The Cloning And Polymorphism Of MHC Gene From Epinephelus Akaara

The major histocompatibility complex molecules(MHCâ… and MHCâ…¡) are fundamental components of imumune response to foreign protein antigens,and occur extensively in all vertebrate organisms.In this study,using molecular techniques such as PCR,RT-PCR,SMART-RACE,SSCP,we report the cloning and polymorphism of three MHC genes from Epinephelus akaara;one of the important aquaculture species in China.The results are shown as follows:1.It was the first time that the complete cDNA sequences of three important non-specific immune genes,MHC classⅠαchain,MHC classⅡαchain and MHC classⅡβchain were obtained from the grouper.The whole MHC classⅠαchain cDNA sequence has 1720bp,which consists of:a 3'UTR(untranscribed region), promoters,a peptide-binding region(α1 andα2),an immunoglobulin-like region (α3),a transmembrane region,a cytoplasmic region and a 5'UTR.The MHC classⅡαchain has 1870bp,which consists of:a 3'UTR,promoters,a peptide-binding region(α1),an immunoglobulin-like region(α2),a transmembrane region,a cytoplasmic region and a 5'UTR.Likewise,the MHC classⅡβchain has 1350bp, which consists of:a 3'UTR,promoters,a peptide-binding region(β₁),an immunoglobulin-like region(β₂),a transmembrane region,a cytoplasmic region and a 5'UTR.2.The tesed three genes have classic MHC 3D molecular structure,and exhibit abundant polymorphisms in the peptide-binding region.Bothα1 andα2 domains of the classⅠαchain consist of an alpha-helical region and a beta sheet of four strands in antiparallel orientation,forming a deep gorge,while theα₃ domain consists of several beta strands.Bothα₁ andβ₁ of MHCâ…¡were composed of an alpha-helical region and a beta sheet of four strands in antiparallel orientation,whileα₂ andβ₂ of MHCâ…¡form a sandwich-like structure made of two antiparallel sheets,each consisting of several strands.3.The above 3 MHC genes are all expressed in brain,heart,spleen,liver, head-kidney,intestines,muscle,blood,thymus,gonad,gill and stomach,and higher levels of expression and polymorphism were observed in immune organs such as head-kidney,spleen and thymus.However,in organs such as gills and muscles that more likely to be frequently exposed to bacterial antigens,the expression and polymorphism levels are lower in MHCâ… while still elevated in MHCâ…¡.This pattern demonstrates the adaptability of MHC molecules in both expression and function.4.Using the SSCP technique,we established a protocol to separate the peptidebinding domain of MHC genes.By this protocol,one can enhance the detection efficiency of the MHC gene polymorphisms enormously.The SSCP primers and the protocol developed in this research will be helpful for the future study in detecting polymorphic sequences of MHC in groupers including E.akaara.5.Phylogenetic trees were also constructed based on the Neighbour-Joining(NJ) methods with the IGC region of these three genes.Similar tree topologies for all three genes confirm the monophyly of the Teleosteans,and also demonstrates that MHC-â… and MHC-â…¡diverged before ancestral fish gave rise to higher vertebrates. Therefore,the amino acid sequences of the IGC region of these three genes are potentially informative for phylogenetic studies in fishes.