Evaluation Of The Regulation Of Intracellular Redox Homeostasis On The Circadian Rhythm Of The Cyanobacteria Synechococcus Elongatus PCC 7942

Diurnal changes in environmental factors such as light or temperature can cause corresponding changes on living organisms’behaviors,as well as their physiology or metabolism.Time cues generated by an endogenous timekeeping system,the circadian clock,are the primary reason for organisms to anticipate daily changed environmental factors and trigger following relevant metabolic adjustments.Cyanobacterium Synechococcus elongatus PCC 7942 is one of the most frequently used model species in the study on the regulatory mechanisms of circadian clock.The circadian clock in S.elongatus PCC 7942 uses the translation product clock protein as a transcription factor to provide feedback to control the transcriptional rhythm of core clock genes,The output pathway conducts endogenous time cues from central clock to regulation processes of downstream genes transcription or coordination the circadian ordering of the life activities in cells.The research works of current dissertation is listed as follows.In this work,wild-type train S149,ΔCikA the knockout cyanobacteria strain of CikA,the key gene in cricadian clock input pathway,as well as the knockout cyanobacteria strains of key genes in the maintaining process of introcellular redox homeostasis,such as GS,TR and 2-Cys Prx genes,were used as experimental materials.The differences among growth phenotypes of these abovementioned cyanobacteria strains were measured under intracellular redox status disturbed conditions triggered by exogenous application of oxidizing agent（methyl viologen,MV）or reducing agent（sodium thiosulfate,ST）.The results showed that there are no remarkable differences have been detected among the growth figures of various KO（knockout）strains under noraml constant growth conditions compared to that of the wild-type S149 strain.Similarly,growth phenotypes of the mutant strains were comparable to those of the wild-type strains treated by 100 m M ST,and therefore no statistically significantly different were found across strains under ST treatment.On the contrary,the growth ofΔGS,ΔTR andΔCikA single knockout mutant lines was significantly inhibited by MV compared to wild-type cyanobacteria S149 at a final concentration of 1μM MV treatment.d2Cys/d CikA double knockout lines had a growth phenotype comparable to d2Cys but significantly better than d CikA single knockout lines.d2Cys/d GS double knockout mutant strains could hardly survive under MV treatment conditions.These results suggest that there should be an additive effect between2-Cys Prx and GS genes under oxidative stresses（but also maybe redundancy under normal cultivated conditions）in the regulation of REDOX homeostasis in cyanobacteria,and these 2 genes also play very complex roles in the functional regulation of circadian clock input pathways.Then the clock genes C and CikA genes were assayed in wild-type S149,ΔGS,ΔTR and Prx family gene knockout mutants to analyze temporal expression patterns of clock genes.The results show that C and CikA genes exhibit a robust rhythmic oscillated pattern in all tested cyanobacteria strains under normal growth conditions.When treated by 1μM MV,the amplitude of the C gene expression was significantly reduced in theΔCikA mutant,while the C gene expression lost its rhythmic oscillated expression pattern in d2Cys/d CikA.In theΔGS-2Cysprx double knockout strain and theΔTR-2Cysprx double knockout strain,the C gene lost the rhythmic oscillation phenotypes too;in wild-type cyanobacteria S149,the biological clock rhythmic oscillation pattern of the temporal expression of the CikA gene was lost and the amplitude was significantly reduced.In theΔGS mutant,CikA gene expression exhibited a phenotype of temporal fluctuations but loss of rhythmicity.The rhythmicity of CikA gene expression was also completely lost inΔ2-Cys Prx.These results revealed that 2-Cys Prx or GS gene affected the expression of CikA a key gene charging of the input pathway of clock,in a similar manner.However,the knockout of key genes maintaining the redox homeostasis,such as 2-Cys Prx,GS or TR could seriously obstructs the rhythmic phenotype of the clock in S.elongatus PCC7942 cells.The deficient of both the key gene for REDOX homeostasis and CikA worsen the circadian rhythm phenotypes,sometimes even let to an arrhythmic phenotype.Finally,the phenotype of Prx-SO_2/3 rhythmic marker in various redox homeostasis maintaining gene knockout mutants（ΔGS,ΔTR）was explored under 1μM MV treatment.The results showed that the MV treatmen promoted the endogenous ROS contents in tested cyanobacterial cells,then boosted the overaccumulation of Prx proteins to scavenging of ROS in cells and severely disturbed the maintenance of Prx-SO_2/3marker’s rhythmicity.