Abstract：Bacillus, an important biocontrol resource, widely exists in nature. The establishment of a simple and efficient genetic manipulation technique for Bacillus is helpful to the study of related molecular mechanism. In this study, the recombinant plasmid pBD1 was constructed by molecular biological methods, which can be used for gene knockdown in Bacillus. In order to verify the gene knockdown efficiency of pBD1 in Bacillus, the small guide RNA (sgRNA) of serine protease gene bace16 in B. nematocida B16 was designed and inserted into pBD1 to construct bace16 low-level expression vector, which then electrotransfected into B16 to compare the expression difference of bace16 in the mutant and wild type. The results showed that reversible Bacillus knockdown vector pBD1 was successfully created based on dCas9, and bace16 low-expression mutant strain was obtained by qRT-PCR and enzyme activity tests. The bace16 expression could be complemented to the level of wild type when the inducer ITPG (isopropyl β-D-thiogalactoside) was removed. The present study constructed a reversible gene knockdown recombinant plasmid suitable for Bacillus, and established the gene knockdown and complementary system of Bacillus, which could provide basic information for the study of gene function of Bacillus.
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