广谱拮抗菌B962II的分子鉴定及GFP标记-华北农学报.PDFVIP

华北农学报 ·2 0 0 9 , 2 4 ( 5 ) : 18 8 19 1 广谱拮抗菌 B96II 的分子鉴定及 GFP 标记 郝变青 ,马利平 ,乔雄梧 ( 山西省农业科学院 山西省农药重点实验室 , 山西 太原 　030031) 　　摘要 :用 PCR 方法扩增的广谱拮抗菌 B96II 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B96II 分类地位为枯草芽孢杆菌 B acillus 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 pNW33NgfpB96IIN 通过原生质体法转化 B96II ,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 B96II 的广谱 抑菌活性没有影响 。 关键词 :广谱拮抗菌 ;分子鉴定 ;绿色荧光蛋白;原生质体转化 ( ) 中图分类号 :S432 . 4 　　文献标识码 :A 　　文章编号 :1000 - 7091 2009 05 - 0188 - 04 Identif ication of Broadspectrum Antagonistic Strain B96II and GFP Tagging HAO Bianqing ,MA Liping ,QIAO Xiongwu (Shanxi Key Laboratory of Pesticide Science ,Shanxi Academy of Agriculture Sciences ,Taiyuan 　030031 ,China ) Abstract :16S rDNA of broadspectrum antagonistic strain B96II was amplified by PCR and sequenced . Using BLAST software ,comparison results showed B96II was possibly identified as B acillus subtilis combining its morphologi cal ,physiological and biochemical characteristics. In order to construct Escherichia coliB acillus shuttle vector containing gfp and B96II self promoter ,digested fragments of total DNA of B96II that was digested by S au3A I were ligated to pNW33Ngfp that was digested by B am H I ,and the product was transformed into E. coli DH5αcompetent cells. Proto plast transformation method was used to gain tagged strain by gfp and GFP was well expressed in tagged B96II under flu orescence microscope . B96IIgfp remained st