Transference Of Tilapia Lysozyme-C3Gene In Zebrafish

Tilapia is one of the most important fish species cultured in China. In recent years,Streptococcus infections in tilapia outbreak in many areas in China includingGuangdong, Hainan, Fujian and Guangxi provinces, which restricts the sustainabledevelopment of China’s tilapia industry seriously. Therefore, it is urgent to improve thedisease resistance of tilapia. Transgenic technology provides a new way for fishbreeding. In this study, tilapia Lysozyme-C3, which showed higher activity againstStreptococcus agalactiae was chosen as the target gene, and the inducible Hsp70promoter of tilapia isolated previously in our lab was used to construct transgenicexpression vector. The expression of the transferred gene in zebrafish was observed.The results will be helpful for further research on construction of transgenicanti-disease tilapia.1. Comparisons of the lytic activities of several fish and shrimp lysozymesGenetic engineering recombinant technology was used to prepare recombinantproteins for three types of C-type lysozyme of tilapia, grass carp C-type and G-typelysozyme, and Penaeuse monodon C-type lysozyme. Turbidimetric method wasapplied to analyse their bacteriolytic activities against8species bacteria, includingStreptococcus agalactiae and Aeromonas hydrophila. The results indicated that all ofthe6recombinant lysozymes possessed bacteriolytic activities against eight kinds ofbacteria but various bacteriolytic activities were observed. Tilapia C3lysozyme had thestrongest bacteriolytic activity against Gram-positive bacteria Streptococcus agalactiaefollowed by grass carp C-type and G-type lysozyme. All recombinant lysozymes hadstrong bacteriolytic activity against gram-negative bacteria Pseudomonas aeruginosa.Tilapia C-type lysozyme had a stronger bacteriolytic activity against Aeromonashydrophila with the strongest bacteriolytic activity of C1compared with that of P.monodon C-type lysozyme, grass carp C-type and G-type lysozyme. The results of thisstudy could be used for the selection of lysozyme with strong bacteriolytic activitywhich could be applied in aquaculture production and the application of transgenictechnology to develop resistant varieties target genes. 2. Construction of Lysozyme-C3gene expression vectorTgf2expression vector pTgf2-Hsp70-eGFP contains tilapia Hsp70promoterwhich has been confirmed to have a strong drive activity. In this study, the openreading frame of tilapia Lysozyme-C3gene was obtained by high-fidelity PCRamplification and was used to replace eGFP gene in pTgf2vector in order to obtainpTgf2-Hsp70-C3expression vector. Then primers were designed to amplify theHsp70-C3-SV40fragment. After that the fragment was insert to pTgf2-Hsp70-eGFPvector, and expression vector pTgf2-Hsp70-eGFP-Hsp70-C3was successfullyconstructed for the transgenic zebrafish.3. Producing and testing of transgenic zebrafish with Lysozyme-C3The Tgf2-Hsp70-EGFP-Hsp70-C3expression vector and transposase mRNA wereco-injected into the eggs of zebrafish using microinjection method. About12hours afterinjection, heat shock treatment was performed at37℃for1h and then incubated at28℃for12h. After that, laser confocal microscopy observation was carried out to detect theexpression of fluorescence. Afterwards, zebrafish individuals which were detectedpositive fluorescence expression were picked out. A total of37tail Lysozyme-C3zebrafish survived while580eggs hatched successfully. The RT-PCR assay wasconducted to estimate tissue expression level of transferred gene. Results showed thateGFP gene was detected in liver, muscles, intestinal tract and skin of the F1transgeniczebrafish individuals while Lysozyme-C3gene was only detected in liver. Consistentwith this, both GFP and Lysozyme-C3protein were detected in liver of transgeniczebrafish by using Western blot, which meaned that the transferred gene could be ableto detected on both RNA and protein levels in liver. All these results confirmed that thetilapia lysozyme gene inheriting from parents expressed successfully in zebrafish F1generation. Comparison of the bacteriolytic activity of lysozymes in muscle and liver ofthe wild type and transgenic F1generation indicated that the transgenic zebrafish hadstronger activity. This implied that the transgenic zebrafish may have a stronger abilityto resist Streptococcus agalactia infection compared to wild type.