| Hibernation is the most dramatic example of phenotypic plasticity displayed by mammals.During mammal hibernation,there will be a lot of physiological changes,of which metabolic changes is the most remarkable.A global depression in metabolism and shifts in the fuel utilization from carbohydrate metabolism to lipid metabolism are found during hibernation.All of the physiological changes during hibernation are considered the results of differential gene expression.For example,studies on ground squirrels and bears reveal that many genes involved in metabolism are differentially expressed during hibernation.Especially,genes involved in carbohydrate catabolism are down-regulated during hibernation,while genes responsible for lipid ?-oxidation are up-regulated.Bats(order Chiroptera)are the only mammals capable of sustained flight.Because of bats' high energy consumption when active,winter hibernation is the optimal strategy for bats.However,studies on the expression changes of metabolism-related genes during hibernation are still lack.In addition,there are a few of studies on the adaptive evolution of hibernation-related genes though it is another important aspect of molecular mechanism of hibernation.The main reason leading to this is lack of a pair of closely related species,one that is able to hibernate,and one that cannot,and the greater horseshoe bat,Rhinolophus ferrumequinum,of which the northeast group strictly hibernate during winter while the southwest(e.g.Yunnan)group are active all the year,provides a good opportunity for this study.In this study,we compared the transcriptomes of liver,an important metabolic organ,of the torpid and active greater horseshoe bats from hibernating group using RNA sequencing method to reveal the differential expression of metabolism-related genes during bats'hibernation at the transcriptome level.In addition,since studies have shown that increased cell proliferation activities during hibernation is an important compensation mechanism to deal with cell injury and death,ZBED1 gene controlling cell proliferation was selected to study its expression changes during hibernation using quantitative PCR and adaptive evolution in the greater horseshoe bat using DNA cloning method.Thus we can determine the expression pattern of ZBED1 gene during hibernation,and adaptive divergence of this gene between hibernating and non-hibernating greater horseshoe bats.As a result,a total of 1358 genes were identified as differentially expressed between the liver transcriptomes of summer-active and winter-torpid greater horseshoe bats.In these genes,we found that most genes involved in metabolism were down-regulated during torpor,amounting to 60%of down-regulated genes in the torpid bats,while only approximately 1%of up-regulated genes in the torpid bats were metabolism-related genes.We also found that genes encoding many key rate-limited regulated enzymes of glycolysis(GCK,HK1,PFKFB3,PFKFB1,and PYGM)were expressed at lower levels during torpor,and genes encoding enzymes involved in gluconeogenesis and synthesis of glycogen(GYS1,GYS2 and G6PC)was over-expressed in the torpid bats.Those genes involved in fatty acid beta-oxidation,and fatty acid transport(ACOT12,ACOX1,EHHADH,SLC27A6,FABP5,FABP1)were up-regulated in torpid bats,while genes involved in lipid biosynthesis(AGPAT2,ACSF3)were down-regulated.These results account for metabolic depression during torpor and the shifts in the fuel utilization,i.e.switching from carbohydrate-to fat-based metabolism.Inaddition,we also found that many genes encoding heat shock proteins and genes related to stress response(HSPH1,DNAJB4,HSP90AA1,HSPA4L.HSPA8,HSPB7,HSPB1,HSP90B1,OXSR1)were over-expressed during torpor,which provided molecular evidences for the liver protective mechanism under various stresses,such as thermal stress and oxidative stress,during torpor.Finally,amounting to 20%of pathways significantly annotated by up-regulated genes were immune-related pathways,indicating enhanced immune function in the liver of hibernating greater horseshoe bats.This result also indicated that liver is also an important immune organ besides an critical metabolic organ.In addition,we investigated the relative mRNA expression levels of ZBED1gene.encoding an critical transcription factor associated with cell proliferation,in 80 samples from five tissues(heart,kidney,liver,brain and skeletal muscle)of the greater horseshoe bat across pre-hibernation,deep hibernation,and post-hibernation.Significantly increased expression(P<0.05)of ZBED1 gene during deep hibernation was only observed in the brain and skeletal muscle tissues,but not in the other tissues studied,indicated cell proliferation was enhanced in these two tissues.suggesting a tissue-heterogeneity of the cell proliferation.Higher stress was inferred suffered by these two tissues,and increased cell proliferation may compensate for tissue damages under stressful conditions during hibernation.In addition,in five tissues studied,the expression of ZBED1 gene was most in the skeletal muscle all the times,which indicated that cell proliferation is most intense in the skeletal muscle.Finally,we investigated the adaptive evolution of ZBED1 gene in the greater horseshoe bat and found 39 haplotypes of 46 individuals from 13 populations.There were 64 mutation sites among these haplotypes,of which seven mutations were consistent in the hibernating groups(northeast and central-east groups),while different from the non-hibernating groups.This indicated the genetic divergence between hibernating groups and non-hibernating groups,and sequence convergence of ZBED1 gene of hibernating groups.In the phylogenetic analysis,the result also showed that the haplotypes of the ZBED1 gene from the Northeast group and the Central-East group did not form two clades,but were clustered into one clade,with a high bayesian posterior probability(1.0),which is different from the mtDNA result.In addition,in the site model of positive selection analyses,several positively selected sites were detected;in the clade model,we found divergent selection pressures of the ZBED1 gene among the three mtDNA groups of the greater horseshoe bat,the average ?values of Northeast clade and the Central-East clade were 2.07667 and 1.07229,respectively,indicating a strong positive selective pressure on the Northeast group and a slight positive selective pressure on the Central-East group.Nevertheless,in the Southwest group,we detected no evidence for positive selection.These results suggested adaptive divergence of ZBED1 gene between hibernating groups and non-hibernating groups of the greater horseshoe bat. |
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