Molecular Evolution Of The Hemoglobin Gene Family In Vertebrates

Hemoglobin?Hb?is an erythrocyte protein that belongs to the globin superfamily,that plays an essential role in sustaining aerobic metabolism by transporting O₂ from the respiratory exchange surfaces into the cells of respiring tissues.Studies have shown that in addition to participating in aerobic metabolism,Hb is also involved in nitric oxide metabolism and antibacterial activity.In jawed vertebrates?gnathostomes?,Hb isa tetrameric protein composed of two?-chain subunits and two?-chain subunitsoriginating from a tandem duplicate event in the common ancestor of all gnathostomes.Gene diagnostics for a particular genomic region indicated that there are three hb gene clusters in vertebrates?the acronym for the two syntenic genes flanking the hb genes?.Vertebrates,as a subphylum of Chordata,are the largest in number,the most complex,and have the highest evolutionary status with a developed oxygen circulation and metabolic system.Previous studies about the evolution of the Hb gene family mainly focused on a single species or taxa of vertebrates,resulting in data on vertebrates as a whole being relatively scarce.The evolutionary profile of the hb gene family in all vertebrates needs to be mapped immediately.Meanwhile,gene conversion poses as a challenge in identifying the homologous relationship between hb genes.Currently,relevant studies are limited to mammals and birds and other groups have not been studied.Therefore,we used CHAP2?Cluster History Analysis Package?to conduct gene conversion analysis on vertebrates.This study selected 97 species of vertebrates?including 1 species of Cyclostomata,6 species of Chondrichthyes,1 species of Sarcopterygii,30 species of Actinopterygii,7 species of Amphibia,11 species of Reptilia,16 species of Aves,and 25 species of Mammalia?to identify the hb genes.We also conducted comparative genomics and evolutionary analysis to obtain a more comprehensive and systematic result.The main results are as follows:1.Identification and distribution of Hb gene family members in vertebratesIn order to accurately identify the hb gene sequences in the vertebrate genomes,we performed homologous predictions which were verified via Blast2 v2.2.Through statistics and classification of the obtained sequences,we finally identified 879 Hb gene sequences from 97 vertebrate genomes.The results revealed that there is little difference in the number of Hb gene family members between reptiles and birds?7members?,while the number of Hb gene family members in mammals varies greatly from 2?sloth,Choloepus hoffmanni?to 14?goat,Capra hircus?.Compared with other tetropods,except for the two-lined salamander?Rhinatrema bivittatum?,there are more members?about 12 gene family members?in amphibians.In fish,different groups showed significant differences.Compared with other fishes,Tetraodontiformes species such as Fugu?Takifugu rubripes?and Tetraodon?Tetraodon nigroviridis?;four Perciformes species namely Boleophthalmus pectinirostris,Periophthalmus magnuspinnatus,Periophthalmodon schlosseri and Scartelaos histophorus possess relatively fewer members of the Hb gene?about 4?,while Astyanax mexicanus,Pygocentrus nattereri?about 20?,and Esox lucius?about 30?possess relatively more members.Through synteny and homology analysis,combined with CHAP2 analysis,it can be inferred that the increased numbers of hb genes in these three species arose due to different gene duplication mechanisms.In addition to fish-specific genome duplication?FSGD?,gene tandem duplication and gene duplication of long fragments were also observed in Teleostei.2.Structural characteristics of Hb gene family members in vertebratesThe prediction results of conserved motifs and gene structures in vertebrate Hb genes showed that most of the vertebrate Hb genes contained 9 motifs?except the Chrysemys picta HBA gene containing 6 motifs and the Lepisosteus oculatus HBA gene containing 8 motifs?.Compared with gnathostomes,the agnatha Hb gene contained special motif5,motif7,and motif9,in line with previous studies showing that the agnatha Hb genes evolved independently.Additionally,three motif segments of the coelacanth?Latimeria chalumnae?were predicted to repeat at segment 1?including motif4,motif6,and motif2?,segment 2?including motif2 and motif1?,and segment 3?motif3 and motif2?and therefore produce longer protein coding regions?CDS?.Simultaneously,based on gene structure predictions,it was found that most vertebrate Hb genes mainly consisted of two introns and three exons,except for human?Homo sapiens?HBZ and HBQ genes consisting of only two exons and one intron and coelacanth Hb genes consisting of 9 exons and 8 introns,suggesting that Hb is conserved in different groups of vertebrates.3.Synteny analysis of the Hb gene family in vertebratesResults of synteny analysis revealed that the NPRL3 gene exists upstream of the Hb gene in all vertebrates,indicating that the Hb genes of cyclostomes and gnathostomes originated from whole-genome duplication in the last recent common ancestor of all vertebrates.In Teleostei,synteny analysis showed that the Hb genes are located on the LA locus produced by FSGD.In amphibians,the Hb genes are located on two different segments of the chromosome,one with the?-and?-globin genes and the other with the multiple?-globin genes.Although the?-globin gene undergoes gene transposition in amniotes,the amniote Hb gene synteny is more conserved than that in fishes and amphibians.Combined with the distribution of the Hb gene on the chromosome fragment of the coelacanth,it can be seen that the?-globin gene of amniotes is derived from the ancestral hb gene on the MN locus.4.Evolution rate analysis of the Hb gene family in vertebratesBased on the maximum likelihood method,PAML software was used to calculate the dN/dS of Hb genes among various groups of vertebrates.The results showed that post-FSGD,the teleost Hb genes on different loci possess distinct evolution rates.The Hb gene on the MN loci demonstrated a higher evolution rate than that on the LA loci.Meanwhile,we found that the dN/dS value of the globin gene was relatively low in amniotes.Moreover,we found that the dN/dS value of the Hb gene was the lowest in birds.Compared with genes without gene transposition,genes with one gene transposition had a higher evolution rate.Therefore,we hypothesized that the higher evolution rate of the amniote Hb gene is related to gene transposition.5.Analysis of X-orthology and N-orthology of the Hb gene family in vertebratesCHAP2 software was implemented for gene conversion analysis.We found that the amniote?-globin gene is affected by gene conversion,whereas in ray-finned fish and amphibians,there is no gene conversion.Combined with gene identification and evolutionary pattern,we noted that gene conversion tended to occur in amniotes with fewer Hb gene members.In mammals,the?-globin gene is affected by more gene conversion events than the?-globin gene and more gene conversion occurs between the HBB and HBD genes.Concurrently,the results of both X-orthology and N-orthology showed that in mammals,HBAT1,HBAT2,and HBAT3 are inclined to fuse.The chimerism of the hb gene in birds is less than that of mammals and CHAP2analysis results showed that their chimerism complexity is lower than that in mammals.There was no gene conversion in the avian?-globin gene,but the frequency was high in the two embryonic avian?-globin genes??and??.Compared with previous studies,we found that birds had more gene conversions in adult?-globin genes?genes?^H and?^A?.The pattern of gene conversion in reptiles was similar to that in birds.No gene conversion was found in the reptilian?-globin gene,but the frequency of occurrence was high in the reptilian?-globin gene.To summarize,our study enriched the basic data available on the Hb gene family through gene identification and analysis of Hb gene content and characteristics in 97vertebrate genomes.Through comparative genomic analysis,the structures,synteny,and evolution rates of Hb genes in vertebrates were determined.Additionally,we analyzed the homologous relationship between Hb genes caused by gene conversion and discussed the role and influence of gene conversion in the evolutionary process,laying a foundation for further analysis of Hb genes.