Study On The Expression Rhythmicity Of Prx Genes In The Output Pathway Clock Genes Knockout Cyanobacteria Mutants

Synechococcus elongatus PCC 7942?S.elongatus PCC 7942?is the simplest model organism for circadian rhythms researches.The rhythmic oscillation of cyanobacterial central clock composed by KaiA/B/C proteins is relied on the Post-Translational Oscillation?PTO?mechanism of KaiA/B/C protein themselves.This rhythmic oscillation can be reconstructed in vitro in the presence of ATP.The clock genes in the signal input pathway and the signal output pathway of a circadian clock system could module the rhythmic of the circadian clock by a Transcription-Translation Feedback Loop?TTFL?mechanism.Through the signal input pathway,the cyanobacterial clock system can sense the ambient time clues and transmit it to the central oscillator of core clock.Then the signal output pathway can transmit the endogenous temporal signals generated by the core clock to the downstream genes regulated by circadian clock.The rhythmic expression of output pathway clock genes is essential for the effective conduct of basal metabolic activities such as photosynthesis in cyanobacteria.Both the light signal in the environment and the redox signal in the cell can entrain the phase of the circadian clock.Among the cyanobacterial cells involved in the maintenance of intracellular redox homeostasis are mainly redox proteins such as Peroxiredoxins?Prx?,Thioredoxin?Trx?and Glutaredoxin?Grx?.Previous studies have shown that the typical2-Cys Prx(Prx-SO_2/3 rhythmic marker)in cyanobacteria has circadian rhythm oscillations at the redox state level.The redox state rhythmic oscillation of the Prx-SO_2/3/3 rhythm marker is different from the previously mentioned PTO and TTFL mechanisms,which are maintained by a non-transcriptional Oscillator?NTO?mechanism.Thioredoxin reductase?TR?plays a key role in the regulating of redox of oxidized Prx protein,and Glutathione synthase?GS?is a cofactor for 1-Cys Prx in the Prxs family.Prx is ubiquitously distributed across archaea,bacteria and eukaryotes,which also has maintained a high degree of conservation in evolution.Therefore,the NTO mechanism of the Prx-SO_2/3/3 rhythm marker based on it is also widely existed.Since the endogenous circadian timekeeping mechanisms of organisms based on TTFL and PTO are not conserved in the organisms from different evolutionary categories.Further research on the NTO mechanism of the common Prx-SO_2/3 rhythmic markers across species,as well as the relationship between the NTO timekeeping mechanism and TTFL and PTO timekeeping mechanisms has displayed an important theoretical value in the modeling mechanism of circadian clock.In this thesis,the model cyanobacteria S.elongatus PCC 7942 was used as materials,the circadian rhythmic phenotypes of the input pathway or output pathway clock genes and the Prxs genes were studied in detail at the transcriptional levels.The research results indicated that theTTFL,PTO mechanisms and NTO mechanism in the regulation of circadian clock are not independent to each other.Therefore,the interaction of CikA and 2-Cys Prx proteins were found and both of these two proteins have involved in the conducting of circadian rhythmic phenotype of the cyanobacterial clock.Nevertheless,the problem about whether the protein interaction between CikA and 2-Cys Prx is direct or not is still keep unkown.Main research contents of this thesis are listed as following:1.The optimal internal reference genes of S.elongatus PCC 7942 RT-qPCR were screened under abiotic stresses,which have laid a technical foundation for the subsequent studies of the expression pattern of the circadian clock output gene and the Prxs family gene at the transcriptional level.The results show that the combination of prs and secA is the most suitable reference gene for all stress conditions and high light stress.The combination of secA and ppc,rimM and rnpA,rnpA and ilvD were most stable under NTC,NaCl and H₂O₂stress conditions,respectively.rimM is only stably expressed under certain conditions and should be carefully selected.16S and rnpB are not suitable as reference genes in S.elongatus PCC 7942.2.A series of single gene knockout mutants were constructed for the outport pathway clock genes of Kai clock and the Prx-SO_2/3 rhythmic marker encoding gene 2-cys prx.The expression patterns of these genes were evaluated in different mutants by RT-qPCR.The results showed that knockout of cikA and sasA genes directly led to the loss of rhythm expression of the rpaA gene.Knockout of 2-cys prx gene slightly disturbed the amplitudes of cikA,rpaA,and 1-cys prx gene,or delayed their peak time.The transcriptional rhythm of1-cys prx,prxQ-A1 and prxQ-B,was also found.Knockout of the 2-cys prx causes a loss of their rhythmic expression phenotypes.In terms of 2-cys prx itself,which did not show expression rhythms.Surprisingly,knockout of 2-cys prx increased the amplitude of the cikA expresison which suggested that 2-cys prx functioned on the upstream of cikA during the regulating process of circadian clock rhythmicity.3.The 2-cys prx,cikA,TR and GS single-knocked cyanobacteria strains and combined double-knocked cyanobacteria strains were constructed,while the periodical length changes of their circadian clocks wre also assayed under different light densities.The results showed that both high light stress and low light stresses could significantly shorten the periodical length of the circadian rhythm in target cyanobacteria mutants.Knockout of the 2-cys prx gene?d2Cys for short?did not significantly affect the periodical length of the cyanobacteria central clock.The TR knockout cyanobacteria?TR knockout strain,dTR for short?is enormously shortened only under normal light intensity,and the GS knockout cyanobacteria?GS knockout strain,dGS for short?has little effect on the cyanobacterial circadian clock.The double-knocked cyanobacteria strains of TR and GS combined with the 2-cys prx gene?referred to as d2Cys/dTR and d2Cys/dGS,respectively?showed a significantly phenotype with shortened core clock period.These results indicated that TR and GS and 2-cys prx gene are involved in 3 different signal pathways that are independent of each other,but can undergo functional compensation,and participate in the input pathway of the environmental clock signal to the circadian clock.4.Detection of interaction between 2-Cys Prx and CikA proteins was performed by Bimolecular Fluorescence Complementation?BiFC?method and yeast two-hybrid assays.The results of BiFC assay showed that there were excitation fluorescence signals of eYFP in tobacco chloroplasts which are generally believed to evolve from cyanobacterium cells and also shared great similarity in the physiological bases to cyanobacterial cells.However,the yeast two-hybrid assay results did not support any detect the interaction of between2-Cys Prx and CikA proteins.Based on these results,the present thesis established a hypothesis to propose a possible explanation of the seemingly contradictory findings from either BiFC or yeast two-hybrid assays.The above mentioned hypothesis is as follows,the intracellular redox state based on photosynthetic electron transport is very likely to be a prerequisite for the interaction of 2-Cys Prx with CikA protein.Anyway,this hypothesis still need more solid experimental evidences to test and confirm.