| The circadian rhythm exists in most organisms and is involved in almost all life activities.Circadian rhythm disorders affect the body homeostasis and cause a series of diseases.Exploring regulatory mechanisms of circadian rhythm is an important part in the field of chronobiology and chronomedicine.Subterranean rodents live in the invisible burrow system,where the light signal was weak and unstable,so that their morphology,behavior and physiology have evolved adaptive response.Mandarin voles(Lasiopodomys mandarinus)as a typical subterranean rodent and its aboveground sibling species Brandt’s voles(Lasiopodomys brandtii)are ideal animal models for circadian rhythm comparative study.In the present study,we conducted comparative biology with Mandarin voles and Brandt’s voles to investigate their behavior and physiological rhythm changes under different photoperiod conditions.To determin the key clock regulatory factors,genes related to circadian rhythm regulation were screened out from the genomic data of two kinds of voles and subjected to functional pathway enrichment analysis.After then,The expression differences of circadian clock genes in central and peripheral tissues of two kinds of voles were analyzed under different photoperiod conditions.Finally,the evolutionary study of core circadian clock genes was performed systematically to reveal the circadian rhythm regulatory mechanism in Mandarin and Brandt’s voles.The major results are as follows: 1.Effect of photoperiod on the activity rhythm in two kinds of volesThe results of wheel activity analysis of Brandt’s and Mandarin voles showed that both voles in 12l:12D and darkness conditions remained significant nearly 24 hours activity rhythm(P < 0.001),activity in subjective night were significantly higher than subjective day activity(P < 0.001),and peak activity focused on the subjective light/dark conversion time;The 24-hour average activity intensity of Brandt’s voles in 12L:12D photoperiod was significantly higher than darkness photoperiod(P < 0.001),while the activity intensity of Mandarin voles showed no significant difference between the two groups(P = 0.847).2.Effects of photoperiod on melatonin secretion rhythm in two kinds of volesThe melatonin secretion results of and Mandarin and Brandt’s voles showed that melatonin of Brandt’s voles had significant 24-hour cosine rhythm under two photoperiod conditions(P < 0.05),while Mandarin voles were not significant.(P > 0.05),but the secretion level was significantly different(12 L: 12 D,χ 2 = 20.617,df = 5,P = 0.001,darkness: χ 2 = 16.191,df = 5,P = 0.006);The peaks of melanin expression in voles were concentrated in subjective nighttime.3.Screening and functional enrichment analysis of circadian clock genes in two kinds volesThe results of circadian clock genes screening enrichment analysis regulation of two voles showed that there were 125 related circadian rhythm genes in Brandt’s voles and 120 genes in Mandarin voles;and the functional signal pathway of the two voles were similar.Both participated to the entrainment of initial rhythm of the cycle,rhythmic behavioral physiology and molecular regulation,hormone levels,neural signaling pathways and longevity regulatory pathways,Mandarin voles also participate in the biological process of blood glucose regulation;At last the functional enrichment results of the core circadian clock genes were consistent.4.Effects of photoperiod on expression of core circadian clock genes in SCN in two kinds of volesCircadian clock genes of Brandt’s and Mandarin voles expressed in SCN showed that Bmal1(P = 0.219)and Per1(P = 0.132)genes in Mandarin voles and Per2(P = 0.432)gene in Brandt’s voles had non-significant 24 hours cosine rhythm in 12L:12D photoperiod,other genes remained significant cosine rhythmic expression(P < 0.05),all genes in darkness group showed significant cosine rhythmic expression(P < 0.05);Genes peak phase of Brandt’s voles in darkness group showed delay results compared with 12L: 12 D group,while genes peak phase of Mandarin voles in darkness group appeared advanced.The variation values(CV)of circadian clock genes showed no significant difference of Brandt’s and Mandarin voles in two photoperiod groups(Brandt’s voles: t = 0.166,df = 4,P = 0.876;Mandarin voles: t = 0.368,df = 4,P = 0.732).5.Effects of photoperiod on expression of core circadian clock genes in different tissues in two kinds of volesThe results of core circadian clock genes expression in voles showed that genes expressed in cosine rhythm showed different numbers under different tissues.Genes expressed significant cosine rhythm in Brandt’s voles were concentrated in the hypothalamus and kidney tissues,and the number of significant cosine rhythm genes in eye was significantly decreased in darkness group.Significant cosine expression genes in Mandarin voles mainly concentrated in hypothalamus and liver tissues,and cosine expression genes number were increased in eye in darkness group.The peak phase of the core circadian genes of the two voles were differs in different tissues.After dark photoperiod treatment.In hypothalamus,the peak phase of core circadian clock genes(except Per3)of Brandt’s voles were advanced,peak phase of genes(except Bmal1)in eye were delayed,and peak phaseof genes were resetted in liver and kidney tissues.When Mandarin voles were treated in darkness photoperiod,the peak phase of genes in hypothalamus(except Clock,Per1)and eyes(except Per3)was advanced,and peak phase were also reset in liver and kidney tissues.The circadian core clock genes expression of two voles differed in different tissues after photoperiod treatment.In Brandt’s voles,the CV value of circadian genes in hypothalamic were non-significantly between two groups(t =-0.821,df = 6,P = 0.443),and CV values in Mandarin voles under darkness group were significantly higher than 12L:12D photoperiod(t =-5.284,df = 6,P = 0.002).In eye tissue of Brandt’s voles,CV values of genes were significantly increased in the darkness group(t =-3.190,df = 6,P = 0.019),and CV value of genes in Mandarin voles showed no significant difference between the two groups(Z=-1.690,P=0.091).CV value of genes in liver of Brandt’s voles showed non-significant difference in two groups(t =-1.377,df = 6,P = 0.218),and CV value of genes in liver were significantly increased under darkness conditions of Mandarin voles(t =-4.192,df = 6,P = 0.006).The CV values of Brandt’s voles(t=-3.018,df=6,P = 0.023)and Mandarin voles(t =-3.888,df = 6,P = 0.008)in kindey tissue were significantly higher in darkness treatment group.6.Adaptive evolution of core circadian clock genes in subterranean rodentsThe results of adaptive evolution show that the classification status of subterranean rodents in circadian clock genes phylogenetic tree were consistent with species in Cyt b phylogenetic tree.Most of the positive selection sites detected by Clock were located in the C-terminal TAD structural domain,positive selection sites of Per3 mostly located in C-terminal structural domain,and positive selection sites of Cry1 were located in the non-conserved structural domain at C-terminal.Conclusions1)Both voles maintained significant circadian activity rhythm nearly 24 hour,the average activity intensity of 24-hour in Brandt’s voles increased under light conditions,while the activity intensity of Mandarin voles was not affected by light signals.Melatonin play less role in regulation of circadian rhythm in Mandarin voles.2)Circadian clock genes enrichment of the two voles showed similar pattern indicate that the circadian clock played important role in regulation of circadian rhythm.3)The endogenous rhythm regulation system of Brandt’s and Mandarin voles played important role in regulation of circadian rhythm;4)Circadian regulation of Brandt’s and Mandarin voles coordinated by central and peripheral organs,but the visual system in Mandarin voles in circadian regulation was weakened.Peripheral organs participate not only regulated by central system,but also have their own biological clock regulation mechanism5)Core circadian clock genes showed conservative evolution during evolutionary process,reflect its importance functions.And different subterranean rodents species formed different adaptive evolution to dark environment.As a typical subterranean rodent,Mandarin voles had a significant 24-hour circadian activity rhythm,which was less affected by the photoperiod and melatonin.However for Brandt’s voles,a typical aboveground one,the 24-hour activity rhythm were activated by light,and melatonin played an important role in rhythm regulation.Circadian clock genes enrichment of the two voles shows the similar pattern.And evolutionary analysis of the seven core circadian clock genes also indicates that the circadian clock genes are conserved.Their expression in central and peripheral tissues also indicates that circadian clock genes play a important role in the regulation of rhythms.In conclusion,this study demonstrated that Mandarin voles could be used as a typical rhythm regulation model for subterranean rodents since the structure of the circadian clock genes was relatively conservative andactivity rhythm was regulated by the expression of the endogenous circadian clock genes,which is less involved in endocrine regulation,.Brandt’s voles show typical activity rhythms of species in high latitudes,which 24-hour activity rhythm was significant,affected by endogenous circadian clock genes,external photoperiod,endocrine participation. |
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