| The phenomenon of circadian rhythm exists in almost all living organisms and is one of the most basic characteristics of living organisms.It can regulate a variety of physiological and biochemical processes in organisms and enable organisms to adapt to changes in the external environment.Bats are one of the most mysterious research groups in mammalian research because of their unique nocturnal habits.Our study focused on the Asian particolored bat(Vespertilio sinensis)as the research object,mainly using transcriptome sequencing technology,supplemented by fluorescence quantitative PCR verification experiments,combined with adaptive evolution analysis methods,etc.,to carry out a systematic study of the expression genes in the brain region of Asian particolored bat.First,we set up four sampling time points with equal time intervals in one day,respectively collected Asian particolored bat brain tissue under different physiological activity states and performed transcriptome sequencing and analysis,and then obtained four gene set with different expression levels.These four sampling time points correspond to the four physiological states of bats,namely rest,sleep,wake,and activity.A total of six sets of differentially expressed genes were obtained through pairwise analysis and comparison of gene sets of four time points.In the comparison of the six groups of differentially expressed genes,we focused on the analysis of the two groups of differentially expressed gene sets with large differences in physiological activity states,namely after feeding vs before feeding(Rest vs Wake)and Sleep vs Activity.The enrichment results of the two comparisons showed that:compared with before feeding,genes with high expression levels in Asian particolored bats were significantly enriched in pathways related to drug metabolism;the genes with high expression levels in the wake state were significantly enriched in a variety of internal secretion physiological pathways;compared with the active state,genes with high expression levels in Asian particolored bats were significantly enriched in ribosome-related pathways in the sleep state;while compared with the sleep state,the Asian particolored bats in the active state had a variety of signal transduction pathways and circadian rhythm-related pathways were enriched by genes with high expression levels.The above results indicate that there are obvious differences in the physiological processes dominated by genes in bat brain regions in different physiological states.Second,we performed trend analysis and rhythmic expression gene analysis on all differential genes co-expressed at four time points in the bat brain region,and screened out a significant expression trends 6(27 genes)and 6 rhythmically expressed genes.According to the trend analysis results,the 27 included in module 6have a significant expression trend in one day: the expression levels of these genes are in the middle in the rest state of the bat,and the gene expression levels gradually decrease to the lowest in the sleep state,and then the expression levels of these genes increased gradually,and finally reached the highest level in the active state.Therefore,we speculate that the change trend of gene expression contained in module 6 is consistent with the biological habits of bat nocturnal emergence.Then,we carried out enrichment analysis on the genes in module 6,and the results showed that these genes were significantly enriched in various endocrine,signaling pathways,and circadian clock-related pathways.The above results reveal that the bat nocturnal activity pattern requires changes in the expression of genes involved in related physiological activities.In addition,according to the trend analysis and enrichment analysis results,we also found an important circadian clock gene,Per1 gene involved in multiple circadian rhythm-related terms and pathways,which may be an important circadian clock gene in the brain region of Asian particolored bats.Finally,we conducted a detailed adaptive evolution analysis of the Per1 gene in the bat brain region,and constructed two adaptive evolution datasets.One is a dataset of 23 mammals with different circadian activity patterns obtained by alignment based on the transcriptome sequence of the bat Per1 gene,with a total of 765 bp.The other is a dataset based on the full-length sequence of the coding region of the Per1 gene,with a total length of 3927 bp,including 22 mammals with genomic information that exhibit different circadian activity patterns.From the tree construction results,the phylogenetic trees constructed by the two datasets have high support,and are basically consistent with the real species tree,and show similar topological structures,indicating that the gene is evolutionarily conservative.However,by performing multiple model positive selection analyses on the Per1 gene,the results showed that the ω values of all nocturnal animals,namely the bat clade and the rodent clade were significantly larger than the ω value of the diurnal species clade,indicating that the Per1 gene is in the nocturnal species are subject to strong selective pressure.This study using the Asian particolored bats as the main research object,and systematically studies the rhythmic expression genes in its brain region by means of transcriptome sequencing technology,fluorescence quantitative PCR experiments and various molecular evolution analysis methods.The gene expression and dominant physiological process at different time nodes are different,and Per1 may be an important biological clock base.Our results help to reveal the physiological basis and evolutionary mechanism of the special nocturnal habit of bats,and provide new data references and research ideas for other non-model organisms related to circadian rhythms. |
