Generation Of Period2 Mutants By TALEN And Its Circadian Roles In Zebrafish

As an endogenous mechanism of physiological regulation, the circadian clock plays a very important role in the whole organism physiology and behavior. According to the epidemiological study, the biological clock disorder is often accompanied by physiological, mental, immune, and cancer. In vivo, the circadian clock forms a 24 hours of oscillation mainly replied on the transcription-translation loop. First, the CLOCK:BMAL complex,as a positive regulatory factor, acts on the E-box of the target gene promoter to activate the negative regulatory factor and the circadian clock genes. A heterodimer formed by the negative regulators PER and CRY enters the nucleus and interacts with the positive transcription factors. The heterodimer lifts the activation of CLOCK:BMAL complex, and thereby a cycle is completed. Per2 as an important negative regulator, plays an indispensable role in the circadian clock system. The zebrafish as an important clock model organism for the study because of its lack of mutants, is greatly limited its field of research in the circadian clock. TALENs,which is a recently rising mutant construction technology, has been applied like a raging fire in zebrafish. In this paper, we used TELENs to construct the zebrafish mutants and studied its effect on the circadian clock in the system itself and reproduction in the per2 mutant.In this study, using TALENs technique we successfully obtained zebrafish per2 mutants, which were 11 base pairs of the deletion and 13 base pairs of the insertion. With the help of the technologies of Q-PCR, transfection and transgenic fish we found that in zebrafish per2 mutant, the rate limiting enzyme of melatonin biosynthesis gene aanat2 was up-regulated, there have been significant changes in the expressions of circadian clock core genes of which per1a, per1b, per3 gene expressions in DD and LD conditions have been remarkably down-regulated, and cry1b,cry2a and cry2b are significantly up-regulated. Whet’s more, bmal1b also appears significantly down.According to the results of the cell experiment, Per2 can inhibit and activate aanat2 and bmal1b respectively through E-box and RORE, which means it’s a dual role. Using cells and transgenic fish we found, the role of Per2 on bmal1b is activated through Rora rather than Rev-erba. In the mouse system, we discovered that the same phenomenon also exists in mammals, and Per2 through RORE to regulate the expression of bmall gene also occurred in the process of evolution of fishes to mammals.We also found that per2 is also tissue-specific in regulating the circadian clock. All these suggest that Per2 plays a very important role in the circadian clock of zebrafish.We also studied the effects of Per2 on reproduction,metabolism,and development in zebrafish, the results found reduction in body weight and body length in the per2 mutant, and the lower hatching rate and change of fullness illustrated that per2 participates in mediating growth and metabolism in zebrafish. According to the detection of the adult sex hormones in vivo, the concentration of testosterone and estradiol were significantly decreased in thhe per2 mutant. What’s more, the quantitative results indicated that in the per2 mutant the expression of cypl9a, which catalyzes androgen into estrogen catalytic enzyme (aromatase), changed obviously. And there is a significant increase in the methylation of two cypl9agene promoter in per2 mutant. To sum up, the disorders per2 affects the methylation aromatase gene which results in the reproduction of the per2 mutant.Conclusion:As one of the core parts of the clock genes, Per2 plays an important role in the circadian clock in zebrafish. Per2 has a dual role, for it can inhibit and activate aanat2 and bmal1b respectively through E-box and RORE, In addition,the role of Per2 on bmal1b is activated through Rora rather than Rev-erba. We also found that per2 is also tissue-specific in regulating the circadian clock. Moreover, the disorders per2 affects the methylation aromatase gene which results in the defect in reproduction of the per2 mutant.