枯草芽孢杆菌B11基因文库的构建及与拮抗物质的生物合成相关基因的克隆

Construction of genomic library of Bacillus subtilis strain B11 and cloning of genes involved in biosynthesis of antagonistic substance

枯草芽孢杆菌菌株B 11对广泛的植物病原真菌和细菌都具有拮抗作用。以柯斯质粒pW EB∷TNC为载体构建了枯草芽孢杆菌菌株B 11的基因文库,文库含9 000个克隆。文库克隆中插入的DNA片段平均为42.1 kb,该文库含有菌株B 11基因组中任一基因的概率为99.99%。采用平板活性检测法筛选文库,筛选到1个对茄青枯假单胞菌菌株P 13具有拮抗活性的文库克隆GXN 9527,该克隆的重组质粒pGXN 9527含有50 kb的菌株B 11的DNA。文库克隆对革兰氏阴性植物病原细菌如水稻黄单胞菌水稻变种也具有拮抗活性,而对革兰氏阳性细菌如地衣芽孢杆菌和植物病原真菌如尖孢镰刀菌西瓜专化型、立枯丝核菌、水稻稻灰梨孢菌则没有拮抗活性。分别含有pGXN 9527的18、12、9、8 kb B amHⅠ片段的亚克隆对P 13均没有拮抗活性,说明编码该拮抗物质的生物合成基因很可能成簇存在。

Bacillus subtilis strain B11 displayed antagonistic activity against a wide variety of plant pathogenic fungi and bacteria. A genomic library of the strain Bll containing 9 000 clones was constructed. The average size of the inserted DNA in recombinant plasmids was 42.1 kb and the probability of harboring any gene in the genome of the strain Bll was 99.99%. The library was screened for antagonistic activity against Ralstonia solanacearurn strain P13 and a clone carrying a 50 kb inserted DNA was isolated, which was designated as GXN9527. Plate assay showed that the GXN9527 could inhibit the growth of plant pathogenic Gram-negative bacterium Xanthomonas oryzae pv. oryzae, but not inhibit the Gram-positive bacterium Bacillus licheniformis or the phytopathogenie fungi Fusarium oxysporum f. sp. niveum, and Rhizoctonia solani. The subclone containing an individual 18 kb, 12 kb,9 kb or 8 kb BamH I fragment of pGXN9527 did not exhibit antagonistic activity against strain P13, indicating that genes involved in biosynthesis of antagonistic substance （s） might be clustered.