Phylogenetic Analysis Of The Immunoglobulin Heavy Chain Genes In Aves

Immunoglobulins(Igs)are pivotal molecules of animal adaptive immune system in jawed vertebrates.Although evolutionarily just as ancient as IgM,it has been thought for many years that IgD(encoded by the ? gene)is absent in birds.Restricted by techniquical means,previous studies on avian immunoglobulins mainly focused on a few species,which indicates that the above conclusion might be questionable.In this thesis,we have comprehensively analyzed the immunoglobulin heavy chain isotypes expressed in the ostrich,emu and zebra finch.The ostrich(Struthio camelus),representing the most primitive living aves,belongs to the Ratitae order,which are phylogenetically close to the crocodilians.Based on the available genome data and high-throughput transcriptome sequencing of immune-relates tissues(spleen,small intestine and the bursa of Fabricius),we demonstrated that the ostrich possessed a functional ? gene,two ? gene(?1 and?2)and two ? gene(?1 and ?2),in addition to the a gene.The full length cDNAs of these genes were amplified using 3' RACE and 5' RACE,and the phylogenetic trees were constructed by using the amino acid sequences of immunoglobulin heavy chain constant regions.Northern Blotting and Quantitative RT-PCR(qPCR)reactions were performed to analyze the expressed IgD form and its expressional characteristics in different tissues at the RNA level.Combining BAC library screening,genome walking and long PCR amplification,we showed that the partial ostrich IgH gene locus was organized as?1-?-?-?2.Furthermore,through BLAST searches against the recently released 48 bird genomes,?sequences were clearly shown to be present in many other avian species,demonstrating that the ? gene is widely distributed among birds and is only absent in certain avian species.To further validate the above findings,we chose emu(Dromaius novaehollandiae)and zebra finch(Taeniopygia guttata)as subjects to analyze their Ig classes.Closely related to ostrich,emu is another primitive bird species.Our results showed that the emu also expressed six IgH isotypes,including two ?,two ?,one ? and one ? genes.Phylogenetic analyses revealed that both subclasses of the emu ?,and ?genes were orthologous to those of ostrich,respectively.Zebra finch belongs to the Passeriformes,which is the most successful bird order in terms of species diversity and numbers.Only three genes encoding immunoglobulin heavy chain isotypes,IgM,IgA and IgY,were identified in the zebra finch.Phylogenetic analyses suggest that the subclass diversification of both ? and ? genes occurred during the early stages of bird evolution but after their divergence from nonavian reptiles.In summary,we demonstrated that an IgD-encoding gene is functionally transcribed in the ostrich and is also present in many other bird species,thus invalidating the commonly held notion that the ?gene is absent in birds.Additionally,this study also suggests that the common ancestors of modern birds should have at least six IgH isotype encoding genes including ?1,?2,?,?1,?2 and ?;and some of these genes have been evolutionarily lost in modern birds.Our findings provide significant new clues to enhance our understanding of IgH gene evolution in tetrapods.