CDNA Cloning, Polymorphism Analysis And Tissue Expression Of MHC Iα And β₂m Gene Of Nile Tilapia （Oreochromis Niloticus）

Major histocompatibility complex (MHC) is a gene family whichrelates to immune response, and exists in all the vertebrates. MHC geneencodes a class of cell surface transmembrane protein that can binds toendogenous and exogenous antigens and then transfers them to Tlymphocytes, stimulating the body’s immune response. Based on thetechniques of homology cloning and RACE-PCR, MHC genes werecloned from Nile tilapia (Oreochromis niloticus). Cloning and sequencingmethod were used to study the MHC α and β2m cDNA polymorphism.qPCR assay were developed to assess the mRNA expression of MHC αand β2m in healthy tilapia’s tissues (brain, gill, liver, stestines stomach,kidney, heart, spleen, skin and muscle) and the tissues of tilapia infectedwith Streptococcus agalactiae. The main results are as follows:1. Cloning, polymorphism analysis and tissue expression of MHC αcDNA of Nile tilapiaThe full-length of MHC α cDNA was1553bp, and ORF (openreading frame) was1053bp in length. Sequence analysis showed that deduced protein sequence of MHC α included a leader peptide, a MHC-Idomain, a IGc1domain and a transmembrane region. The putative proteinof MHC α has26.86%-74.0%identity with those of other teleosts andmammals. Nile tilapia MHC α presents a close relationship withMaylandia zebra in the phylogenetic trees, with a high identity of74.0%.While the identity with mammals is low, such as mus musculus that isonly26.86%. Six different MHC α cDNA sequences were obtained fromsix individuals. They were named as allele Orni-UAA*0101,Orni-UAA*0102, Orni-UAA*0103, Orni-UAA*0104, Orni-UAA*0105and Orni-UAA*0106. They have multiple single nucleotidepolymorphisms which were generated from the base conversion or (and)transversion in the alleles, encoding different amino acid sequences. Thisindicated that MHC α cDNA has abundant polymorphism. qPCR resultsshowed that the highest expression level of MHC α was detected inspleen and heart, and moderate expression in gill, kidney and intestine,whereas the lowest expression was detected in brain, liver, stomach, skinand muscle. Different changes were found in the expression levels ofMHC α mRNA in the gill, kidney, heart and spleen of Nile tilapia afterchallenged with Streptococcus agalactiae. The findings provide usefulinformation for further investigation to select disease resistant molecularmarker of Nile tilapia using MHC Iα as candidate gene.2. Molecular characterization, polymorphism analysis and expression analysis of β2-microglobulin in Nile tilapiaTwo cDNA sequences of β2m gene of Nile tilapia were cloned. Thefull-length of two β2m cDNA were900bp and906bp respectively, ORFof two cDNA were351bp in length. The putative protein has37.07%-90.52%identity with those of other teleosts and mammals. Sixdifferent β2m cDNA sequences were obtained from six individuals. Theycan be divided into two main types, six subtypes. They were named asallele Orni-β2m*0101、Orni-β2m*0102、Orni-β2m*0103、Orni-β2m*0104Orni-β2m*0201and Orni-β2m*0202. QPCR results showed that thehighest expression level of β2m was detected in kidney, spleen and heart,moderate expression in gill and intestine, whereas the lowest expressionwas detected in skin and muscle. Different changes were found in theexpression levels of β2m mRNA in the gill, kidney, heart and spleen afterchallenged with Streptococcus agalactiae.