Effect Of Clock1a Mutation On Rhythmic Recruitment Of Neutrophils In Zebrafish

Biological clock is an endogenous mechanism,which enables the body to predict the changes of daily environment,thus coordinating various physiological activities and behavioral rhythms,so as to help individuals survive better.Biological rhythm has an important influence on animal immune response and growth,development and reproduction.The molecular mechanism of biological clock mainly depends on a transcription/translation feedback loop regulated by cells.CLOCK in this loop can form hetrodimers with BMAL1 in the nucleus,which can activate the transcription of negative regulatory factors of target genes including E/E’-box elements in their promoter and enhancer regions,such as per and Cryptocyanin(cry)gene families.Per and Cry protein form another heterodimer,which is then transferred to the nucleus and interacts with the CLOCK:BMAL1 complex to inhibit its transcription activation,thus forming a feedback loop.In this study,the clock1a homozygous mutant(clock1a-/-)was successfully constructed by CRISPR-Cas9 technology,and hybridized with the green fluorescent protein Tg(lyz:EGFP)strain labeled with neutrophils to obtain the clock1a homozygous mutant labeled with green fluorescent protein.The validity of clock1a mutation was identified by restriction enzyme identification,gene sequencing and qRT-PCR detection.Based on the construction of clock1a-/-zebrafish,the growth and physiological state of clck1a-/zebrafish were further studied,and it was found that clock1a-/-did not affect the growth and morphology of zebrafish larvae.By monitoring the circadian rhythm of zebrafish larvae under normal photoperiod and continuous darkness,it was found that the movement rate of clock1a-/-zebrafish decreased under normal photoperiod,and the circadian rhythm disappeared under continuous darkness.In the study of the expression level of clock1a-/zebrafish juvenile downstream clock gene,it was found that the expression levels of bmal1b,per1b,per2 and cry1ba genes changed significantly under normal light and continuous dark conditions.All the above results show that clock1a-/-makes zebrafish lose its circadian rhythm.On this basis,we further studied the effects of clock1a-/-zebrafish on neutrophil migration and cytokine expression after its circadian rhythm disappeared.The results showed that clock1a-/-could significantly increase the migration of neutrophils to inflammatory sites during the day and night,and in this process,clock1a-/-promoted the expression level of cytokines,which was consistent with the migration trend of neutrophils.These results indicate that the biological clock gene clock1a can regulate the migration of neutrophils and the expression level of cytokines,thus affecting the immune response.In this study,we successfully constructed a zebrafish strain with homozygous mutation(clock1a-/-)of the biological clock gene by using CRISPR-Cas9 technology,and proved that clock1a can regulate the migration of neutrophils and thus regulate the immune response,which can further understand the role of biological clock in the immune response process,deepen the understanding of this field,and provide theoretical reference for the treatment of diseases related to biological clock disorder in animals and the research of biological clock in poultry animal husbandry immunity and disease resistance.