Objective: We studied the influence of spoIIID gene deletion on the activity of cry1Ac, cry3A, cry4A and cry8E gene promoters in Bacillus thuringiensis and compared the activity among these promoters in spoIIID mutant (HD-delta SpoIIID).
Methods: We constructed 4 promoter fusions with lacZ gene and transformed them into wild-type strain HD-73 and HD-deltaSpoIIID to analyze their transcriptional activity. We constructed a spoIIID gene mutant (HD-(deltaASpoIIID with deletion of the cry1Ac-harboring native plasmid based on HD-ASpoIIID strain. We constructed four promoter fusions with crylAc gene and transformed them into HD-ASpoIIID and HD(-)-deltaSpoIIID to perform Cry protein quantization and bioassay.
Results: By Beta-galactosidase assay we found that the activities of the four promoters were, in decreasing order, Pcry8E > Pcry1A > Pcry4A > Pcry3A in both HD-73 and HD-deltaSpoIIID strains. The deletion of spoIID had no effect on transcriptional activity of PcrylAc and Pcry8E. The transcriptional activity of Pcry3A in HD-deltaSpoIIID was slightly higher than that in HD-73. The transcriptional activity of Pcry4A in HD-deltaSpoIIID was decreased compared to HD-73. The Cry1Ac protein production directed by Pcry1Ac was as much as Pcry8E in HD-deltaSpoIIID and higher than that by Pcry4A and Pcry3A in accordance with the bioassay result.
Conclusion: The cry8E gene promoter is the strongest promoter among four promoters in spoIIID gene mutant at transcriptional level. The Cry1 Ac protein production directed by Pcry1Ac is almost equal to that by Pcry8E.