Detecting The Circadian Rhythms Of Clock Proteins In Microcystis Aeruginosa And Screening Of Their Interaction Proteins

The circadian clock is an endogenous cellular mechanism that allows organisms to temporally regulate gene expression and thereby regulate complex biological processes as a function of time. Temporal organization of cellular functions allows organisms to capitalize on the environmental condition at any given time of day. In any organism, biological phenomena defined as a circadian clock have three characteristic features: a free running periodicity of about 24 h, phase resetting by environmental cues, and temperature compensation of the period.Cyanobacteria are the simplest organisms known to have the circadian clock. kai genes and their expression products KaiA, KaiB and KaiC are the core of circadian clock of cyanobacteria. The KaiC phosphorylation cycle which is regulated by the interactions among Kai proteins generates the circadian rhythms. The oscillation of KaiC phosphorylation is the pacemaker of the cyanobacterial circadian clock. it controls the expression of relevant genes including the kai genes to generate physiologically functional circadian oscillation.In order to study the rhythms of Kai proteins in Microcystis aeruginosa PCC7820, kai genes were cloned into pET-His vector and then expressed in E.coli BL21 (DE3), the purified products His₆-KaiA, His₆-KaiB and His₆-KaiC were used to immunize mice to prepare antisera respectively. The antisera were then applied to western blot. The circadian variation in Microcystis cellular amounts of each Kai protein was determined every 4h under LL by western blot . The analysis of the western blot indicated that the abundances of KaiB and KaiC displayed high amplitude rhythms for at least 3 days under LL. The peaks of KaiB and KaiC levels occurred at CT 16 to 20 and CT 8 to 12, respectively. The phosphorylation of KaiC also showed robust circadian rhythm under LL. In contrast, KaiA level were relatively high at all phases, displaying little rhythm.We demonstrated heterotypic(KaiA-KaiB, KaiA-KaiC and KaiB-KaiC) and homotypic interactions(KaiA-KaiA and KaiB-KaiB) of Kai proteins by pull-down, heterotypic interactions were retested by far-western. The result of SDS-PAGE indicate that both KaiA and KaiB can form dimmer even in the presence of SDS. Furthermore, the signal of bands from far-western indicate the interaction between KaiA and KaiB is much weaker than that between KaiC and KaiA or KaiB, implying that KaiB may not interact with KaiA directly in vivo. Three interacting proteins of KaiB were screened in Microcystis aeruginosa by pull-down, one of them was identified as HSP70-2 and others were unknown. By the same way, HSP70-2 was also obtained in Synechocystis sp. PCC 6803, furthermore, a hypothetic transmembrane protein named MP was screened from total membrane proteins of Synechocystis sp. PCC 6803. we supposed HSP70-2 may be directly involved in heat resistance of circadian rhythm, and carry out the temperature compensation of circadian rhythm by interacting with KaiB. KaiB may anchor to membrane by interacting with the hypothetic transmembrane protein.