Study On The Functions Of Prx Gene In Intracellular Antioxidation And The Sustaining Of Kai Circadian Clock In Cyanobacterium

The central oscillator of circadian clock in cyanobacterium, Synechococcus elongatus PCC 7942, is composed of kaiA, kaiB, kaiC and its coded proteins. Many researches confirmed that this self-sustained intracellular circadian clock, so-called Kai clock, have play the pivotal role in the generation and calibration of the time clue for the circadian rhythm in cyanobacterial metabolism. Some proteins,such as SasA?LabA?CikA?RapA, are considered to be the key switches that have operated the output pathways of Kai clock. These output pathways would both regulate the rhythm of central oscillator by a transcriptional-translational feedback loop (TTFL) method, and deliver the corrected phase information of circadian clock to the downstream genes.Peroxiredoxins,or Prxs in short, are families of thiol-specific antioxidant protein, which have be convinced to exert an important function in the whole antioxidant defense system of cyanobacteria.The genome DNA of S. elongatus PCC 7942 encodes 6 different prx genes. Among all the Prxs proteins translated from these prx genes, the cyclical changes redox state of Prx-2Cys are proved as a conserved non-transcriptional rhythmic marker.The main purpose of present work is to reveal the functions of Prxs proteins in antioxidation and its rhythmic phenotype by both physiological traits analysizing and molecular biology methods.To understand how the prx-2cys gene contributes to the process of excessive reactive oxygen species (ROS) scavenging, some physiological testing have been carried out in the cyanobacterium that had been grown under several stressed conditions,such as excessive H2O2 treatments,UV-B radiation and high intensity radiation stresses.By comparing the differences of physiological treats and relative surviving rates between the wild type(WT) and prx-2cys knockout (?~2cys) strains which had be grown under above mentioned various stresses, a conclusion could be drawn that the physiological traits and relative surviving rates of ?2cys are significant lower than that of WT, which indicates that the prx-2cys gene plays a pivotal role in the total antioxidation system of S. elongatus PCC 7942.Some efforts also have been exert to test the relationship between the circadian clock driven by Kai proteins and the PRX-SO2/3 marker that have been thought to be a new conserved rhythmic marker:(1) The total RNA preparation methods in S. elongatus PCC 7942 were optimized.By testing different treating periods of lysozyme, initial cell amount, the removal method of genomic DNA, and the purification method of cDNA, the present work have established a proper RNA extraction method which is suitable for the submerged cultured cyanobacteria. Our results suggested that 1.1 mg (dry weight) cyanobacterial cell treated by lysozyme for 10min were the best material for the total RNA extraction experiments by a RNA extraction Kit from TAKARA (Dalian, China).(2)Detecting of the expression patterns of clock genes by a Florescent real-time quantitative PCR (qPCR) method. A qPCR method was employed to test the expression of several clock genes in time course samples which had been cultured under different light intensity.The results demonstrated that under the natural light condition, the rhythmic phenotype of the central clock genes were not affected by whether the prx-2cys gene were knocked out in S.elongatus PCC 7942 cells. But the expression patterns of the genes operating the output pathway were severely affected.Though the expression patterns of the output pathway genes were affected both in WT and ?~2cys strains under a high light stressed condition, the rhythmic phenotype of the central clock gene expression patterns nearly disappeared in WT strains, while this rhythmic phenotype was restored in ?~2cys cells by an interesting unknown method.(3) To build double knockout cyanobacterium mutants, which would provide material for the following researches,4 different prx coupled with prx-2cys gene double knockout vectors were constructed individually. Firstly, a kanamycin resistance expression cassette were cloned and inserted into the gene coding regions of 4 prx genes (prx-QA1?prx-QA2?prx-QA3? prx-QB) to establish individual single prx gene knockout vectors. Then these above mentioned knockout vectors were used to transfer to the ?~2cys cells, a prx-2cys gene knockout cyanobacterium mutant with chlorampenicol resistance, to obtain the desired double knockout strains.