Cloning, Expression And Transference Analysis Of Oreochromis Niloticus γIFN

Oreochromis niloticus belongs to Percifomes, Cichlidae. It has been recommendedas an aquaculture fish species world-wide by FAO, and is now one of the mostimportant cultured fish in China. However, disease caused by Streptococcus agalactiaehas become a serious problem recent years, so it is necessary to carry out the researchabout the immune defense mechanisms of tilapia. γIFN is one of the importantnon-specific immune factors in veterbrates as well as in fish. This study cloned γIFNgene of the tilapia，and detected the expression levels in various tissues and organsbefore and after Streptococcus infection by Quantitative real-time PCR, in order tounderstand the role of γIFN in Nile tilapia immune response. In order to evaluate theeffectiveness of goldfish Tgf2transposon system in transgene fish a RFP genetransferred zebrafish was established. The main contents are as follows:1. Cloning and expression analysis of Oreochromis niloticus γIFN. γIFN is a majorcytokine regulating both innate and cell-mediated immune responses. It plays a keyregulatory role in the immune system, in addition to having a broad-spectrum antiviralfunction. In this study, the γIFN cDNA was obtained from nile tilapia, Oreochromisniloticus. The length of γIFN cDNA was690bp, and its ORF was621bp which encoded206amino acids. Bioinformatics analysis showed that nile tilapia γIFN gene encoding aprotein molecule that contains a signal peptide and a nuclear localization signal. Theprotein containing10α-helix,13β-fold, with a strong hydrophilicity. Homologyanalysis showed that it possessed the highest similarity with Epinephelus coioides(61.5%). Real-time quantitative PCR demonstrated that O. niloticus γIFN mRNA wasubiquitously expressed in all the tested tissues, and is highly expressed in spleen, butlowly expressed in muscle, heart and skin. Challenge of O. niloticus with Streptococcusagalactiae, results in a significant increase of the expression of γIFN in the spleen, skin,gills and kidney. The result implied that γIFN might play an important role in theimmune system. In addition, a fusion protein of γIFN was prepared with pET30c system,for further study of its function.2. Application of Tgf2transposon system in construction of RFP transgeniczebrafish. Improving the efficiencies of gene transfer and integration are the key points in transgenic fish research. Tgf2transposon system, which had been developed in recentyears, has been used to construct the transgenic zebrafish in this study. The Tgf2transposon donor plasmid pTgf2-EF1α-eGFP was re-constructed by replacement of thetransgenic elements, EF1α-eGFP with the myosin light chain2promoter (mylz2) andred fluorescent protein (RFP) gene, mylz2-RFP. The recombinant Tgf2transposon donorplasmid, pTgf2-Mylz2-RFP was supposed to sever as a transposon donor plasmid andwould specifically express red fluorescent protein in muscle tissue. The donor plasmidand the transposase mRNA were co-injected into zebrafish fertilized eggs. A total of972eggs were microinjected and803larvae survived, with a positive rate of76.6%.The red fluorescence (RFP) gene transgenic zebrafish (F0) was cultivated. Among them10individuals of sexual maturity transgenic zebrafish were mate with the wild-typezebrafish respectively. F1individuals that show red fluorescence on their body surfacewere obtained from one of the mating pair. The integration efficiency was10%. Themature RFP positive F1individual was then mating with the wild-type fish, and69%positive rate of F2was obtained. This study shows that transgene mediated by the Tgf2transposon could effectively improve the efficiency of gene transfer and integration,suggesting that Tgf2transposon would be applied in construction of transgenic fish aswell as in other transgenic fish research.3. Construction of γIFN gene expression vector and transferred into zebrafish. TheTgf2transposon donor plasmid pTgf2-hsp70-eGFP was re-constructed by replacementof the transgenic elements, eGFP with γIFN gene. The recombinant Tgf2transposondonor plasmid, pTgf2-hsp70-γIFN was supposed to express γIFN gene which wasdriven by the promoter of hsp70. The donor plasmid and the Tgf2transposase mRNAwere co-injected into zebrafish fertilized eggs. A total of3000eggs were microinjectedand1480larvae survived. The microinjected fish were cultivated and reached sexualmaturity.2out of8individuals produced positive F1when mated with wild-type fish.This study lays the foundation for further anti-disease ability analysis of the transgeniczebrafish, as well as for preparation of transgenic Oreochromis niloticus.