| period(per)genes as canonical clock genes are highly conserved among fruit flies,zebrafish and mammalian.Fruit flies have one per gene,mammals have three Pergenes,Per1,Per2 and Per3,while zebrafish have four per genes,per1 a,per1b,per2 and per3.Zebrafish per1 a and per1 b are co-orthologs of mammalian Per1,derived from the third-round teleost genome duplication(TGD).The function of the zebrafish per1 b gene in the circadian clock has been systematically analyzed in our laboratory,but the function of per1 a in the zebrafish circadian clock is still unknown.In order to examine whether per1 a is regulated by the circadian clock,real-time quantitative PCR results show that the per1 a gene has rhythmic expression in wild-type zebrafish and is down-regulated in the homozygous clock1 a mutant.To investigate the role of Per1 a in the regulation of the circadian clock,we used the per1 a mutant line generated by our lab using CRISPR-Cas9 technology.The per1 a homozygous mutant(per1a-/-)larvae show no significant differences in the period,phase,and amplitude of locomotor in constant dark(DD)condition compared to wild types.While the locomotor activity of per1 a mutant is significantly decreased under LD condition.The period is similar to WT,amplitude is decreased and phase is advanced under LL condition.In addition,we generated a pineal gland-specific knockout per1 a transgenic fish line Tg(U6x: sgRNA(per1a);LC);Tg(exorh: Cas9;CG2).In LD condition,the activity of pineal gland-specific knockout per1 a transgenic fish line is significantly decreased compared to control.In DD condition,however,the phase,amplitude and period of pineal gland-specific knockout per1 a transgenic fish line behavior are notchanged compare to control group.And in LL condition,period short 0.5h,amplitude is decreased and phase is delayed.We conclude that per1 a mutant has a little effect for zebrafish circadian behavior.So we want to know whethere per1 a mutant affect the transcriptional level in zebrafish.We performed deep sequencing-based transcriptome analysis of homozygous per1 a mutant and wild-type control larvae.The results showed that circadian genes per1 a,per1b,per2,per3,cry1 aa,are down-regulated during the day(CT2),revealing that Per1 a may play an important role in zebrafish circadian clocks.Further we used qPCR to examine the effect of per1 a mutation on the transcript level of zebrafish,results showed that the circadian genes of per1 a,per1b,per2,per3,cry1 aa and cry1 ab are down-regulated in the per1 a homozygous mutant under both LD and DD conditions.These results demonstrate that per1 a has little effect on the zebrafish behavioral rhythm,but plays an important role in the transcriptional regulation of the circadian clock system.To validate this conclusion,we further generate heat shock-inducible per1a-overexpression transgenic fish line Tg(hsp70l;per1a;CG2).Quantitative PCR showed that per1 a,per2,per1 b,per3,cry1 aa and cry1 ab are up-regulated in per1 a overexpressing transgenic fish lines,contrary to the results from per1 a mutant.Futhermore,the locomotor activities of per1a-overexpression transgenic fish line is significantly increased under LD condition,the amplitude of locomotor behaviors is significantly decreased,even though the phase and period of locomotor behaviors are not changed compared to control group under DD condition.And in LL condition,period increase 1.2h,amplitude and phase are similar to control.So,Per1 a effect zebrafish locomotor rhythm and transcriptional level.Therefore,we suggest that Per1 a plays an important role in the zebrafish circadian clock. |
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