转“全鱼”溶菌酶基因大菱鲆的研究

用大西洋条鳕(Macrozoarces americanus)抗冻蛋白启动子opAFP promoter和牙鲆(Paralichthys olivaceus)c型溶菌酶基因构建“全鱼”溶菌酶基因元件opAFP—ly，首次应用电脉冲精子介导法将该基因片段导入到大菱鲆(Scophthalmus maximus)受精卵内，获得原代转基因大菱鲆。先后采用单因子方法和正交实验方法，重复实验确定了最适导入条件：脉冲电压为400V／cm，脉冲次数为5，脉冲宽度为25ms，外源DNA浓度为50mg/L。利用最适导入条件，实现基因元件大规模转移。采用PCR技术对1月龄的原代转基因大菱鲆仔鱼的染色体DNA进行分析，结果表明外源基因的整合率可达28％。

Electroporated sperm mediation of an "whole fish" lysozyme gene construct into Scophthalmus maximus

Lysozyme gene from the Japanese flounder(Paralichthys olivaceus),driven by the opAFP promoter,was first transferred to turbot(Scophthalmus maximus)eggs by electroporated spermmediation.The treated frywere sampled and analyzed by polymerase chain reaction(PCR).Single-factor experiment and orthogonal design experiment were used to determine the optimal parameters for the uptake of foreign DNA into the turbot sperm cells during electroporation.The electroporation conditions(field strength of400V/cm,5pulses,pulse length of25ms and foreign DNA concentration of50mg/L)was applied for mass gene transfer.As high as28%of treated1-mouth-old turbot fry contained foreign genes by PCR analysis.