Identification And Functional Analysis Of Acetylcholine Receptor And IL-17 Genes In Pinctada Fucata Martensii

The cholinergic immune regulatory system mediated by neurotransmitter acetylcholine play an important role in inhibitting the release of inflammatory cytokines,which invovled in regulating the immune response of organisms.In this study,the genes of nicotine acetylcholine receptor(nAChR)and inflammatory cytokine interleukin-17(IL-17)which are important constituent molecules of cholinergic immune regulatory system in P.f.martensii were identified by analyzing published genomic sequences,and their functions were preliminarily analyzed by various bioinformatics and molecular biological methods.The main results are as follows:1.Identification and comparative analysis of acetylcholine receptorsCompared with vertebrates(Homo sapiens and Danio rerio),nAChR gene was expanded in in molluscs,annelids,and coelenterates.Compared with gastropods and cephalopods,nAChRs expanded most significantly in five species of bivalves(Pinctada fucata martensii,Crassostrea gigas,Mizuhopecten yessoensis,Bathymodiolus platifrons,Modiolus philippinarum)ranging from 99 to 217,of which P.f.martensii was the most abundant with 217.Intron-exon structure analysis revealed that the intronless nAChRs existed widely in molluscs.There were 120 intronless genes in the nAChRs gene of P.f.martensii.In addition,there are a large number of tandem repeats of nAChRs genes in molluscs,140 nAChRs genes in P.f.martensii are tandem repeats.In summary,there has been a large expansion of nAChRs in molluscs,probably due to reverse transcriptional translocation and tandem replication.2.Analysis of sequence variation of nAChRsThe sequence length and domain combination of nAChRs genes in P.f.martensii are diversified,and the ligand-binding domain(LBD)sequence has a large degree of variability.Multiple sequence alignments showed that Cys-loop sequences in LBD of P.f.martensii were less conservative.27 ? nAChRs were identified in P.f.martensii,and 15 of them had mutations or deletions in key amino acids at the ligand binding site,and the same phenomenon was observed in C.gigas.Homologous modeling showed that there were 6 genes lacking the ?1 helix and 5 genes lacking the?-fold,in which Pm448.7,Pm10013868 and Pm10016494 simultaneously deleted the?1 helix and the ?-sheet.The M4 and MX helices of the transmembrane region of Pm10031648 were deleted.In addition,the Tyr residues in 15 ? nAChRs Loop A and Loop C were deleted or replaced.For example,the Tyr residues in the Loop A structure of genes such as Pm10031648,Pm448.7,and Pm10016494 were deleted.3.Functional analysis of nAChR:Transcriptome analysis of different stages of development of P.f.martensii,C.gigas,M.yessoensis,B.platifrons and M.philippinarum revealed that four nAChRs in P.f.martensii were highly expressed in fertilized eggs,and 32 nAChRs were highly expressed in D-type larvae,and some nAChRs are highly expressed at other developmental stages.The expression pattern of nAChRs in C.gigas and M.yessoensis was similar to that of P.f.martensii.Similar to the transcriptome analysis of different tissues of C.gigas,M.yessoensis,B.platifrons and M.philippinarum,nAChR is highly expressed in the mantle and gill of P.f.martensii.The full-length cDNA sequences of five nAChRs genes(Pm10012059,Pm10024952,Pm10020990,Pm10008204,and Pm93.653)were obtained by gene cloning.qRT-PCR analysis showed that all five genes were highly expressed in the blastocyst and gastrula stage.The results of in situ hybridization of Pm10012059,Pm10024952,Pm10020990,and Pm10008204 showed that there were obvious positive signals in the blastocyst,gastrula,and trochophore.The nAChRs genes(Pm10029677,Pm10029677,and Pm10000530)specifically expressed in the late developmental stages had significant positive hybridization signals in the soft tissue of D-larva,eyed larvae,and spat.The expression profiles of mantle edge(ME)at 0 h,4 h,6 h,12 h,24 h,36 h and 48 h after shell injury show that 99 nAChRs(rpkm > 1)were detected.Compared with control group(0 h),41 nAChRs were up-regulated significantly,15 genes were up-regulated significantly at 4 h and 6 h,10 genes were up-regulated significantly at 36 h and 48 h,and 4 genes were up-regulated significantly at 4 h,36 h and 48 h after shell injury.The intron-exon structure analysis of 99 nAChRs(rpkm > 1)revealed that there were 58(58.6%)nAChRs genes with0-2 introns.4.IL-17 gene identification and functional analysis:Compared with vertebrates(H.sapiens and D.rerio),the IL-17 gene expands in molluscs.The largest number of P.f.martensii,there are 15.Phylogenetic analysis found that IL-17 was infiltrated in invertebrates.Intron-exon structure analysis revealed the presence of a large number of intronless IL-17 genes in P.f.martensii.In addition,7(47%)of P.f.martensii were tandem repeat genes.Transcriptome analysis of different tissues and developmental stages of P.f.martensii showed that PmIL-17 expression has tissue and developmental stage specificity.PmIL-17-2 and PmIL-17-4have similar expression patterns,which are highly expressed in the eyed larvae and mantle central,and PmIL-17-7 are highly expressed in the trochophore and gonads.The PmIL-17-10 gene is highly expressed in juvenile and gill,PmIL-17-12 is expressed with higher amounts in D-larva,gill,and hepatopancreas,while PmIL-17-13 is highly expressed in gastrula and mantle edge.The above studies indicate that the nAChR and IL-17 genes undergo a large expansion in P.f.martensii,which may be due to tandem repeats and reverse transcriptional transposition,and gene expansion leads to sequence variation and functional differentiation of nAChRs and IL-17.