生防假单胞菌FD6电击转化条件的优化  

Optimization of electroporation conditions of biocontrol Pseudomonas protegens FD6

作　　者：王诚 刘银银 施伟凡 温德宇 张清霞[1] WANG Cheng;LIU Yinyin;SHI Weifan;WEN Deyu;ZHANG Qingxia(College of Horticulture and Plant Protection,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学园艺与植物保护学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2021年第4期117-122,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(32072471、31772210)。

摘　　要：不同细菌电击转化效率各异,为筛选防御假单胞菌(Pseudomonas protegens) FD6电击转化最佳条件,选取具卡那霉素抗性标记的质粒pBBR-rpoD,从电场强度、电击时间、质粒添加量、电击悬浮溶液和细菌生长阶段5个方面优化电击转化条件。结果表明:当细菌培养至D600 nm值1.4左右时,在电场强度为12 kV·cm^(-1)、电击时间为4 ms、质粒添加量为200 ng,并以1 mmol·L^(-1) HEPES作为电击悬浮溶液时转化效率最高,可达1.1×10^(5)转化子·μg^(-1)(DNA)。此外,HEPES浓度的改变并未对转化效率产生影响。这一研究建立了一套防御假单胞菌FD6的高效电击转化体系,为今后该菌株在分子水平上的遗传操作提供了重要参考。Previous studies suggest that different bacteria have different electroporation transformation efficiencies. Plasmid pBBR-rpoD with anti-kanamycin marker was selected as the vector to screen the optimum conditions for electroporation transformation. The electro-transformation conditions were optimized from the following five aspects: the electric field strength, pulse time constant, plasmid amount, electroporation buffer and bacterial growth phase. The results indicated that the optimum transformation efficiency of 1.1×10^(5) transformants·μg^(-1)(DNA) was achieved at field strength of 12 kV·cm^(-1) with pulse time constant of 4 ms, when cell density reached D_(600nm)=1.4, 200 ng DNA, and 1 mmol·L^(-1) HEPES served as electroporation buffer. In addition, different concentration HEPES did not affect the transformation efficiency. In this study, a set of high-efficiency electroporation transformation system has been established for P.protegens FD6, which will provide important references for later genetic experiments of strain FD6 at the molecular level.

关 键 词：防御假单胞菌 生防细菌 电击转化 转化效率 

分 类 号：Q939.112[生物学—微生物学]