Genomic Structural And Phylogenetic Analyses Of T Cell Receptor Genes In The Alligator Sinensis

Involved in cellular immunity,T lymphocytes play important roles in animal adaptive immune system,such as undertaking signal transduction,activating B cells,and killing target cells,thus regulating immune response.To fulfill these roles,T lymphocytes depend T cell receptors(TCR)to recognize antigen-MHC complex.Four conventional TCR types of α,β,γ and δ genes have been shown to be present in all jawed vertebrates,forming membrane-bound heterodimers comprising αβ and γδchains,respectively.There has also been other atypical TCR gene types found in lower mammals,birds,amphibians and cartilaginous fish(TCRμ,VHδ(V genes in TCRα/δ locus homologous to IGHV),NAR-TCR).Although reptiles represent a key evolutionary position linking amphibians,birds and mammals,very few studies have been conducted on the TCR genes of this group of animals.Representing primitive reptiles,crocodilians are the closest relatives of birds,and are widely believed to have a strong immune system.In this study,we have determined the genomic structure and expressed repertoire of TCR genes in the Chinese alligator(Alligator sinensis),a reptilian species indigenous only to China.Firstly,we used the chicken TCR constant region sequences to conduct BLAST searches against the Chinese alligator whole genome shotgun sequence(WGS)assembly database.Based on the information obtained,PCR primers were designed and used to identify 17 BAC(bacterial artificial chromosome)clones from a Chinese alligator genomic BAC library,and 9 clones of them were fully sequenced.According to the sequencing data,the alligator TCRβ locus was shown to span approximately 500 Kb of genomic DNA,whereas the TCRy locus spans 109 Kb.TCRα/δ genes are distributed in two loci,where locus A spans about 1 Mb DNA,and locus B spanning approximately 400 Kb.Combining the transcriptional data obtained from the RACE(rapid amplification of cDNA ends)experiments with the Southern blotting results,we showed that the alligator TCRβ locus was organized as Vβ(39)-Jβ1-ψVβ1-Dβ2-Jβ2(11)-Cβ2-Vβ(4),whereas the TCRy locus was organized as Vγ(18)-Jγ(9)-Cγ.The TCRα/δ locus A is composed of V(104)-Dδ1(3)-Jδ1(3)-Cδ1-Jα1(91)-Cα1-VHδ(2)(including 80 Vα/δ and 26 VH8 gene segments),and the locus B contains V(39)-Dδ2(3)-Jδ2(3)-Cδ2-Vδ-VHδ-Jδ3-ψCδ3-VHδ(3)-Dδ4-Jδ4-Cδ4-VHδ(2)-Dδ5-Jδ5-Cδ5-Jα2(63)-Cα2(including 37 Vα/δ and 11 VHδ gene segments).RACE results showed that the locus A and B were adjacent to each other,as they could share variable genes in expression.The expression data showed no evidence supporting somatic hypermutation occurred in the expressed TCR genes.However there are a plenty of N-and P-nucleotides in the CDR3 region.The TCR8 chains could use at least 50%of the Vα segments and most of the functional VHδ segments,as well as multiple D segments,which all together could increase their CDR3 diversity significantly.Additionally,quantitative PCR showed that the expression level of γδ TCR was similar to that of αβTCR in peripheral lymphoid tissues,while the expression level of γδ TCR was higher in mucosal tissues.The alligator TCRβ and TCRy loci exhibit conserved syntenic structre with their genomic counterparts in other species.The alligator TCRα/δ loci contain a large number of VH8 segments,which are co-expressed with Cδ segments.The VHδ segments in the alligator TCRα/δ loci can be divided into three groups:the first group(VHδ Ⅰ)is highly similar with the VH8 segments in birds(VH Clan Ⅰ);the second group(VHδⅡ)is similar with the VHδ and Vμ.segments in platypus and opossum(VH Clan Ⅲ);the third group(VHδ Ⅲ)is most similar to crocodilian IGHV(immunoglobulin heavy chain variable region)(VH Clan Ⅰ,Ⅱ,Ⅲ),but the majority of VHδ Ⅲ members are pseudogenes.Based on sequence alignments,phylogenetic analyses and syntenic genes analyses,a hypothetic evolutionary process of the amniotes TCRα/δ loci was proposed.In this model,the common ancestor of reptiles,birds and mammals is thought to have one TCRα/δ locus,which contains VHδ Ⅰ and VHδⅡ segments(VHδⅠ may have occurred after the divergence of mammals and reptiles).Along with the evolution of mammals,the VHδⅡ genes were retained in monotremes and marsupials,but lost in placental mammals.Cδ segment duplications should have occurred in the ancestor of reptiles and birds to create more Cδ segments(Cδsegments are divided into three groups:CδⅠ,CδⅡ and CδⅢ in reptiles).Whereas birds retain VHδⅠand CδⅠ,and generated a new VHδ-Cδ locus by genomic duplication,VHδⅠ and VHδⅡ were maintained in crocodilians with the newly appearance of VHδⅢ.Apparently,there should have been a duplication of the entire TCRα/δ locus during the speciation of crocodilians.While the alligator TCRα/δlocus B is more structurally conserved,the locus A should have been duplicated from the locus B.However,the expression level of genes in the TCRα/δ locus A in lymphoid tissues was much higher than the genes in locus B.In summary,we have thoroughly analyzed the genomic structure,expressional patterns and phylogenetic characteristics of Chinese alligator TCR genes,and proposed a hypothetic evolutionary process of the amniotes TCRα/δ loci,which provide important clues for the study of origin and evolution of VHδ and TCR genes in tetrapods.