Screening Interacting Proteins Of Clock Protein KaiC From Microcystis Aeruginosa By The Yeast Two-hybrid System

Circadian clock is an endogenous timing system for regulating biological metabolism and behavior,which have been observed ubiquitously in various organisms. Cyanobacteria are the simplest organisms exhibiting circadian rhythms which enhance their fitness in a day/night alternating environment.The kai gene cluster,which is composed of three genes,kaiA,kaiB and kaiC,has been identified as essential time-keeping component.The protein-protein association among three Kai proteins and other proteins may be a critical process in the generation of circadian rhythms.KaiC protein is the key part in the biochronometer mechanism of cyanobacteria,The phosphorylation state of KaiC affects the interaction between KaiC and other proteins,which makes many gene expressions rhythmicity..In order to study the function of clock gene KaiC of Microcystis aeruginosa,the yeast two-hybrid technique was used to screen the interacting proteins with KaiC.After kaiC was subcloned into bait plasmid pGBKT₇,the recombinant plasmids pGBKT₇-kaiC were transferred into AH109 and its expression product were assayed. The result indicates that the KaiC protein was not toxic to AH109 and could not activate the reporter gene.This lay a basis for further screening of proteins interacting with KaiC from the genome of Microcystis aeruginosa.To isolate proteins interacting with KaiC from M.aeruginosa,the genomic DNA library of Microcystis aeruginosa was constructed and then screened by two-hybrid system.The total of 11 positive clones interaction with KaiC were picked out.Then, C99 and C196 proteins were further studied.C196 protein of Microcystis aeruginosa was expressed in E.coli.Affinity purification of GST-C196 from cell extracts,and subsequent far-western analysis confirm heterotypic interaction between KaiC and C196 protein in vitro.Thus,we speculated that C196 might be a protein related to the regulation of the Microcystis aeruginosa biochronometer.Because the C99 sequence of Microcystis aeruginosa was unknown,C99 from Synechocystis sp.PCC 6803 instead of Microcystis aeruginosa was used for further research.The pull-down and the yeast two hybrid technique both show no interaction between C99 protein of Synechocystis sp.PCC 6803 and KaiC of Microcystis aeruginosa in vitro or in vivo.