Screening Interacting Proteins With Microcystis Aeruginosa Clock Protein KaiA By The Yeast Two-hybrid System

Cyanobacteria are the simplest organisms known to have the circadian clock. The clock gene cluster kai of cyanobacterium, which is composed of three genes, kaiA, kaiB and kaiC, has been identified as essential time-keeping component. Disruption of each of the kai genes abolishes circadian rhythmicity. The protein-protein association among the Kai proteins and other proteins may be a critical process in the generation of circadian rhythms. The role of KaiA is probably as a conduit for information from the input pathways, to receive environment information and dock with the clock complex, stimulating KaiC autophosphorylation and inducing a conformational change. Interaction among Kai proteins and other proteins and the phosphorylation state of KaiC protein are central to the timekeeping ability of the Kai Oscillator.The two-hybrid system is a powerful technique for detecting protein-protein interactions. To study kaiA function and find its interacting protein, the clock gene kaiA was cloned from Microcystis aeruginosa by PCR and then subcloned into the bait vector pGBKT₇ and prey vector pGADT₇of yeast two-hybrid system. After being verified by sequencing, the recombinant plamid pGBKT₇-kaiA/ pGADT₇- kaiA and pGBKT₇-kaiA/pGADT₇ were co-transferred into yeast strain AH109 respectively and its expression products were assayed whether it can affect the growth of yeast cells and activate the reporter genes. The result shows that KaiA protein was not toxic to AH109 and could not activate the reporter gene. The results also demonstrated homotypic interaction of KaiA in transformed yeast cells. The constructed plasmid pGBKT₇-kaiA may serve as bait vector of yeast two hybrid GAL4 system to fish KaiA interaction protein from genomic library of Microcystis aeruginosa . Therefore, two genomic libraries pGADT₇-Sau3AI and pGADT₇-HapII were constructed and then...