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Cloning and Functional Analysis of Quorum Sensing Gene ComQ in Bacillus Subtilis Strain NCD-2

Author: WangPeiPei
Tutor: DongJinGao；MaPing
School: Agricultural University of Hebei
Course: Plant Pathology
Keywords: Bacillus subtilis Quorum sensing system comQ Electroporation Conversion efficiency
CLC: S476.1
Type: Master's thesis
Year: 2011
Downloads: 82
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Abstract


Bacillus subtilis (Bacillus subtilis) NCD-2 strains effective control of cotton verticillium wilt, clearly the biocontrol mechanism will help to improve its biocontrol effect. In this study, carried out to explore the factors that affect the strains of B. subtilis NCD-2 genetic transformation and optimize its conversion conditions, efficient electroporation system of the strain. Its conversion efficiency by comparing five different sizes and plasmid replicon carrying different sources, found no clear correlation between the plasmid size and NCD-2 strains shock conversion efficiency; carry replicon derived from B. coagulans the the plasmid pNW33N, the highest NCD-2 strains electroporation efficiency can reach 3.6 × 10 4 cfu · μg -1 DNA replicon sources affect the conversion efficiency an important factor. PNW33N plasmid different resistance, electric field strength and culture shock conversion efficiency under time study results show that the strain is transferred to the growth medium, 37 ℃, 150 r · min -1 cultivate 3.5 h, the resistance value 200Ω, electric field strength of 14.0 kV · cm -1 conditions into the most efficient, able to reach 6.1 × 10 4 cfu · μg -1 DNA. The optimized conversion system laid the foundation for the smooth conduct of the B. subtilis NCD-2 strain of molecular genetics research. Bacterial quorum sensing systems (quorum sensing, QS) bacteria based on the specific concentration of signaling molecules to monitor environmental changes, and regulatory gene expression to adapt to environmental changes, a phenomenon. Regulation of bacterial biofilm formation, antibiotic production into traits and spore formation in Bacillus subtilis, QS system. The QS system comprising four genes, i.e. comX, comQ, comP and ComA wherein ComX is a 10 amino acid peptide, after ComQ modified group of the histidine kinase COMP secreted into the extracellular membrane binding, COMP autophosphorylation and phosphorylation transfer to ComA, phosphorylation of ComA control of the transcription of the feeling state forming a series of encoding Factor. The complete genome sequence of B. subtilis NCD-2 strain analysis, from the positioning and the cloned the QS system gene comX, comQ comP and comA. Using homologous recombination technology on the B. subtilis NCD-2 strains comQ gene mutagenesis to get the comQ gene deletion mutant body strain M6. Lipopeptide extract extract of B. subtilis NCD-2 wild-type strain and mutant strains M6. Tablet antagonistic measurement results show that significantly reduced in the mutant strain M6 lipopeptide extract of the antibacterial effect of Botrytis cinerea. The lipopeptide extracts by matrix-assisted laser parsing mass spectrometry (MALDI-TOF-MS) identification analysis showed that: mutant strains M6 than NCD-2 wild-type strains lipid peptide class material FengycinB the generated amount significantly with reducing; M6 than NCD-2 multi- The generate a lipopeptide substances IturinB. Therefore, to speculate comQ the FengycinB generation has a positive role on generation of IturinB has a negative regulatory role. Mass spectrometry are also found in the NCD-2 wild-type strain and mutant strain M6 lipopeptide extract containing a Class unknown substance for the larger molecular weight, but its concentration in NCD-2 wild-type strain was significantly higher than that of mutant strain M6. ComQ gene for this group of substances produce has an important impact. NCD-2 wild-type strain and mutant strains M6 biofilm formation qualitative detection and quantitative detection, the results show that quorum sensing system comQ gene mutant strains M6 biofilm forming ability decreased, suggesting the formation of biofilms has positive regulatory role. In this study, the understanding of the quorum sensing system function in NCD-2 strain of the biocontrol premise basis, and to provide a basis for clear NCD-2 strain of the biocontrol mechanism.

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CLC: > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens
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