Study On Stimulated Expression Of Silent Gene Cry2Ac By Cody Protein

Bacillus thuringiensis (Bt) is a gram-postive bacterium which widely exists in nature. Bt can form spore during the mid-stationary phase and also can produce insecticidal crystal protein (ICPs) poisonous to larval of many insects. ICPs is widely used in pest control due to the high toxicity. CodY is a global transcriptional regulatory factor in gram-positive bacterium regulating the transcipiton of gene involved in spore formation and stationary phase. These genes usually are induced under the poor nutrition conditions in order to improve adaptability of bacterium. Expression of most cry genes is dependent on the formation of spore. There is little reaserch about expression of cry2Ac gene in Bt. cry2A gene usually doesn’t express in wild type bacterium such as cry2Aa and cry2Ab and cry2Ac gene. In this research, we find that when we over-express the transcriptional regulatory factor CodY in YBT-881, large amount of Cry2Ac protein was produced. So in this study, we discussed much about cry2Ac, tried to explain the relationship of CodY protein and the cry2Ac gene that may lay a good foundation of the regulatory networks about CodY in Bacillus thuringiensis.1. Most of the cry gene was located in plasmid, few in chromosome. cry2Ac gene was proved to be positioned on the plasmid of YBT-881using Southern blot method.2. Part of the upstream sequence was obtained using a genome walking technology TAIL-PCR, bioinformatics analysis of the sequence was made, we found no similarity with the cry2Ac operon sequence reported. It showed that the structrue of cry2Ac gene in YBT-881is pretty different with the operon structure reported. Of course, the whole upstream sequence of cry2Ac also needs more reaserach.3. RT-PCR experiment of codY and cry2Ac was performed in genetically engineered starin YBT-881-L1in which CodY protein is over expressed and the wild type YBT-881. We found that in log phase and stationary phase, codY was transcribed in both strains, cry2Ac gene was only transcribed in YBT-881-L1but not transcribed in YBT-881correspondingly. It suggests that CodY may impact cry2Ac gene in transcriptional level not in translational level. 4. In this study, we established the platform about discussing the interaction of CodY protein with the target gene using bacterial one-hybrid reporter system. Through this platform, we found that there is no interaction with CodY and the promoter of cry2Ac gene. When codY gene and cry2Ac gene was co-transformed into BMB171, still no Cry2Ac protein was produced. Western blot analysis suggests that in BMB171codY+cry2Ac+, hardly any protein was expressed. This further proved that CodY doesn’t affect cry2Ac gene directly, it may control the expression of Cry2Ac protein through regulating other proteins.