗菌蛋白.质谱鉴定结果表明,抗菌蛋白与B.amyloliquefaciens FZB42内切葡聚糖酶同源性最高.根据质谱结果克隆了该抗菌蛋白的编码基因,该基因的核苷酸和氨基酸序列与B.amyloliquefaciens FZB42的内切葡聚糖酶的核苷酸和氨基酸序列同源性分别达99％和100％.拮抗香蕉枯萎病菌的芽孢杆菌LX1可作为潜在的防治香蕉枯萎病的的生防制剂,其抗菌蛋白基因也可通过遗传工程应用于香蕉枯萎病的防治.%To isolate the microorganisms against fusarium oxysporum f.sp. cubense race 4 (Foe 4) from saline-alkali soils in Hainan Province. The microorganisms against Foe 4 were isolated from different saline-alkali soils in Hainan Province. The morphological, physiological and biochemical characteristics of strain LX with the highest antagonistic activity against Foe 4 were assayed. Antifungal protein was isolated and purified from the fermentation supernatant liquid of LX strain, and the coding gene of the antifungal protein was cloned according to the result of protein mass spectrum identification. A Bacillus amyloliquefaciens with strong antagonistic activity against Foe 4 was isolated and identified from saline-alkali soils in Hainan Province. Antimicrobial tests indicated that the crude proteins from fermentation supernatant liquid of LX strain possessed antagonistic activity against Foe 4. Antifungal protein with highest antagonistic activity against Foe 4 was isolated and purified by Sephacryl S-200HR gel filtration chromatography and DEAE Sepharose Fast Flow ion exchange chromatography. Mass spectrometry identification of the purified antifungal protein showed that amino acids sequence showed high sequence identity with endoglucanase of B. amyloliquefaciens FZB42. The coding gene of the antifungal protein was cloned by PCR, and the nucleotide sequence was sequenced and blasted in GenBank. The results showed that nucleotide sequence and amino acid sequence of the antifungal protein gene had 99% and 100% homology with the nucleotide sequence and amino acid sequence of endoglucanse gene form B. amyloliquefaciens FZB42 strain. B. amyloliquefaciens strain LX1 with antagonistic activity can be used as biocontrol agents to control banana wilt disease and thernantifungal protein gene can be applied into the control of banana wilt disease by genetic engineering.