Analysis Of Evolution And Expression Profiling Of Immune-related Genes In Amphioxus

Amphioxus is existing basal chordate,belonging to Cephalochordates,Amphioxichthyes,Amphioxiformes,and Amphioxidae.The existing amphioxus mainly consists of three genera(Branchiostoma,Epigonichthys,and Asymmetron),Branchiostoma of which includes the most species,and is widely used as a proxy for common ancestors of cephalochordate and vertebrates.Therefore,amphioxus is an ideal model for investigating the scientific problems involving the evolution of chordates,including comparative immunology and evo-devo biology.Currently,with the genome and transcriptome sequencing of multiple amphioxus species,many scientific problems to be solved and interesting studies will be conducted,e.g.the molecular phylogenetics of amphioxus and analysis of immune-related gene from perspectives of genome-wide sequence variation and gene expression in amphioxus,based on big omics,etc;additionally,with the help of these plentiful genetic resource,developing tools widely used in amphioxus research to promote development of related fields,such as molecular marker development,selection of reference gene used in quantitative real-time PCR(qRT-PCR),and design of various probes.Comparing with other animal groups,the existing species of amphioxus are less specie numbers,complexity and diversity in taxonomic units.However,amphioxus phenotype among different species are very similar,and the different species of amphioxus distributed dispersedly in the world.Therefore,systematic classification of amphioxus is a challenging task.In addition to those above mentioned,the amphioxus are sister group of both vertebrates and urochordates,and their evolutionary status locate at base of Chordata.Most studies thus focused on molecular phylogenetic relationship between amphioxus and other animal groups,such as vertebrates,deuterostome and urochordate,but molecular phylogenetic relationship within the amphioxus remains largely unknown.Phylogeny within amphioxus is blurry,which has brought some difficulties for conducting other related studies,such as the evolution and expression analysis of immune-related genes in amphioxus.In previous studies,only phylogenetic trees based on mitochondrial DNA(mtDNA)were used to explore molecular phylogeny among multiple amphioxus species(>three species),and infer their speciation history.So far,no studies based on big-omic data have been conducted to investigate molecular phylogeny and speciation history within the multiple amphioxus species,and to discuss the evolutionary characteristics of protein-coding gene(PCGs)sets among them.This study first determined phylogenetic relationships among amphioxus,and then selected the amphioxus species locating at suitable evolutionary status,and conducted comparative genomic analysis to explore the evolutionary characteristics of immune-related genes within amphioxus from the perspective of sequence variation;in addition,mechanism of immune response was investigated based on expression level of immune-related genes in amphioxus.Here,transcriptome of Branchiostoma japonicum was sequenced,as well as filtering transcriptomic data(Asymmetron lucayanum)were downloaded.These two transcriptomes were de novo assembled,and then through combining with genomic and transcriptome sequences of other three species of amphioxus(B.belcheri,B.lanceolatum,B.floridae),and genomic sequences of other 14 species containing vertebrates,we constructed 19-way maximum likelihood(ML),Bayesian(BI)and PhyloBayes trees.The trees show a consistent topology among three algorithm,and each node label presented a high bootstrap value(>80%).Molecular dating analysis including several time constraints revealed a divergence time of?115 Mya(million years ago)for Asymmetron-Branchiostoma;?102 Mya for B.belcheri+B.japonicum-B.lanceolatum+B.floridae;?0.90 Mya for B.lanceolatum-B.floridae;?0.74 Mya for B.belcheri-B.japonicum;these divergent time frame is nearly coincident with Tethys formation caused by earth plate activity,indicating that the amphioxus speciation are related to paleogeologic event.In addition,from?587 Mya to?540 Mya,less than 50 Mya,the origin of vertebrate,cephalochordate,deuterostome and urochordate subphyla provided some evidence for the existence of Cambrian explosion from the perspective of the molecular clock.We analyzed the evolutionary characteristics of amino acid domains among five species of amphioxus,and then identified 4,814 ancient amphioxus domains,and 366,492,361,427,255 species-specific domains in A.lucayanum,B.belcheri,B.floridae,B.lanceolatum and B.japonicum,respectively.GO(Gene ontology)enrichment analysis revealed these specific domains mainly involving immune response,apoptosis,and lipid metabolism,indicating that pathogens and changes in metabolic requirements are an important pressure for driving the evolution of amphioxus species-specific domains.The phylogenetic tree showed overall slow evolution rate in most amphioxus,but several involved in innate immunity,stand out as evolving relatively quickly.This may be due to the lack of an developed adaptive immune system and the relatively high levels of bacteria in the inshore waters as cephalochordates inhabit.Amphioxus is ideal animal for studying comparative evolution of tissues,organics and immune system for vertebrates.qRT-PCR is an accurate and reproducible method for gene expression analysis.Choice of reference genes(RGs)is necessary to obtain reliable qRT-PCR results for target genes.In this study,we analyzed thirteen candidate RGs in different developmental stages and tissues in amphioxus.Four independent(geNorm,NormFinder,BestKeeper and delta Ct)and one comparative algorithms(RefFinder)were used to evaluate the RG stability in qRT-PCR.The results showed top two stable RGs were the following:(1)S20 and 18S in thirteen developmental stages,(2)EF1A and ACT in seven normal tissues,(3)S20 and L13 in challenged intestine and hepatic caecum with lipopolysaccharide(LPS),and(4)S20 and EFIA in challenged gill with LPS.The expression profiles of two target genes(EYA and HHEX)in thirteen developmental stages were analyzed,which confirmed the reliability of chosen RGs.This study identified optimal RGs that can be used to accurately measure gene expression under these conditions,which will benefit evolutionary and functional genomics studies in amphioxus.It is an important step for understanding of immune mechanism in amphioxus by exploring changes in genome-wide gene expression profiling between post-and pre-immunization of amphioxus.To understand the immune responses of amphioxus to LPS,five ribosomal RNA(rRNA)-depleted libraries of amphioxus were constructed,including one control(0 h)library and four treatment libraries at 6,12,24,and 48 h post-injection(hpi)with LPS.The transcriptome of Branchiostoma belcheri was analyzed using strand-specific RNA-seq.A total of 6161,6665,7969,and 6447 differentially expressed genes(DEGs)were detected at 6,12,24,and 48 hpi,respectively,compared to expression levels at 0 h.We identified amphioxus genes active during the acute-phase response to LPS at different time points after stimulation.Moreover,in order to better visualize the resolution phase of the immune process during immune response,we identified 6057 and 5235 DEGs at 48 hpi by comparing with 6 and 24 hpi,respectively.Through qRT-PCR analysis of 12 selected DEGs,we demonstrated the accuracy of the RNA-seq data in this study.Functional enrichment analysis of DEGs demonstrated that most terms were related to defense and immune responses,disease and infection,cell apoptosis,and metabolism and catalysis.Subsequently,we identified 1330,485,670,911,and 1624 time-specific genes(TSGs)at 0,6,12,24,and 48 hpi.Time-specific terms at each of five time points were primarily involved in development,immune signaling,signal transduction,DNA repair and stability,and metabolism and catalysis,respectively.As this is the first study to report the dynamic change in gene expression profiles of amphioxus following LPS challenge at multiple time points,it provides gene expression information for further research into the evolution of immunity in vertebrates.We still do not know about how to microRNAs(miRNAs)regulate antiviral immune response of amphioxus.In this study,we sequenced six small RNA libraries(three biological replicates were included in the treatments challenged by the viral mimic,poly(I:C)(pIC)and control groups,respectively)from Branchiostoma belcheri.A total of 151 known miRNAs,197 new miRNAs(named novel_mir,including nine conserved miRNAs)were identified by deep sequencing from the six libraries.We primarily focused on differentially expressed miRNAs(DEMs)after pIC challenge.Next,we screened a total of 77 DEMs,including 27 down-and 50 up-regulated DEMs in response to pIC challenge.Furthermore,we used real-time quantitative PCR(qRT-PCR)to verify the expression levels of 10 randomly selected DEMs.Target genes likely regulated by DEMs were predicted,and functional enrichment analyses of these targets were performed using bioinformatics approach.MiRNA targets of DEMs are primarily involved in immune response,diseases,cancer and regulation process,and could be largely linked to 14 immune-related signaling pathways,including NF-kappa B,NOD-like receptor,RIG-I-like receptor and endocytosis.The present study for the first time explores key regulatory roles of miRNAs in the innate antiviral immune response in amphioxus,and will provide insight into the molecular basis of antiviral immunity and evolution of immune-related miRNAs.