Verification And Screening Of Cyanobacteria 2Cys-PRX Protein Interaction Factors

Cyanobacteria is the simplest model organism in the current scientific research on circadian rhythm,especially Synechococcus elongatus PCC 7942,which is the first cyanobacteria strain to establish the classical Kai circadian clock molecular regulation model.At present,all known clock regulation models include three key components:core oscillator,input pathway and output pathway.In the case of S.elongatus PCC 7942,the circadian clock core oscillator is constructed by three clock genes(KaiA,KaiB and KaiC),and their expression products,and is regulated by a Transcription-Translation Feedback Loop(TTFL).Under the regulation of this negative feedback loop,cyanobacteria cells under constant conditions will produce and maintain the circadian oscillation of KaiC protein phosphorylation state with a cycle of about 24h.At present,it is generally believed that the circadian rhythm oscillation of KaiC protein phosphorylation state is the generation of endogenous time information of the circadian clock.Not only that,when purified KaiA,KaiB and KaiC proteins,ATP and Mg²⁺in appropriate proportions are mixed in vitro,KaiC protein circadian oscillations of phosphorylation level can be separated from the transcription/translation regulation process,and occur at the level of post-translational modification(PTO).The input system of the circadian clock is responsible for sensing external time information from the environment and transmitting it to the core oscillator.The core oscillator generates endogenous time cues consistent with the external time through the aforementioned TTFL and PTO mechanisms,and controls the circadian rhythm of peripheral genetics,metabolism and physiological activities through the output system of the circadian clock.The output pathway transmits the endogenous time signal generated by the core oscillator to the downstream genes regulated by the circadian clock,and then regulates downstream life activities such as gene expression,replication,protein post-translational modification,biological metabolism,and neural excitability.In general,the TTFL mechanism is responsible for the input of environmental time cues and the generation and output of endogenous biological rhythm signals,which are crucial in the circadian clock regulation of cyanobacteria,while the PTO mechanism is responsible for regulating the expression of the core clock element KaiBC operon,which regulates the precision of the clock by maintaining the clock phase.Reactive oxygen species(ROS)is one of the important by-products of photosynthesis.In order to eliminate the excessive accumulation of reactive oxygen species in the cell,cyanobacteria have evolved a variety of antioxidant mechanisms.Among them,the Peroxiredoxins(P rxs)play a key role in maintaining redox homeostasis in the intracellular environment.Previou studies have shown that the 2-Cys Prx protein encoded by the cyanobacteria genome can exhibit the phenotype of circadian rhythmic oscillation in the redox state.This rhythmic oscillation phenomenon is also known as the Prx-SO2/3 rhythmic label.In addition,some studies have shown that the Prx-SO2/3 rhythmic label can maintain the circadian phenotype of oscillation in kaiA mutant cyanobacteria cells without significant changes.It is a circadian rhythmic phenomenon that maintained by the non-transcriptional Oscillator(NTO)mechanism.Different from the TTFL and PTO mechanisms mentioned above,the basic theoretical questions of the biological clock,such as the generation and maintenance mechanism of the NTO time retention mechanism,and its relationship w ith the TTFL and PTO time retention mechanism,have not yet been answered,and related research is urgently needed.In the preliminary research work,the S.elongatus PCC 7942 was used as the research object,and the two-molecule fluorescence complementation method(BiFC)and yeast two-hybrid method were used to detect the interaction between 2-Cys Prx and CikA protein.The BiFC experimental results show that there is an interaction between 2-Cys Prx and CikA,while the yeast two hybrid experiment results s how that there is no interaction.The results of the BiFC and yeast two-hybrid experiments seem to be contradictory,but it preliminarily shows that this interaction is related to the specific redox state in the organism's cells.In order to further understand the interaction between these two proteins,on the one hand,this paper uses the pull-down method to indirectly verify the interaction,and on the other hand,it also uses the cyanobacteria in situ Co-IP to directly verify the interaction.We also constructed a cDNA library of C yanobacteria,and made a preliminary attempt to screen more potential interacting proteins of 2-Cys Prx or CikA in C yanobacteria by yeast two-hybrid method.