Development Of Molecular Markers And Cloning Of C1Inhibitor Gene In Tilapias

Tilapias, members of the family Cichlidae, are an important economicfish species in the world. Cichlid fishes (Cichlidae) have been widely usedto study evolutionary biology of fish as model species because of theirdiverse characteristics of morphology, behavior and ecology. As the factthat molecular markers are not adequately available for researches ofgenetics and evolutionary biology and disease of tilapia recently broke out,in the present studies, we developed general microsatellite molecularmarkers (SSR) for Nile tilapia (O. niloticus) and blue tilapia (O. aureus),and determined the complete mitochondrial genomes of three species oftilapias (O. niloticus, O. aureus, black-chin tilapia (Sarotherodonmelanotheron)), as well as conducted cloning and expression analysis ofcomplement1inhibitor gene (C1INH) in O. niloticus. The aims of thestudies are:(1) to provide molecular marker tools for studying genetics andevolutionary biology in tilapias,(2) to make theoretical basis for improvingaquaculture industry in tilapias. The details of those studies are presented asfollows:1. Molecular markers development of tilapia I: Determination ofcomplete mitochondrial genome of three species of tilapiasCichlid fishes have played an important role in evolutionary biologyand aquaculture industry. Nile tilapia (Oreochromis niloticus), blue tilapia(Oreochromis aureus), Mozambique tilapia (Oreochromis mossambicus)and black-chin tilapia (Sarotherodon melanotheron) are useful models instudying evolutionary biology within Cichlid fishes, and are also mainly cultured species in aquaculture industry with great economic importance.In this section, the complete nucleotide sequence of the mitochondrialgenome for O. niloticus, O. aureus and S. melanotheron were determinedand phylogenetic analyses from mitochondrial protein-coding genes wereconducted to explore their phylogenetic relationship within Cichlids. Themitogenome is16,625bp for O. niloticus,16,628bp for O. aureus and16,627bp for S. melanotheron, which contain the same gene order and anidentical number of genes or regions with the other Cichlid fishes,including13protein-coding genes, two rRNA genes,22tRNA genes andone putative control region. Phylogenetic analyses using three differentcomputational algorithms (maximum parsimony, maximum likelihood andBayesian method) show O. niloticus and O. mossambicus are closelyrelated, and O. aureus has remotely phylogenetic relationship from abovetwo fishes.2. Molecular markers development of tilapia II: Development ofgeneral SSR markers for Oreochromis niloticus and O. aureusNile tilapia (Oreochromis niloticus) and the hybrid of Nile tilapia (♀)×Blue tilapia (♂) are currently cultured fishes in tilapia industry. However,their genetic assessment and germplasm identification need more molecularmarkers. The microsatellite markers have been developed for O. niloticusbut the commonality for two species was not assessed.In this study, the SSR markers were developed based on the DNA ofNile tilapia.256positive clones were detected and207clones weresequenced.159of207were found with repeat motif and96pairs ofprimers were designed. We evaluated the utility of these loci using31samples of wild Nile tilapia and25samples of wild blue tilapia. Finally,27general SSR markers were obtained from96pairs of primers designed. Themean allele number was5.78(Nile tilapia) and5.04(Blue tilapia); themean observed heterozygosity was0.6022and0.4199; the mean expectedheterozygosity was0.7436and0.5928, respectively. There were4loci deviating from Hardy–Weinberg Equilibrium (HWE) in the Nile tilapiapopulation and7in the blue tilapia population (P<0.05). The results in thissection indicated these loci would be useful in indentifying geneticresources and studying population genetics in tilapia.3. Cloning and expression analysis C1INH gene In Nile tilapiaTilapia is one of important cultured species in china and it has beenrarely diseased. However, the diseases of tilapia have recently broken outaccompanied by the technology development of high density aquaculture intilapia. At present, researchers want to solve the problem through thedevelopment of antibacterial vaccine and the study of the immune systemof tilapia. The complement system, which is consisted of a series ofproteins, is one part of the immune system. Its main functions believed aredestroying and clearing up the antigen and cell binding with antibody. Thecomplement1inhibitor (C1INH) was cloned and its expression profileswere analyzed in this chapter.We designed primer based on the similar species’ C1INH gene and thenpartial sequence C1INH was obtained, finally,3’RACE and5’RACEtechnologies were used to obtain the full sequence information of C1INH.The cDNA length of this gene was2,128bp with101bp5’UTR,236bp3’UTR and1791bp open reading frame (ORF) coding597amino acids.The predicted protein weight was67KDa. The results found that thecomplement C1inhibitor factor were differently expressed within differenttissues, the expression level in the liver tissue is the highest, followed by inthe gills, heart, brain and kidneys. This result of the expression in differenttissues is consistent with the expression pattern of C1INH genes in miceand humans.