Identification And Characterization Of The Three Circadian Clock Genes In The Crayfish Procambarus Clarkii

Circadian rhythms are an endogenous timing system,relying on the core clock genes of cycle(Cyc),clock(Clk),timeless(Tim),period(Per),cryptochrome(cry),etc.to drive numerous physiological and behavioural processes followed a rhythm of approximately 24 h and ensure an accurate adaptation to external daily rhythm.In this study,three of the main circadian clock genes cycle(PcCyc),clock(PcClk),cryptochromel(PcCry1)in the red swamp crayfish(Procambarus clarkii)were cloned.The expression patterns of mRNA in the three clock genes under different light cycles were examined by fluorescence quantitative PCR.Polyclonal antibody against PcCYC protein was prepared.The expression of PcCYC protein in brain tissue of crayfish was detected by western blotting and immunohistochemistry.Based on the information of the transcriptome of crayfish which has been uploaded by NCBI(Accession code.SRP044128),the full-length or partial cDNA sequence of the major clock genes PcCyc,PcClk,PcCryl in the red swamp crayfish was cloned using RACE(rapid amplification of cDNA ends)technique.The function and physical and chemical properties of the predicted protein sequences were analyzed by bioinformatics.Real-time quantitative PCR technology was used to explore the mRNA expression of the circadian clock genes PcCyc,PcClk,PcCry1 in brain,eyestalk and liver tissues of the crayfish under different light conditions(12LD,24DD and 72DD)respectively,and to explore circadian rhythm of clock genes in crayfish.The results showed that the PcCyc and PcClk genes had similar mRNA levels under the similar light conditions.It is proved indirectly that the PcCYC and PcCLK proteins form the heterodimer complex and regulated the transcription of all other genes within the CCN(the core-clock network).Under the illumination change,the mRNA expression of PcCry1 gene was significantly different.The results showed that the PcCry1 gene could sense the light signals and convert the light signals into optical signals,and participate in clock gene regulation.In addition,the polyclonal antibody titer of PcCYC protein was 1:80000,and the results of western blotting detection were positive.It is an effective antibody for immunohistochemistry detection.