Clone And Expression Analysis Of Clock Gene Cry In The Diapause-induce Process Of Silkworm, Bombyx Mori

Circadian rhythm is the most common behavior and basic feature of organism. Photoperiod and temperature are two important zeitgebers that affect the expression of circadian genes.While most researches about temperature induced mechanism just focus on Drosophila,and there are fewer reports on Lepidoptera.Cry is one of the core clock genes which have been identified,and Cryptochromes(CRY) are photoreceptors that regulate entrainment by light of the circadian clock in plants and animals.They also act as integral parts of the central circadian oscillator in Drosophila brains.It has been proved that CRY is indispensable in both temperature- and photo- entrainment in Drosophila.Two cry gene families cry1 and cry2 have been birth during the evolvement in insects.Dm-cry has been identified as cry1,and Apis only have cry2.While cry1 and cry2 are both expressed in some Lepidoptera.Bombyx mori is a model organism of lepioloptera,and whose genome sequencing has been finished.It is possible to investigate cry1 and cry2 in one insect species by using silkworm as experimental material,and help us to understand the intaction and functional difference between the them.Bombyx mori has reached industrial production scale due to its economic importance.Diapause is a basic physiological phenomenon in B.mori.It is recognized as an important trait that affects the sericulture production and pest control.Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs.It is of great theoretical value and practical significance to study the molecular mechanism circadian gene cry in the process of silkworm diapause,which can help to further illustrate the molecular mechanism of dispause in B.mori.We colned the 2 homologous genes of cry,named Bm-cry1 and Bm-cry2,and analysised the protein structure and molecular evolution of them.Further more,we investigated the expression profiles of the two genes under different photoperiod and temperature incubation conditions of eggs and parental egg,and discussed the action mechanism of circadian rhythm gene cry from the dispause regulation point.The ORF of Bm-cry1 which code 525 amino acids is 1 578 bp.Bm-cry1 includes 12 exons and 11 introns.The ORF of Bm-cry2 which code 730 amino acids is 2 193 bp. Bm-cry2 includes 9 exons and 8 introns.Protein analysis show that molecular weight of Bm-CRY1 is 60 241.96,isoelectric point is 6.01.Molecular weight of Bm-CRY2 is 83 609.91,isoelectric point is 8.93. Both Bm-CRY1 and Bm-CRY2 do not show significant signal peptide sequence which indicate that they are Non-secretory protein.N-glycosylation site,Protein kinase C phosphorylation site,Casein kinaseⅡphosphorylation site,Tyrosine kinase phosphorylation site,N-myristoylation site are identified in both Bm-CRY1 and Bm-CRY2.However,Bm-CRY2 has cAMP- and cGMP-dependent protein kinase phosphorylation site,Amidation site and Cell attachment sequence which Bm-CRY1 does not have.The Protein homology analysis showed that Bm-CRY1 is of high conservative in different species.The N-terminal of Bm-CRY2 has the higher conservative,C-terminal lower,showing that the 3' region of ORF in Bm-cry2 is the variable region of C terminal of the Bm-CRY.Furthermore,Bm-cry1 and Bm-cry2 both have two conservative domain of DNA photolyase and the FAD-binding domain.This indicates that the two genes are members of enzymes DNA photolyase/cryptochrome super gene family. Analysis of molecular evolution indicates that Bm-cry1 and Bm-cry2 are closely phylogenetic with Lepidoptera insects as Antheraea pernyi,whereas far away from vertebrate,which is consistent with traditional theory.Diptera insects such as Drosophila only have cry1 and Hymenoptera insects such as Apis mellifera only have cry2.While the circadian clock mechanism of Lepidoptera insects depend on exist of both cry1 and cry2,which may reflect characteristics of both Diptera and Hymenoptera.The offspring eggs are completely dispause under the 25℃and constant illumination incubation condition(25LL) of parental egg,and they are completely non-dispause under 15DD(15℃,constant dark),and mixture of dispause and non-dispause in 20LD(20℃,12h illumination and 12h dark).The Bm-cry2 gene expression level of eggs in 25LL is significantly lower than 15DD;it is also lower than 20LD.The expression level of Bm-cry2 in 15DD is higher than 20LD in the early incubation stage(7 days before),but it is obvious in the late.To further investigate the Bm-cry2 expression in different moment every day of the sensitive period(7d,8d,9d) during the process of embryogeny,we found that it fluctuated feebly in a period approximate to 24 h under 25LL condition,while the expression of Bm-cry2 showed up obvious rhythmicity under 20LD and 15LL condition,and the fluctuate period are approximate to 42h and 96h respectively.This result clues that low-temperature and short photoperiod incubation conditions will change the rhythm of Bm-cry2 expression and delayed the phase of Bm-cry2.The lower temperature and the shorter illumination was given,the more accumulated temperature of finishing a expression period was needed.The offspring eggs of B.mori are all diapause due to the incubation condition of parental eggs under 25℃and continuous illumination(25LL),after treated by hydrochloric acid when they were layed about 20h,Bm-cry2 keep high transcription activity during the activation process of embryo.The expression of Bm-cry2 was lower in the offspring eggs under 25℃than under 15℃.This indicates that after offspring eggs went into the diapause stage,the transcription lever of Bm-cry2 was falling subsequently.We put the eggs under different incubation temperature and found that continuous illumination may heighten the expression lever of Bm-cry2 in the offspring eggs of B.mori.Collaboration effection of illumination and temperature on offspring eggs only function during the early incubation stage.Expression analysis of Bm-cry2 in different tissue of B.mori larva showed that Bm-cry2 had higher transcription level in head than in fat body,which indicate that Bm-cry2 may play a different role in central clock and periphery clock.