Construction And Application Of A Constitutive And Efficient Expression And Secretion System For A Rice Epiphytic Bacillus Pumilus

Application of native or genetically modified microbes in protecting plant from pests and pathogens gains a lot of attention, especially the microbes which are the natural inhabitants of plant leaves or roots. The engineered epiphytes could produce foreign proteins successively on the surface of plant leaves and roots, making crop capable of suppressing pests or pathogens. The environment destroy which is a great public concern can be avoided using this strategy instead of insecticide, pesticide or other chemical in control of the plant disease. Among the biocontrol microorganism, Bacillus plays a very important role in the suppression of various plant pathogens and pests. A rice epiphyte DXOl was isolated from cultured rice leaves and identified as an isolate of Bacillus pumilus. In order to genetically modify this rice epiphyte into recombinant antifungal microbe, we did a series of research work on the bacterium as followed:The promoterless gfp gene was transcriptionally fused to a strong Bacillus pumilus promoter pCP01 functional fragment F1, which was subcloned from the genome of strain DX01 by PCR technique, and then inserted into an Escherichia coli-Bacillus shuttle vector pHY300PLK to form expression vector pHY300-Flgfp. The DXOl cells carrying the plasmid pHY300-Flgfp can produce bright green fluorescence when they were examined by using fluorescent microscopy. Through investigating the GFP-expressing-cell percentage and mean fluorescence intensity (MFI) of B. pumilus DX01 cell population harboring pHY300-F1gfp at different growth stages with FACS analysis, promoter Fl was confirmed to have strong transcription activity and be a vegetative-phase constitutive promoter.To establish a high-efficiency expression and secretion system for Bacillus pumilus, the function of three signal peptides in DXOl were tested. B. subtilis levansucrase (sacB) gene and subtilisin gene signal sequences and B. pumilus DXOl RNase gene signal sequence could all direct the secretion of report protein, E. coli β-lactamase, from DX01, but the signal sequence of B. subtilis sacB was the most effective. They can also drive the secretion of green fluorescence protein (GFP) from DX01. Expression and secretion of GFP in DX01 were confirmed by Western blotanalysis.The anti-fungal protein 1 (Rs-AFP1) cDNA was derived from radish cultivar "Chuanxihong" by reverse transcription PCR, then the gfp gene with a mutated terminator codon was fused to its 5' end and a T7 terminator was fused to its 3' end. The fused gene was inserted into the expression and secretion vector that included F1 promoter and the signal sequence of B. subtilis sacB to form the vector pHY300-F1LgAFP1. The fused protein was confirmed to be able to express and secrete in B. pumilus DX01 cells by Western blotting analysis. Rs-afp1 gene with a T7 terminator was inserted into the vector pHY300-F1LgAFP1 replacing the fused gene gfp-Rs-afp1 to form the vector pHY300-F1LAFP1. Comparing with the wide-type DX01, the antifungal activity of the DX01 carrying the plasmid pHY300-F1LAFP1 was enhanced, which showed that Rs-AFP1 protein could functionally express and secrete in B. pumilus DX01 cells.