用PCR方法扩增的广谱拮抗菌B96-Ⅱ的16S rDNA经序列测定和BLAST同源序列比较，结合传统的形态观察、生理生化特征鉴定，确定了B96-Ⅱ分类地位为枯草芽孢杆菌(Bacillus subtilis)。将含有绿色荧光蛋白基因和氯霉素抗性基因标记的大肠杆菌一枯草芽孢杆菌穿梭表达载体(pNW33N-gfp-B96-Ⅱ-N)通过原生质体法转化B96-Ⅱ，获得表达GFP的4株标记菌株，在荧光显微镜下发出明亮的绿色荧光。室内平板抑菌试验结果表明，GFP标记对B96-Ⅱ的广谱抑菌活性没有影响。
16S rDNA of broad-spectrum antagonistic strain B96-II was amplified by PCR and sequenced.Using BLAST software,comparison results showed B96-II was possibly identified as Bacillus subtilis combining its morphological,physiological and biochemical characteristics.In order to construct Escherichia coli-Bacillus shuttle vector containing gfp and B96-II self promoter,digested fragments of total DNA of B96-II that was digested by Sau3A I were ligated to pNW33N-gfp that was digested by BamH I,and the product was transformed into E.coli DH5αcompetent cells.Protoplast transformation method was used to gain tagged strain by gfp and GFP was well expressed in tagged B96-II under fluorescence microscope.B96-II-gfp remained strong antifungal activities against 12 kinds of plant pathogentic fungi as B96-II.The results provided fundamental possibility for the further study on the ecological behavior of broad-spectrum antagonistic bacterium B96-II.
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