Establishment Of Genetic System For Bacillus Marinus B-9987and Function Of Regulatory Gene Bmb1102Involved In Secondary Metabolism

Bacillus marinus B-9987was isolated from the rhizosphere of Suaeda salsa collected in the intertidal zone of Bohai Bay of Eastern China. The fermentation broth of B. marinus B-9987exhibited significant inhibition against plant pathogens such as Pyricularia oryzae and AIternaria solani both in vitro and in vivo, suggesting that B-9987has remarkable producibility of bioactive compounds, In order to study the secondary metabolism and their regulatory mechanism on molecular level, two sections of works were described in this thesis.The first section is the establishment of genetic system for B. marinus B-9987.In order to perform genetic manipulation in B. marinus B-9987, the first step is to establish an efficient method to introduce the heterologous DNA into B-9987to self-replicate or replicate with the B-9987chromosome via either homologous recombination or site specific integration. No genetic system has been established for B-9987. In this thesis, the procedures for B-9987protoplast formation, regeneration and transformation were investigated, and a protoplast-electroporation method was developed with the efficiency of3×104CFU/μg using pHT3101as plasmid DNA.The second section is function of the regulatory gene bmb1102that involved in secondary metabolism of B-9987.Bioinformatics analysis and comparative genomics analysis of the B-9987genome revealed a bmb1102gene probably involved in the regulation of the secondary metabolism of B-9987. bmb1102consists of585bp and its encoding protein shows strong homology to regulatory proteins of TetR-N family. There is a typical HTH (helix-turn-helix) super family DNA binding motif. The members of this family usually are involved in the regulation of multi-drug efflux pumps or antibiotic biosynthetic pathways via their binding to the target genes. Therefore, in the thesis, bmb1102was cloned and a recombinant plasmid for double crossover mutation was constructed. By using electroporation method, Δbmb1102double crossover mutants were obtained, thereby, the function of bmb1102was characterized.The above results laid the foundation for genetic manipulations and investigations on the regulatory mechanism of secondary metabolism of B-9987.