The Relationship Of Regulatory Factors Of Spores And Cry Protein Expression In Bacillus Thuringiensis

The most distinctive features of the bacteria Bacillus thuringiensis (Bt) is that they are Gram-positive, Bacilli which produces crystal toxins during spore formation and vegetative growth. The crystal toxins have been found to be active against agronomical insects pests in the orders such as Lepidoptera, Coleoptera, Diptera, Homoptera, Hymenoptera, Orthoptera and other non-insect pests. The crystal is composed of insecticidal crystal proteins which is encoded by cry or cyt genes. Though there have been in-depth research on the mechanism of regulation of expression of different types of cry gene, the activity of different promoter has not been reported. This study compares the activities of the promoters of crylAc, cry3Aa, cry4A, cry8Ea genes in the mother cell regulatory factors SpoⅢD mutants and conducted a preliminary analysis of the sRNA of spore formation gene.The enzyme assay of β-galactosidase activity showed that the deletion of spoⅢD had no effect on transcriptional activity of PcrylAc and Pcry8E. The transcriptional activity of Pcry3A in HD-ΔSpoIIID was found to be slightly higher than that in wild type HD-73. The transcriptional activity of Pcry4A in HD-ASpoIIID was observed to be lower compared to HD-73. The activities of the four promoters were found to be Pcry8E>PcrylA>Pcry4A>Pcry3A in both HD-73and HD-ASpoIIID strains which shows a decreasing order trend.660nm Protein Assay Kit was used in assaying the total proteins in the supernatants of HD-AspoⅢD and HD--ΔSpoⅢD, and the same amount of protein samples were taken for SDS-PAGE electrophoresis. The results showed that there was the same amount of total protein in HD-AspoⅢD and HD--ASpoⅢD strains. The Cry1Ac protein production directed by Pcry1Ac was as much as Pcry8E in HD-ΔSpoIIID and higher than that of Pcry4A and Pcry3A in accordance with the bioassay result. The analysis of the activities of the expressed CrylAc against diamondback moth were consistent with the level of protein production directed by different promoters.In order to analyze the impact of wild type promoter expression of cry1Ac and the expression by different cry promoters, a spoIIID gene mutant (HD--ΔSpoⅢD) with deletion of the cry1Ac-harboring native plasmid was constructed based on HD-ASpoIIID strain. In HD--AspoⅢD bacterial strains, with the same amount of protein, the CrylAc protein production directed by PcrylAc was as much as Pcry8E in HD-ASpoⅢD and higher than that by Pcry4A and Pcry3A in accordance with the bioassay result. This is consistent with the protein expression in HD-ΔspoIIID bacterial strains. This finding shows that the presence of cry1Ac gene and its wild type promoter is of no significant impact compared to other types of cry gene promoters, but the differences is the amount of Cry1Ac protein expressed.Considering the high translational and expression level of CrylAc under the control of cry8E promoter, the expression of exogenous gene in the HD-ΔspoⅢD can be successfully controlled by cry8E promoter. This not only lay the foundation for building a highly efficient expression system, but also provides expressive elements for the development of new pesticides.From bioinformatics analysis of the sequencing data of30nt fragment RNA of mixed samples from SM medium of the wild-type strains at T0, T3, T7time points,15candidate sRNA fragments which resembles the genes for spore formation e.g. spoⅢD were found. The sRNA was preliminarily identified by RT-PCR analysis of the candidate sRNA transcriptional activity, as well as the relationship between the upstream and downstream genes.