Hui Tian1, Bo Liu1, Juan Yang1, Chengfang Zhou2, Xinxin Xu1, Yuhong Zhang1, Zhijun Lu3, Wei Zhang4. 1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, China. 2. Beijing Plant Protection Station, No. 9 Beisanhuan Zhonglu, Beijing, 100026, China. 3. Beijing Plant Protection Station, No. 9 Beisanhuan Zhonglu, Beijing, 100026, China. 13693683207@163.com. 4. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, China. zhangwei02@caas.cn.
Abstract
OBJECTIVES: To construct a genetic transformation system for Bacillus velezensis NSZ-YBGJ001 and identify the origin element in an endogenous plasmidpBV01 for curing pBV01 by plasmid incompatibility. RESULTS: A plasmid pUBC01 was constructed, and then an electrotransformation system for B. velezensis NSZ-YBGJ001 was developed, which reached ~ 1000 transformants per microgram of pUBC01 DNA. Additionally, a 7276-bp circular plasmid pBV01 with a G + C content of 37.5% was isolated from B. velezensis NSZ-YBGJ001 and analyzed via sequence analysis. To cure pBV01, an incompatible plasmid pBV02 harboring the replication element of pBV01 was developed and functionally replicated in both Bacillus subtilis WB600 and B. velezensis NSZ-YBGJ001. pBV01 was cured through introduction of pBV02 into B. velezensis NSZ-YBGJ001 after serial subculturing for approximately 40 generations. Finally, another plasmid, pBV03, was constructed based on pBV-ori, and exogenous genes in pBV03 could be efficiently expressed in B. subtilis. CONCLUSIONS: The results of this study, including the genetic transformation system, plasmid-curing strategy, and exogenous gene expression, will support genetic manipulation of B. velezensis to promote its application in biocontrol and industry.
OBJECTIVES: To construct a genetic transformation system for Bacillus velezensis NSZ-YBGJ001 and identify the origin element in an endogenous plasmidpBV01 for curing pBV01 by plasmid incompatibility. RESULTS: A plasmid pUBC01 was constructed, and then an electrotransformation system for B. velezensis NSZ-YBGJ001 was developed, which reached ~ 1000 transformants per microgram of pUBC01 DNA. Additionally, a 7276-bp circular plasmid pBV01 with a G + C content of 37.5% was isolated from B. velezensis NSZ-YBGJ001 and analyzed via sequence analysis. To cure pBV01, an incompatible plasmid pBV02 harboring the replication element of pBV01 was developed and functionally replicated in both Bacillus subtilis WB600 and B. velezensis NSZ-YBGJ001. pBV01 was cured through introduction of pBV02 into B. velezensis NSZ-YBGJ001 after serial subculturing for approximately 40 generations. Finally, another plasmid, pBV03, was constructed based on pBV-ori, and exogenous genes in pBV03 could be efficiently expressed in B. subtilis. CONCLUSIONS: The results of this study, including the genetic transformation system, plasmid-curing strategy, and exogenous gene expression, will support genetic manipulation of B. velezensis to promote its application in biocontrol and industry.