Function Ressearch Of Serine Protein Kinase In Bacillus Thuringiensis Stain

Bacillus thuringiensis is one of the Gram-positive bacteria which widely exists in the soil, whose prominent feature is to produce insecticidal crystal proteins with specific toxicity to insects following the formation of spores in the process of growth, and it has been found so far that B. thuringiensis is virulent to thousands of pests.Comparative proteomic study based on the different growth periods of B. thuringiensis stain YBT-1520showed PrkA protein was expressed abundantly in the late stabilization period, with obvious phosphorylation map. Moreover, spores and parasporal crystal of B. thuringiensis were also formed during this period, therefore, we infered that PrkA protein had a very close relationship with the synthesis of spores and parasporal crystal of B. thuringiensis. PrkA protein was firstly isolated from B. subtilis, and only serine phosphorylation activity was detected, but its biological function was not clearly understood. So through function verification of PrkA protein I expected to reveal the regulatory role of PrkA involved in the metabolic activity of B. thuringiensis, and I also hoped to uncover the relationship between PrkA protein and the formation of spores and the expression of insecticidal crystal protein in B. thuringiensis in order to have a deeper understanding about the formation mechanism of the parasporal crystal.1. This research was to verify the function of PrkA protein, and we firstly compared the physiological characteristics between the overexpression PrkA strain and the wild strain. During this process, we found that the bacterial cell of the overexpression PrkA strain was harder to sedimentate than that of the wild strain and the overexpression PrkA strain forms the spores and parasporal crystal ahead of time.2. The motif search showed that PrkA contained Sigma-54interaction domain, so interaction validation was needed between PrkA and Sigma-54by GST pull-down and bacterial two-hybrid system, and it proved that there was no interaction between PrkA and Sigma-54.3. In order to find proteins interacting with PrkA, we firstly inserted the prkA target gene into the expression vector pGEX to construct a prokaryotic expression vector plasmid, which was transformed into E. coli expression strain BL21to express and purify the GST-PrkA fusion protein. Secondly, through GST pull-down technique and two-dimensional electrophoresis experiment, I found out proteins potentially interacting with PrkA, and specific protein bands were identified by mass spectrometry. Finally, we conducted the functional analysis of the nine proteins from mass spectrometric identification, and then we could guess that cell settling experiment was related to the regulation of PrkA on GGDEF family protein and the earlier formation of the spores and parasporal crystal was related to the regulation of PrkA on GGDEF family protein and transcriptional regulator TetR.