生防菌株ZA1的GFP基因标记及其功能稳定性测定

为明确生防莫海威芽胞杆菌Bacillus mojavensis菌株ZA1在作物体内定殖与作物和病原物的作用机理,运用电击转化法将绿色荧光蛋白表达载体pGFP78导入菌株ZA1体内,并采用细菌培养法、继代培养法和平板抑制法检测其功能稳定性。结果表明,质粒pGFP78成功导入菌株ZA1,标记后菌株ZA1-gfp经形态学和分子生物学验证均证明菌株ZA1已成功标记,标记菌株ZA1-gfp与菌株ZA1形态一致,在荧光显微镜下呈现明亮的绿色,并且能够提取导入质粒pGFP78,扩展出质粒所携带的启动子78序列。功能稳定性鉴定结果表明,标记菌株ZA1-gfp遗传稳定性达100%;与菌株ZA1的生长速率差异不显著,且不影响菌株的运动性;菌株ZA1对马铃薯枯萎病菌、马铃薯炭疽病菌和马铃薯坏疽病菌的抑制率分别为64.50%、68.35%和66.55%,标记菌株ZA1-gfp对3种病原真菌的抑制率分别为67.46%、76.56%和66.75%,表明外源质粒的导入,不影响菌株的抑菌能力。

Bacillus mojavensis ZA1 labeling with GFP and its functional stability

In order to confirm the interactions between Bacillus mojavensis strain ZA1 with plant-host and pathogens after strain ZA1 was transformed into plant, and the vector of pGFP78 was transformed into biocontrol strain ZA1 by electroporation, and the germiculture, subculture and dual-culture technique were used to test the functional stability of strain ZA1. The results showed that pGFP78 has transformed into strain ZA1 successfully, there was no difference in morphology between positive strain ZA1-gfp and strain ZA1, the ZA1-gfp appeared bright green under the fluorescence microscope, and obtained promoter 78 of pGFP78 by PCR after plasmid extraction from ZA1-gfp. The stability turned out to 100% and there was no obvious difference was found in the growth curves of strains ZA1 and ZA1-gfp. In addition, the results revealed genetic transformation had no effect on the motility of strain ZA1. The inhibition rates of strain ZA1 against Fusarium avenaceum, Colletotrichum coccodes and Phoma foveata were 64.50%, 68.35% and 66.55%, respectively, and the inhibition rates of ZA1-gfp against F. avenaceum, C. coccodes and P. foveata were 67.46%, 76.56% and 66.75%, respectively, which indicated that genetic transformation had no impact on the ability of biocontrol B. mojavensis.