Study On The Cloning, Expression And Function Of The Circadian Clock Gene Timeless Of The Red Pirate

Circadian rhythms are about 24-hour oscillations cycles of life process.Many biological processes maintain its own physiological and behavioral process by the internal clock mechanism to adapt to the environment.The inner clock mechanism actually is the result of clock genes interactions.Timeless is one of the important endogenous circadian clock genes which is together with period,clock and cycle forming Per/Tim transcriptional feedback loop and periodic expression in organism to maintain the circadian rhythm of time and activate the output pathway.Recent studies show that oscillatory mechanisms in different insects are not all similar to Drosophila melanogaster.So in this paper,we explored the function of timeless in Tribolium castaneum and provided more information for insect circadian oscillatory mechanism.This study identified a clock gene timeless in the Tribolium castaneum(Tctimeless).Tctimeless is located in the chromosome 5 of Tribolium castaneum with 9 introns,10 exons and a open reading frame(ORF)-3240 bp encoding a putative protein of 1079 amino acids and its 3 'UTR was 122 bp.Evolutionary analysis showed that timeless only existed in arthropod and echinoderm but lost in Apis mellifera.The qRT-PCR analysis in different development stages showed that the Tctimeless was expressed in each development stage,but significantly highly expressed in the late pupae.And tissue expression profile analysis of Tctimeless showed that in addition to the high expression of head,epidermis,fat body and accessory glands in the male had higher expression.And daily expression pattern of Tctimeless in one circadian cycle(one day)peaked in ZT4 and ZT12 respectively.After pupae RNAi of Tctimeless,Tctimeless rhythmic expression disappeared and rhythmic expression of circadian clock genes Tcperiod,Tccycle and Tcclock also disappeared.After pupae RNAi of the Tccycle and Tcclock respectively,daily expression of Tctimeless disordered.Larvae RNAi of Tctimeless caused all pupae faild to initiate molt and finally died at pupal stage.RNA interference of late pupae showd that fertility had fallen by 40.5%and only 57.8%egg could successfully hatch into larvae.After RNAi of Tctimeless we infected them with gram-positive bacteria Bacillus subtilis(Bs)and gram-negative bacteria Escherichia coli DH5a(Ec).The results showed that after the infection of Bs for 18-36 h,the level of melanization levels in IB was more serious than interference group,and melanization rate of IB was significantly higher than experimental group after the infection of Bs for 30 h.However,after infection of Ec,significant difference was hardly observed.Thus,our dates suggested that Tctimeless showed rhythmicity with 2 peaks.And Tctimeless played important roles in initiating eclosion,reproduction and immunity.Besides,Tctimeless showed interactive regulation with Tcperiod,Tccycle and Tcclock.After Tctimeless RNAi,the expression of circadian clock genes Tcperiod,Tccycle and Tcclock decreased,However after Tccycle and Tcclock RNAi respectively,the expression of Tctimeless was increased.