Construction Of Duox Zebrafish Mutant And Its Effects On The Circadian Clock

The circadian clock is a stable endogenous mechanism formed by the long-term evolution of the body in order to adapt to changes in the external environment.This endogenous mechanism is regulated by the internal clock genes bmal,clock,cry,per,etc.At the same time,external timing factors such as light and temperature can change or reset the circadian clock.Biological rhythm plays an important regulatory role in many life activities such as sleep regulation,hormone secretion and immune response.It is very important to study the regulation mechanism of biological clock for the regulation of body health.Reactive oxygen species(ROS)are important substances involved in the regulation of physiological homeostasis as signaling molecules during cell respiration,and low ROS levels in vivo will destroy learning and memory,reproductive development and cell proliferation and differentiation,and high ROS levels in the body will lead to oxidative stress,aging,cardiovascular disease,and respiratory failure.The nicotinamide adenine phosphate dinucleotide(NADPH)oxidase(NOX)family is involved in ROS production in response to various extracellular signals.As an important member of the NOX family,dioxidase(DUOX)has been shown to be involved in the regulation of homeostatic equilibrium of ROS and the regulation of redox signaling pathways.Studies have shown that ROS levels in vivo are circadian rhythmic,so we speculate that altered ROS levels can regulate the circadian clock.In order to study the effect of ROS on the circadian clock,we first designed the sgRNA by CRISPR-Cas9 technology and targeted knockout the target site sequence of duox in zebrafish embryos by microinjection.The hybrid mutant of duox was identified by enzyme digestion and sequencing.Three months later,the hybrid mutant of single stable mutant type was obtained by hybridization with wild type zebrafish(WT).Three months later,the hybrid mutant was mated with male and female,and the duox homozygous mutant was screened and identified.We evaluated the growth and development of the mutant by observing and recording the tail wagging,heartbeat,body length,morphology and activity of duox-/zebrafish.The kit was used to verify that duox deletion reduced the levels of hydrogen peroxide(H2O2)and ROS in zebrafish.Next,we used behavioral experiments and fluorescence quantitative experiments to detect the effect of duox on the circadian clock.The results of three-day behavioral experiments showed that compared with WT,the activity of duox-/-zebrafish decreased significantly during the day and night under normal light(LD(14 h light:10 h dark)).Under continuous dark(DD)conditions,the cycle of duox-/-zebrafish was significantly shortened and the phase was moved forward.RT-qPCR results showed that compared with WT,the expression levels of bmal1b,per1b,per3 and clock1a in duox-/zebrafish were significantly changed under LD conditions,and the expression peak of clock1a was shifted.Under DD conditions,the expression levels of bmal1b,per1b,per3 and clock1a in duox-/-zebrafish were significantly increased.In order to preliminarily study the regulation mechanism of duox on zebrafish circadian clock,we will take WT and duox-/zebrafish samples for transcriptome sequencing analysis.In summary,we have successfully constructed zebrafish duox homozygous mutants at the animal level and found that duox plays an important role in zebrafish circadian clock regulation.