Effect Of Gamma Radiation On The Biological Rhythm Of Zebrafish(Danio Rerio) Embryos

Objective:This project is to study the biological effects of individual and molecular levels of zebrafish embryos under different doses of radiation(0 Gy,0.01 Gy,0.1 Gy,1.0 Gy).The effects and mechanisms of gamma irradiation on the biological rhythm of zebrafish embryos were analyzed,and the harm of gamma irradiation was explored from the perspective of biological rhythms,which provided a scientific basis for the prevention and control of gamma irradiation biological toxicity.Methods:Using the AB-series zebrafish as a biological model,when the zebrafish embryo developed to 5.5 hours,it was irradiated with different irradiation doses using a biological irradiator.At the same dose rate,the cumulative dose was 0 Gy,0.01 Gy,0.1 Gy,1.0 Gy.Observing Biological effects at different levels of radiation:1.Morphological observation: Observation of the embryo's gross morphology under the microscope,and calculation and statistics of 72 hpf embryo survival rate,hatching rate and malformation rate.2.Behavioral index detection: use zebrafish behavior recorder to observe activity behavior at 5 dpf of juvenile fish,record and analyze its activity amplitude,phase and behavior cycle.3.Melatonin detection: use ELISA kit to detect 72 hpf embryo melatonin concentration at different times of the day.4.Detection of mRNA expression levels of key biorhythmic genes: RT-PCR technology was used to detect mRNA expression of key genes of biorhythm in different dose groups of zebrafish embryos.5.CLOCK protein expression detection: Western blot detection of CLOCK protein expression in zebrafish embryos under different doses.Results:1.Zebrafish embryos developed to 5.5 hours were irradiated with gamma irradiation at 0 Gy,0.01 Gy,0.1 Gy,and 1.0 Gy using a biological irradiator,respectively.Zebrafish embryos were collected at 72 hpf and under a fluorescent upright microscope Observe,make statistics and take pictures.Zebrafish embryos developed to 72 hpf were collected and the fluorescence Observe,take pictures and count under the microscope.The results showed that the mortality rate of 72 hpf zebrafish embryos increased with 0.1 Gy and 1.0 Gy irradiation,the zebrafish embryo hatching rate of the 0.1 Gy dose group increased significantly,and the zebrafish embryo deformity rate increased with 1.0 Gy irradiation.However,mild symptoms include curvature of the tail spine and swelling of the yolk sac.2.After irradiating zebrafish embryos that have developed to 5.5 hours with gamma irradiation of 0 Gy,0.01 Gy,0.1 Gy,1.0 Gy,collect zebrafish juveniles that have developed to 5 dpf,and use zebrafish behavior recorder to detect The activity amplitude,phase and behavior cycle of juvenile fish were observed and counted.The results showed that the amplitude of zebrafish activity in the 0.1 Gy and 1.0 Gy dose groups decreased,the duration of exercise extended,and the cycle shortened.3.After irradiating zebrafish embryos developed to 5.5 hours with gamma irradiation of 0 Gy,0.01 Gy,0.1 Gy,1.0 Gy,respectively,collect zebrafish developed to 72 hpf at different time points T0,T6,T12,T18.For embryos,a group of 40 pieces were centrifuged for centrifugation using an ELISA kit for melatonin concentration detection.The results show that when the radiation dose reaches 0.1 Gy and 1.0 Gy,the melatonin secretion of zebrafish embryos at various time points will be reduced,that is,a certain dose of radiation will reduce the melatonin secretion of zebrafish embryos.The effect is present at different times throughout the day,and is more significant at irradiation doses of 0.1 Gy and 1.0 Gy,and the reduction is highest at zero at night,when the melatonin secretion is the strongest in the day.4.Zebrafish embryos developed to 5.5 hours were irradiated with gamma irradiation of 0 Gy,0.01 Gy,0.1 Gy,and 1.0 Gy,respectively.Zebrafish embryos developed to 72 hpf were collected,and zebrafish embryos arntl1(bmal1),clock1 a,per1b,per2,cry2,nrld2 a and arntl1 a mRNA expression levels.The results showed that the expressions of arntl1(bmal1),clock1 a,and per1 b were significantly up-regulated under the irradiation of 1.0 Gy,and the expressions of per2 and cry2 were significantly down-regulated under the irradiation of 0.1 Gy,and cry2 was at the 0.01 Gy dose.There were already down-regulations under irradiation,while nrld2 a and arntl1 a had no obvious expression changes at each dose.5.Zebrafish embryos developed to 5.5 hours were irradiated with gamma irradiation of 0 Gy,0.01 Gy,0.1 Gy,and 1.0 Gy,respectively.Zebrafish embryos of each group were collected at 72 hpf,and the expression of CLOCK protein in the embryos was measured by Western blot technology.The situation is detected.The results showed that the expression of CLOCK protein in zebrafish embryos of the 0.1Gy and 1.0 Gy groups was up-regulated.Conclusions:1.The 0.1 Gy and 1.0 Gy doses of gamma radiation cause exact damage to zebrafish embryos,but the minor deformity caused may not be the main reason for the increase in mortality.2.The 0.1 Gy dose of gamma radiation will promote the hatching of zebrafish embryos.The low dose radiation stress effect can effectively stimulate the life process,but this can also cause various microscopic damage.3.Irradiation with 1.0 Gy dose of gamma will cause up-regulation of some clock genes and protein expressions,affecting melatonin secretion will directly lead to zebrafish behavioral rhythm disorder and damage to body health.4.The different genes on the core loop of the circadian clock show different sensitivity to gamma radiation.