Electroporated Sperm Mediation Of An "All Fish" Lysozyme Gene Construct Into Scophthalmus Maximus

An "all fish" lysozyme gene was constructed with a c-type lysozyme gene from the Japanese flounder and was drived by antifreeze protein (AFP) gene promoter from ocean pout. Integration of the objective gene was conducted by electroporated toubot sperm into turbot eggs for mass gene transfer. The total RNA was extracted from peripheral mononuclear (PMNC) of Japanese flounder and c-type lysozyme gene as objective gene was obtained by the method of RT-PCR. The objective gene was inserted into the expression vector(opAFP-V)to construct "all fish" lysozyme gene. It's linearized fragments were collected and transformed into turbot eggs by electroporated sperm mediation. Single-factor experiment and orthogonal design experiment were designed to optimize the parameters for the integration of foreign DNA into the turbot sperm cells during electroporation. The survival rate of gastrula, hatching rate and gene transfer were considered. The electropration manipulation parameters (field strength of 400 V/cm, 5 pulses, pulse length of 25 ms and foreign DNA concentration of 50 ug/ml) were applied for mass gene transfer. It turned out that about 28% of treated turbot fry (1 month) contained foreign gene detected by PCR analysis. The preliminary study showed that electroporation sperm mediation was an valid way for transgene study of turbot in practice.