CryK13V基因对绿色木霉原生质体的转化

Transformation of CryK13V gene into protoplasts of Trichoderma viride.

将Cry基因的13位赖氨酸（K）突变成缬氨酸（V）的突变基因CryK13V，构建于pCSN43载体。完成绿色木霉转化载体pCSNTCCm的构建．在研究绿色木霉（Trichoderma viride）原生质体形成和再生基础上，初步研究了绿色木霉转化条件．结果表明，绿色木霉原生质体形成的合适条件为：pH6．98磷酸盐缓冲液加入4mg·mL^-1 glucanex。培养24h的菌丝，40r·min^-1振荡条件下30℃酶解4h，原生质体产量达到4．7×10^7个·mg^-1．原生质体在0．3mol·L^-1肌醇和0．3mol·L^-1。KCl的CM培养基上再生率为14．5％．用限制酶XhoⅠ介导将带有潮霉素抗性标记的CryK13V基因转化到绿色木霉中。转化率为每微克DNA得到1～2个转化子，转化子稳定性和PCR鉴定结果表明，外源基因已转入受体菌绿色木霉中．

The lysine in site 13 of cryptogein protein was mutated to valine（K13V） through PCR sitedirected mutagenesis. The mutant fragment （CryK13V） was confirmed by enzyme digestion and DNA sequencing. The CryK13V gene was expressed in Trichoderrna viride, with constructed vector pCSNTCCm. Transformation was carried out by restriction enzyme XhoⅠ mediated integration and transformants were obtained on the CM media contained 200 μg·mL^-1 hygromycin B, and the transformation rate was 1-2 transformants per microgramme vector DNA. The optimum of isolation, regeneration of the protoplasts from T. viride was that： pH=6.98 phosphate buffer, 4 mg·mL-1 glucanex, hypha cultured 24 hours, digested at 30℃ for 4 hours, and the yields of the protoplasts was 4. 7×10^7 per·mg^-1. On the CM medium containing 0. 3 mol·L^-1 KCl and 0.3 mol·L^-1 Inositol, the regeneration rate was 14.5%.