Analyses On The Expression And Evolution Of Sperm-egg Interaction And Immune Related Genes In Amphibians Based On Transcriptome Data

Most of amphibians have a complex life cycle including aquatic and terrestrial development stages,they live and/or reproduce in various humid environments.In such environments occur various pathogens while external fertilization appears as a big challenge.Yet,reproduction and individuals'immunity appear to play determinant roles in the adaptation to diverse environments.Sperm-egg binding is necessary for successful fertilization in amphibians,and the interaction of proteins on the sperm and egg is vital for the species-specific binding.However,few studies were conducted on the sperm-egg binding protein in amphibians.Meanwhile,amphibians are vulnerable to the invasion of external pathogens and parasites because of the diverse habitats they living in.However,systematic studies on the immune related genes are limited in amphibians.Systematic study of amphibians'sperm-egg binding and immunity genes could enhance our understanding of the molecular basis of reproduction,disease resistance and the ability of adaption to diverse environments.To determine the expression and evolution of the sperm-egg binding and immune related genes in amphibians,we sequenced the total RNA from testis,ovary and skin tissues in twelve species,screened corresponding sperm-egg binding and immune related genes by annotation and performed selection analyses on obtained data combined with data from other species acquired from Genbank.1)In total,23 940 046-35 427 271 raw reads and 19 792 130-34 524 624 clean reads were obtained per sample of transcriptome from testis,ovary and skin.After assembling by Trinity and removing the duplication and redundancy,we obtained the unigene per sample with the N50 values ranging from 893 to 2663 bp.The mapping rate of clean reads onto unigenes ranged from 72.99%to 88.3%.Then we annotated the unigenes against several databases using BLAST method.The range of annotation rate against Nr database across different amphibians was 34.55%-49.04%.Those indicated the high quality of assembly and annotation.Furthermore,to understand the primary biological functions of unigenes,we annotated the unigenes against Gene Ontology(GO)database.The results of GO annotation indicated that the distribution pattern of GO terms in reproduction-related and immune-related transcripts were similar in studied species.2)Expression analyses of sperm-egg interaction showed that zona pellucida(ZP)was the mainly expressed sperm-egg binding gene family on the egg.To verify the quality of transcriptome sequences,we amplified the whole length of ZP3 genes as an example.The results showed that the transcriptome sequences were highly consistent with the experimentally amplified sequences.ZP gene family was also well expressed in testis and,at low level,in skin.The components of the ZP gene family expressed in amphibians' ovary were:ZP1,ZP2,ZP3,ZP4 and ZPAX.Among them,ZP1 gene was for the first time discovered in anurans.Components of ZP genes expressed in ovary were consistent across studied species,suggesting invariable expression among amphibians.They were similar with those found mammals whereas ZP components expressed in amphibian testis differed greatly from those found in mammals.Specifically,the majority of sperm-egg binding related genes expressed in mammals' testis were not expressed in testis of studied amphibians.We found that,only the genes?-1,4-galactosyltransferase(BGalT4),Zonadhesin(ZAN),A disintegrin and Metalloprotease 28(ADAM28),Sperm surface protein17(SP17)and Sperm associated antigen(SPAG)families were expressed in this organ.ADAM28 gene was not specific expressed in ovary and testis,it also expressed in skins;SP17 and ZAN are not specific expressed in testis,they also expressed in ovary,but not expressed in skins.Moreover,the expression of these genes was different among studied species.We screened numerous innate and adaptive immune genes.With a total of 19 antimicrobial peptides families(108 genes)and 195 other innate immune-related genes(including Cluster of differentiation),we found that majority of expressed genes were innate immune genes.Comparing the types of antimicrobial peptides(AMP)expressed in skin between three families,we found no statistically significant difference.Other innate immune genes were expressed in all the three sampled tissues,but with different components.The adaptive immune genes were mainly the major histocompatibility complex(MHC)and Cluster of differentiation(CD)genes.Expressed MHC class I genes were primarily the classical genes(MHC la).Expressed MHC class ? genes were mainly DRA,DQA,DMB,DRB and a chain of regardless position.Expressed sub-region loci of MHC genes were different across species.3)Evolutionary analyses of sperm-egg interaction and immune related genes showed that the ZP2,ZP3,ZP4 and ADAM28 genes all had the homologues.Moreover,the ZP3 genes had at least six distinct homologues genes,which suggested the influence of gene duplication on these genes.Based on the phylogenetic tree,we found that the ZP3 gene evolved firstly.And the ZP1 and ZP4 genes were highly homologous with a shared common ancestor.By detecting positive selection,we found that only ZP1 gene had two positive selection sites,suggesting the important roles of this gene in sperm-egg binding process in amphibians.Furthermore,no positive selection was detected in the complete of experimentally amplified ZP3 gene sequences within(sampled from different regions)and between species.For genes expressed in testis,only SPAG9 gene had one positive selection site.Generally,the roles of gene duplication were stronger than that of positive selection during the evolution of sperm-egg binding related genes.For the evolution of immune related genes,several innate immune genes had homologous genes,and most of the rest genes were independent genes.Positive selection analyses indicated that the antimicrobial peptides and most of innate immune genes were not subject to positive selection.This result suggests that only a few innate immune genes undergone gene duplication and positive selection alone or together,most of innate immune genes were extremely conservative.Among the MHC genes,both the MHC class I genes and the MHC class II genes had at least two gene copies,and both of them showed trans-species polymorphism.Moreover,a total of eight positive selection sites were detected in MHC I genes and one site was detected in the MHC II a chain.Significant positive selection signals were also detected in MHC II ?chain.In addition,it is the first evidence of positive selection of MHC IIDMB with nine positive selection sites.Meanwhile,the MHC IIDRB had 8 positive selection sites.Parts of the CD genes also had different homologous genes.The CD9 and CD81 genes had relatively close relationships,and far away from other CD genes based on the phylogenetic relationship.Furthermore,among the CD genes,CD9,CD81 and CD3g genes,which play important roles in adaptive immunity,had experienced positive selection,suggesting that evolutionary rate is faster in the adaptive immune genes than in the innate immune genes.In conclusion,we obtained high quality transcriptome data of testis,ovary and skin tissues from twelve amphibians.By analyzing the expression of sperm-egg binding related genes in amphibians,we found that the sperm-egg binding related genes were not specific expressed in gonads,some genes also expressed in skin tissues.The components of ZP gene families expressed in ovary were consistent across different amphibians.But the egg-binding genes expressed differently in diverse amphibians.Similarly,the expression pattern of immune genes differed across the twelve species.By analyzing the evolution of sperm-egg binding related and the immune related genes,we found that the influences of gene duplication on the sperm-egg binding related genes were stronger than the effects of positive selection;whereas,the innate immune genes were relatively conservative and the adaptive immune genes evolved relatively rapid.Our study provided a comprehensive supports for understanding the reproduction,disease resistance and the adaptive evolution of amphibians at molecular levels,thus offering a theoretical basis for the protection of amphibians.