枯草芽孢杆菌胞外蛋白酶突变株的筛选及其拮抗作用  

Screening of Extracellular Protease Mutants of Bacillus subtilis and Their Antagonistic Function

作　　者：李然[1] 邢介帅[1] 韩立英[1] 竺晓平[2] 赵蕾[1] 

机构地区：[1]山东师范大学生命科学学院,济南250014 [2]山东农业大学植物保护学院,山东泰安271018

出　　处：《应用与环境生物学报》2008年第2期231-234,共4页Chinese Journal of Applied and Environmental Biology

基　　金：山东省自然科学基金项目(Y2005D06);“十一五”国家科技支撑计划重点项目(No.2006BAD17B00)资助~~

摘　　要：从小麦根际土壤中分离到一株对多种植物病原真菌有拮抗作用且产蛋白酶的枯草芽孢杆菌Bacillus subtilis T2.经紫外线(UV)和硫酸二乙酯(DES)诱变处理,获得具有良好遗传稳定性的蛋白酶正负突变株.与出发菌株相比,培养48h的正突变株胞外蛋白酶活力提高108.5%,负突变株蛋白酶活力降低81·1%,而二者的几丁质酶和β-1.3-葡聚糖酶活力均无显著变化.正突变株对棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)的平板拮抗作用增强,其粗酶液可使该菌菌丝变形膨大成珠状、原生质浓缩,萌发的孢子芽管短且畸形膨大;负突变株对该菌的平板拮抗作用显著降低,粗酶液对该菌菌丝和孢子的形态无明显作用.表明提高生防芽孢杆菌胞外蛋白酶的活力可增强其拮抗病原真菌的能力.Several Bacillus strains were reported to be effective in controlling plant diseases, and the action of extracellular hydrolytic enzymes was considered as one of the main mechanisms involved in the antagonistic process. Strain Bacillus subtilis T2 was a potential biocontrol agent against a broad-spectrum plant pathogenic fungi with the ability to produce extracellular protease. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of extracellular protease derivates. Two protease mutants with genetic stability were obtained by means of UV-irradiation and DES treatments. It was revealed that extracellular protease activity of the positive mutant T2-18 was elevated by 108.5% and the negative mutant T2-2 decreased by 81.1% after 48 h cultivation. Whereas, the activity of chitinase and β-1.3-glucanase did not change significantly compared to the wild type. Moreover, the positive mutant T2-18 proved to be better antagonistic against Fusarium oxys- porum f. sp. vasinfectum in vitro antagonism experiments. Its cell-free culture filtrate showed great inhibition to mycelial growth and conidial germination of the pathogen, of which the swelling, distortion hyphae and shortened germination tubes were observed under light microscope. On the contrary, the inhibitory effect of the negative mutant T2-2 decreased markedly compared to wild type T2, and there was no obvious effect on morphology of the hyphae and conidia. This study suggests the possibility of using mutants with secretion of extracellular protease against plant pathogenic fungi. Fig 5, Tab 2, Ref 15

关 键 词：枯草芽孢杆菌 胞外蛋白酶 诱变 拮抗作用 

分 类 号：S476[农业科学—农业昆虫与害虫防治] Q936[农业科学—植物保护]