摘要
用大西洋条鳕 (Macrozoarcesamericanus)抗冻蛋白启动子opAFPpromoter和牙鲆(Paralichthysolivaceus)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 (Scophthalmusmaximus)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 %。
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.
出处
《海洋科学》
CAS
CSCD
北大核心
2004年第5期8-14,共7页
Marine Sciences
基金
青岛市科技局资助项目
