Proteomics’ Study Based On RPLC Chromatogram Separation Of Bacillus Thuringiensis’ Insecticidal Crystal Proteins

Bacillus Thuringiensis (BT) is the most widely used miscrobial pesticide, whichcovers90%of pesticide production. More than120BT products had been productedin60countris and it had become the hot points in the development and research ofbiological pesticides. In recent years, Proteomics is developing rapidly, its researchtechnology become mature and diversity， specially for the modern analysistechnology such as high performance liquid chromatography and mass spectrometry.In order to efficiently separation and accurately analysis of the Bacillus Thuringiensiseffective insecticidal proteins，HPLC and biomass spectrum technology have beenapplied to analysis the Bacillus Thuringiensis insecticidal powder in domestic market.In chapter1, Bacillus Thuringiensis, insecticidal crystal protein types, it’sstructure, insecticidal mechanism, and research status of crystal proteins, introducesthe proteomics, proteomics research technology had been discribed, and the purposeof this thesis had been put forward.In chapter2, In order to extract BT’s insecticidal crystal protein, the condition ofdissolved and precipitation in solution about BT insecticidal crystal proteins sampleshad been determined firstly, and then the buffer concentration, and precipitation timein dialysis precipitation method had been optimized; finally we decide to precipitatethe BT insecticidal crystal proteins in alkali solution, under the buffer solution(pH4.5)containing1mM EDTA、100mM CH3COONa~CH3COOH, then dialysis5hours indialysis bag.In chapter3, In order to fastly separate insecticidal crystal proteins by RPLC,protein denaturation method had been applied to change the properties of BTinsecticidal crystal proteins in solution, to meet the reversed-phase liquidchromatographic separation condition. Then the target protein components inchromatographic had been collected and enriched and analyzed by SDS-PAGE.In chapter4, MALD-TOF-MS and LTQ ORBITRAP XL MS had been used totest the peptides cutted by trypsin in gel protein band; Then extract primary MSinformation to do Peptide Mass Fingerprint (PMF), and extract tandem MS information to do MS/MS Ion Search. Finally24peptide sequences had beenidentified in Bacillus thuringiensis insecticidal crystal proteins. One peptide sequenceAVNALFTSTNQIGLK only matched to the protein gi169788589-pesticidal crystalprotein cry1Ac [Bacillus thuringiensis serovar kurstaki], the other peptide sequenceEIYTNPVLEDFNGSFR only matched to the protein gi46409861-cry1A type crystalprotein [Bacillus thuringiensis serovar kenyae] too，the rest peptide sequencesmatched to dozens of Cry proteins...