Analysis Of The Insecticidal Crystal Mutants Of Bacillus Thuringiensis YBT1520 By Proteomic Method

Bacillus thuringiensis is the most widely used as biological pesticides, the Gram-positive bacteria, up to now there are 83 subspecies,71 serotypes were found. The high insecticidal capacity is due to its insecticidal crystal proteins, the protein is accumulated from stationary phase, and then assembled into the crystal. Crystal metabolic regulatory mechanism is one of hotspots in the study of Bacillus thuringiensis, because so far the formation mechanism of spore has got a breakthrough, but the metabolic regulatory mechanism has not a reasonable and clear explanation yet. In this work the High virulent strains of YBT1520 was treated by different induced mutagenesis, obtained different crystal mutants, then got the discriminating proteins by using 2-DE, proteins were identified by mass spectrometry. The object is to be associated with the crystals functional genes about metabolic regulatory mechanism.1. Bacillus thuringiensis YBT1520 was treated by wide ion beam, UV+LiCl mutagenesis, microwave+UV compound mutation, NTG+LiCl mutagenesis approach, et al.. Mutant strains were screened, three types of crystal were got, including the shape of smaller crystals, bigger crystals and no crystal.2. The stability of the passages was tested, the result showed that the smaller crystal and crystal-free mutant were stable; the plasmids of YBT1520 and mutants were extracted, the result showed that pBMB12 of the smaller crystal was absent, which existed in the wild. The crystal-free mutant are quite different from the wild. Then their growth curves were measured, the smaller one kept the same tendency with the wild, but the growth curve of the crystal-free fluctuated in stationary phase; then the insecticidal capacity of the wild and the mutants were tested, results showed that the smaller was similar to the wild, the crystal-free mainly has no insecticidal capacity. According to the experimental information obtained and experimental purposes, the smaller crystal mutant was determined for the two-dimensional electrophoresis.3. According to crystal formation time, the 14h,18h,22h three time points were chosen for 2-DE experiments, gel maps were analyzed with the PDQuest software, and mass spectrometry was used to identify different proteins, and 13 credible proteins were found out, there were 8 proteins were up-regulated,5 proteins were down-regulated. Results include 5 classes:metabolic-related enzymes, chaperones, transcription regulatory proteins, translation regulating proteins and proteins of unknown function.