The Expression And Regulation Of Paralichthys Olivaceus Bmal1 And Cry1 And Their Effect On Immunity

There is a circadian clock system in organisms.Organisms form a set of circadian clock system to regulate their behavior and various physiological activities including immune reaction.In mammals,BMAL1,a major clock transcriptional activator,promotes transcription of many genes,including Cry.A large number of studies have shown that the two genes,Bmal1 and Cry1,play important roles in immune regulation.In this study,we investigated thre circadian expression and the immune function of Japannese flounder(Paralichthys olivaceus)Bmal1(PoBmal1)and Cry1(PoCry1).First,we examined whether light had any effect on the resistance of flounder against bacterial infection.The fish were maintained under four different light conditions: LD(12 h light followed by 12 h darkness),DL(12 h darkness followed by12 h light),LL(continuous light)and DD(continuous darkness).The fish were then challenged with Edwardsiella tarda,and bacterial dissemination into kidney and spleen was determined.The results showed that the bacterial recoveries in DD and DL groups were significantly higher than that in LL and LD groups.Different tissues were collected from the fish at 7 time points within a 24 h light-darkness alternating cycle:07:00(denoted by zeitgeber time ZT 0),11:00(ZT 4),15:00(ZT 8),19:00(ZT 12),23:00(ZT 16),03:00(ZT 20),and 07:00(ZT 24,next day).The expression profiles of the pro-inflammatory cytokines of IL-1β,IL-6,IL-8,and TNF-α were detected by q RTPCR.The results showed that the expression trends of the four cytokines were generally similar,all which increased gradually with the prolongation of light stimulation,reaching the peak at the end of light stimulation(ZT 12),and then decreased gradually with the dark phase.The expressions of PoBmal1 and PoCry1 at the 7 time points were also examined.The results showed that,similar to the pro-inflammatory cytokines,the expression of PoBmal1 rose steadily in the photophase and peaked at the end of the photophase,and then declined steadily during the scotophase and reached the lowest level at the end of the dark phase.PoCry1 expression showed opposite trend.Sequence alignment showed that PoBmal1 and PoCry1 shared high identitites with the Bmal1 and Cry1 of other species.PoBmal1 and PoCry1 m RNAs were widely distributed in various tissues of Japanese flounder by q RT-PCR.Bacterial infection inhibited the m RNA expression of PoBmal1 and PoCry1.In order to study the function of PoBmal1 and PoCry1,the two genes were knocked down in vivo.PoBmal1 knockdown significantly reduced the expression of PoCry1,PoCry2,IL-1β,IL-6,IL-8,and TNF-α,indicating that the PoCry genes and the four pro-inflammatory cytokines were positively regulated by PoBmal1.When infected with bacterial pathogen,PoBmal1 knockdown flounder exhibited significantly decreased bacterial dissemination compared with the control fish,and significantly increased expression of inflammatory cytokines.In flounder with PoCry1 knockdown,IL-1β,IL-2,IL-6,IL-8,IL-12 p35,IL-12 p40 a,TNF-α,CXCL10,CCL19 and NOS1 expressions were all upregulated to different degrees,indicating that they were negatively regulated by PoCry1.When infected with bacterial pathogen,flounder with PoCry1 knockdown exhibited significantly decreased bacterial dissemination and upregulated expression of immune-related genes compared with the control fish.Inhibition of PKA in PoCry1 knockdown flounder promoted bacterial infection and repressed the expression of immune related genes.These results suggested that knockdown of PoCry1 likely stimulated the PKA activity in Japanese flounder,which led to the activation of downstream NF-κ B signaling pathway,thereby promoting the expression of the immune related genes and enhancing the antimibribial ability of Japanese flounder.Taken together,the results of this study indicated that light could affect the ability of flounder to resist bacterial infection,and that light also affected the expression of PoBmal1 and PoCry1,which play important roles in regulating the immune response of Japanese flounder against bacterial infection.