Study On The Function And Mechanism Of Two Circadian Clock Genes In Antiviral Immunity Of Decapods

The biological clock is a physiological regulatory mechanism.It exists in organisms and is formed through the participation of multiple genes,which is manifested as a rhythmic response in behavior and physiological functions.The typical biological clock is the circadian rhythm of an organism,which is regulated by external environmental changes such as light.Currently,the close relationship between the circadian clock and immune processes has been widely studied and proven in mammals,but in crustaceans of important economic value,knowledge about this aspect is relatively scarce.Viral diseases caused by White spot syndrome virus(WSSV)infection have caused serious harm to a variety of crustacean aquaculture.Research on important factors that control WSSV replication in the host can provide direction and theoretical support for the effective prevention and control of diseases caused by WSSV.In this study,we firstly constructed a "light-immunity" model for shrimp based on the new finding that the time of WSSV infection affects the survival rate of crayfish Procambarus clarkii,and then used the model to discover and investigate the molecular mechanisms by which light affects the immune response of shrimp against WSSV through circadian clock genes.In the "light-immunity" model,the absolute virus content and replication status of WSSV in crayfish were measured,and the amount of WSSV replication in crayfish was significantly different under normal light(12L/D),full light(LL)and total darkness(DD)conditions,showing that WSSV replication levels in shrimp were higher under full light conditions,followed by normal light conditions and lower under total darkness conditions.The expression of several core circaidan clock genes in different tissues of P.clarkii under three light conditions revealed that light could significantly affect the expression of Clock in different tissues,while the expression of Cryptochrome(Cry)gene was less changed.Compared to the control group,knockdown of the Clock gene in crayfish and shrimp by RNAi technology revealed a significant decrease in viral replication,indicating that CLOCK promotes the replication process of WSSV in the host.Further studies showed that WSSV infection induced the interaction of CLOCK protein with NF-kB analog Dorsal,resulting in increased acetylation and nucleus translocation of Dorsal,chromatin immunoprecipitation and dual luciferase reporter system assays showed that CLOCK could bind certain WSSV immediate early(ie)genes and initiate their transcription.In addition,CLOCK enhanced the binding activity of Dorsal to the viral DNA in the ie genes.Light promotes these processes by up-regulating Clock expression,thereby facilitating virus replication in shrimp,and CRY protein functions as a suppressor of viral replication by inhibiting the nuclear translocation of CLOCK and Dorsal and the binding activity to viral DNA caused by WSSV infection,acting as a reverse regulator in this process.This thesis systematically investigated the expression changes of several core circaidan clock genes in crustaceans under different light conditions,deeply analyzed the functions and molecular mechanisms played by CLOCK in shrimp antiviral immunity,and elucidated the involvement of light and circadian clock gene Cry in regulating the antiviral immunity process of crustaceans through Clock.The results provide insight into the functions of circadian clock genes in crustacean antiviral immunity and theoretical support for the use of environmental factors such as light to control the outbreak of white spot virus disease in shrimp aquaculture.