本实验研究了大黄鱼肌肉生长抑素前肽基因对红鲤的促生长作用.分别通过RT-PCR和PCR从大黄鱼(Larimichtys crocea)和pIRES-EGFP质粒扩增得到了肌肉生长抑素(MSTN)前肽基因及核糖体内部进入位点序列(IRES)-增强型绿色荧光蛋白基因(EGFP)片段,经测序验证正确后,构建了Tol2转座子供体质粒pT2AL200R150G-MSTⅣpropepti de-IRES-EGFP.通过精子介导法(S1、S2组)、电穿孔法(E1、E2组)及基因枪法构建转基因红鲤(Cyprinus carpio),孵化72 h后的鱼苗经荧光显微镜检测,EGFP表达阳性率为:精子介导法S1组38％,精子介导法S2组48％,电穿孔E1组47％,电穿孔E2组53％,基因枪组2％;孵化10d的仔鱼RT-PCR检测EGFP和MSTN前肽基因阳性率为:精子介导法S1组35％,精子介导法S2组45％,电穿孔E1组45％,电穿孔E2组55％,基因枪组1.8％.孵化后75 d转基因红鲤与对照组相比,体长和体重分别提高了21.31％和27.59％.本实验结果表明,精子经高渗、低渗保存剂处理并通过电穿孔作用可大幅提高基因转移效率.%This paper discovered the growth improvement of red carp transferd with the myostatin propeptide gene from Larimichthys crocea. Myostatin (MSTN) propeptide gene from Larimichthys crocea and the internal ribosome entrysite (IRES)-enhanced green fluorescence protein gene (EGFP) fragment from the pIRES-EGFP plasmid were cloned by RT-PCR and PCR, respectively. After confirming their sequences, the recombinant Tol2 transposon donor plasmid pT2AL200R150G-MSTV propeptide-IRES-EGFP was constructed. Three different gene transfer methods, sperm-mediated gene transfer (SMGT), electroporation (E) and gene gun (G) were used to obtain transgenic red carp (Cyprinus carpio). In SMGT, common sperm storage buffer (SI) and hypotonic dilution (S2) were used, respectively. In electroporation, 1 500 V (El) and 2 500 V (E2) were applied, respectively. The rates of EGFP expression were as follows: SI, 38%; S2, 48%; El, 47%; E2, 53% and G, 2% in larvae after 72 of hatching, respectively. The positive rates of reverse transcript-polymerase chain reaction (RT-PCR) for the transgene were as follows: SI, 35%; S2, 45%; El, 45%; E2, 55% and G, 1.8% in larvae after 10 d of hatching, respectively. After 75 d of hatching, the body length and weight of transgenic fish increased 21.31% and 27.59% in comparison with the control, respectively. The results showed that gene transfer efficiency could be improved by treating sperm with hypertonic and hypotonic dilution, and electroporation.
展开▼
