Antibacterial Protein Separation And Analysis And Mass Spectrometry-based Gel Electrophoresis Lysobacter Sp.SNNU513 Extracellular

The secretome, which is one of the most important subjects of proteomics, is all of the protein in extracellular secreted by organism under certain condition. As a hotspot in life sciences, the secretome has significantly biological activity, which has shown closely and coordinately dynamic change with behaviors of the living cell. The continuously development of proteomics has provided a powerful platform for revealing the relationship between physiological function and cell life activity of the secretome.The microbial insecticide with highly efficient, low toxicity and residual has gained considerably ecological and social benefit, and remarkably economic value in agricultural production. The biocontrol bacteria have generated a large number of metabolites with antimicrobial activity or rivalrousness in their growth processes, which can preclude or kill the pathogenicbacteria, and then have a significantly role of biological control. Therefore, the biocontrol bacteria-derived antibacterial material of agricultural activity have a widely application prospect.This thesis obtained a bacterium with a broad spectrum of antimicrobial activity from the roots of Radix Polygalae, which is identified as the new strains of Lysobacter, namely Lysobacter sp.SNNU513(NCBI JF445288.1). Our result suggests that the extracellular material against plant pathogens is predominated by the protein, and its extracellular protein has striking inhibitory activity against Colletotrichum gloeosporioides, Botrytis cinerea et al.Furthermore, in aspect of sample preparation method, sample loading and focusing program, this thesis has explored the method that has been used to optimized the two-dimensional electrophoresis condition of the Lysobacter sp.SNNU513extracellular protein, and has established the ideal two-dimensional gel electrophoresis of the intracellular and extracellular protein. Under the foundation of essential information about extracellular protein, this thesis has rapidly separated the extracellular protein based on the combination of Ultrafiltration and Native-PAGE, and then obtained the antibacterial activity protein bands NO.6and NO.7from the gel. This thesis, thus, has introduced two mass spectrum methods to analysis the obtained protein bands:(1) Two-dimensional gel electrophoresis was used to analysis the protein band6. The18 protein spots derived from2-DE map were digested and then identified using the tandem mass spectrometry. The result has shown that12protein spots were successfully identified.(2) Shot-gun method was proposed to analysis the protein band7. The result has suggested that24protein spots were successfully identified. Over all,4protein spots (e.g. a-Lytic protease, subtilisin savinase, β-lytic protease and soluble lytic murein transglycosylase) from above two methods have the potential antibacterial activity.This thesis has used the new strains of Lysobacter(Lysobacter sp.SNNU513) and introduced the polyacrylamide Gel electrophoresis and mass spectrum methods to successfully identify the antibacterial activity extracellular protein, which has laid a solid foundation for researching the powerfully potential biocontrol agents.