生防菌株ZA1的GFP基因标记及其功能稳定性测定

发布时间：2018-05-18 07:30

  本文选题：莫海威芽胞杆菌 + GFP基因　； 参考：《植物保护学报》2017年04期

【摘要】：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。
[Abstract]:In order to clarify the mechanism of colonization, crops and pathogens of Bacillus mojavensis strain ZA1 in crops, the green fluorescent protein expression vector (pGFP78) was introduced into ZA1 by electroporation, and the bacterial culture method was used. The functional stability was detected by subculture method and plate inhibition method. The results showed that the plasmid pGFP78 was successfully introduced into strain ZA1, and the labeled strain ZA1-gfp was proved by morphological and molecular biological verification to be marked successfully. The labeled strain ZA1-gfp was identical to the strain ZA1 in morphology and appeared bright green under fluorescence microscope. Moreover, the plasmid pGFP78 can be extracted and the promoter 78 sequence carried by the plasmid can be expanded. The results of functional stability identification showed that the genetic stability of the labeled strain ZA1-gfp was 100%, the growth rate of the labeled strain was not significantly different from that of strain ZA1, and it did not affect the motility of the strain. The inhibition rates of anthracnose and gangrene were 64.50% and 66.55%, respectively. The inhibition rates of the labeled strain ZA1-gfp on the three pathogenic fungi were 76.56% and 66.75%, respectively, indicating that the introduction of exogenous plasmids did not affect the bacteriostatic ability of the strains.
【作者单位】： 甘肃农业大学植物保护学院甘肃省农作物病虫害生物防治工程实验室;甘肃农业大学甘肃省作物遗传改良与种质创新重点实验室;
【基金】：国家现代农业(马铃薯)产业技术体系(CARS-10-P18) 国家自然科学基金(31660148)
【分类号】：S476.1
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本文编号：1904970