Molecular characterization of IgM, IgZ and IgD heavy chain genes in orange-spotted grouper(Epinephelus coioides)

Abstract

In the present study,the membrane-bound immunoglobulin M(mIgM),mIgD and secretory IgZ(sIgZ) have been identified by using RACE-PCR from the orange-spotted grouper,Epinephelus coioides.The mIgM and sIgZ are comparable with their counterparts in other teleost in terms of their heavy chain cDNA sequences and gene structures.The constant region of the mIgM had three CH domains,i.e.,CH1,CH2,CH3,and two transmembrane domains,TM1 and TM2.The TM1 exon is spliced into the regular donor site at the 3’ terminus of the CH3 domain.When comparing the amino acid sequence of mIgM with those in teleost fish,it is found that the grouper mIgM CH region shares highest identity with that in the oliver flounder(Paralichthys olivaceus),with CH1,CH2,CH3 having 53% 78% identity with those in the flounder,respectively.The complete transcript of the grouper mIgD contains 3 375 bp,including a 3 006 bp open reading frame,and the constant region of the mIgD consists serially of a μ1 domain,seven δ domains(δ1 to δ7),and a TM region.The degree of amino acid sequence identity,in domain-by-domain comparison with other teleost fishes,reveals an overall higher identity between the IgD in the grouper and that in mandarin fish(Siniperca chuatsi),with 1 to 7 having 75.5%,75.8%,65.4%,76.6%,88.1%,90.6%,82.8% identity,and the transmembrane domain having 82.7% identity,respectively.The sIgZ has a four exon structure in genome,with the introns being 222,129,and 458 bp in length,respectively.The semi-quantitative RT-PCR was performed to examine the expressions of IgZ,IgM,and IgD in various organs / tissues of the grouper.Transcripts encoding the mIgM were detected in all examined samples.IgD was mainly detected in pronephros,kidney,spleen and thymus,with week expression in intestine.IgZ was expressed in lymphoid tissues such as pronephros,kidney and spleen,with relatively low expression detected in gills,thymus and heart.