Cloning And Expression Patterns Of MHC â…¡ Gene And Analysis Of MHC â…¡B Gene Polymorphism In Nile Tilapia

The tilapia aquaculture industry has been severely threatened by streptococcois disease in recent years. Unfortunately, there are not the effective prevention and treatment measures against the disease. Breeding of disease-resistance strain of Nile tilapia(Oreochromis niloticus) maybe is a potential approach to resolve the problem. And the research of molecular heredity foundation of disease resistance is the premise of disease resistance breeding. Major histocompatibility complex (MHC) is a large genomic region serving as peptide receptors in the immune system of many vertebrates, and plays an important role in the initiation and maintenance of the body’s defensive immune. In the present study, the full lengths of MHC IIA and ⅡB genom and cDNA sequences were obtained from Nile tilapia by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. Then according to the sequences of MHC IIA and IIB cDNA, specific primers were designed and Quantitative real-time PCR was used to detect the expression levels in various tissues and organs. Methods of cloning and sequencing were used to understand the relationship between polymorphism of the MHC II genes and the disease resistance of Nile tilapia. This study has identified potential candidate genes for further investigation into the breeding of disease-resistant strain of Nile tilapia. The main results are as follows in this dissertation.1. MHC II genomes and cDNA cloning and sequences polymorphism analysis in Nile tilapiaThe full lengths of MHC IIA and ⅡB cDNA were obtained from Nile tilapia (Oreochromis niloticus) by homology cloning, RT-PCR and rapid amplification of cDNA ends (RACE) approaches. And the sequence of the full-length MHC IIA and ⅡB gene were able to obtained and available in Genbank (Accession No. JN967619and JN967618, respectively). The full length of MHC IIA gene was1614bp, and its cDNA was1076bp in length. Compared with other teleosts, the genomic structure of Nile tilapia MHC IIA contained4exons and3introns, and ORF was723bp, encoded240amino acids. The deduced amino acid sequence of the class IIA molecule shared25.4-64.5%similarity with other teleosts and mammals. However, the full length of MHC IIB was3143bp and its cDNA was1076bp in length. Six exons and five introns were identified in Nile tilapia MHC ⅡB, and ORF was750bp, encoded249amino acids. And the deduced amino acid sequence shared26.9-74.7%similarity with other teleosts and mammals.There are lots of alleles in the Nile tilapia MHC IIA and ⅡB genes. In this research, twelve and ten different cDNA sequences were identified in Nile tilapia MHC IIA and ⅡB genes (Accession No.:JN983472-JN983493), respectively.The identification of MHC genes may improve our understanding of vertebrate immunity and be helpful for breeding disease-resistant strain of Nile tilapia.2. Tissue expression analysis in Nile tilapia MHC Ⅱ geneTo determine the expression of MHC Ⅱ genes in healthy tilapia and bacteria challenged tilapia, primer pairs were designed based on the MHC ⅡB cDNA sequences of Nile tilapia MHC Ⅱ gene sequences. Quantitative real-time RT-PCR demonstrated that MHC Ⅱ genes were ubiquitously expressed in ten tissues of Nile tilapia, but the expression level was different among various tissues. The high levels of MHC IIA transcripts were detected in stomach, and gill and low expression in brain, and muscle. Besides, their transcriptional levels were significantly up-regulated in gill, kidney, intestine and spleen after intraperitoneal injection with Streptococcus agalactiae.3. MHC ⅡB gene polymorphism and its relationship with Streptococcus susceptibility in Nile tilapiaIn order to understand the relationship between polymorphism of MHC ⅡB gene and the disease resistance of Nile tilapia, the MHC ⅡB gene fragment was amplified from40susceptible Nile tilapia individuals and40resistant Nile tilapia individuals, using primer pairs of2b-snp-sf and2b-snp-sr. The length of the PCR products was between775bp-797bp. Among the PCR products of775bp-797bp nucleotides acid,265(33.2%) nucleotide positions were variable in resistant individuals; and in susceptible individuals, the variable positions was255(31.9%). Among the putative101amino acid sequence encoded by the amplification product,32amino acid positions were polymorphic in resistant individuals, and33amino acid positions were variable in susceptible individuals.7different MHC ⅡB alleles which encoded7different amino acid sequences were identified among441clones from80individuals. Most alleles were commonly found in both individuals. Alleles Orni-DAB*0201and Orni-DAB*0401were significantly more prevalent in resistant individuals than in susceptible individuals, with15.5%,5.6%in resistant stock and7.4%,1.8%in susceptible stock respectively. Allele Orni-DAB*0501was only found in resistant individuals.70%in resistant individuals presented2-4alleles, only30%individuals presented1allele. Whereas42.5%in susceptible individuals presented2-3alleles,57.5%individuals presented1allele. These results indicated that individuals possessed more MHC IIB alleles have stronger resistance ability to streptococcus disease. This study laid the foundation for disease resistance breeding of Nile tilapia.