顶头孢霉原生质体制备、再生及带vgb基因的质粒DNA转化条件的研究

对头孢菌素C的工业生产菌种顶头孢霉(Cephalosporium acremonium)原生质体形成和再生条件进行了研究。优化后的制备及再生条件为:在蛋白胨固体培养基上培养110~120 h的菌丝体,30℃条件下经1%蜗牛酶和1%纤维素酶混合液酶解3.5 h,原生质体得率最高可达2.377×107个/mL;在以KC(0.6 mol/L KCl+25mmol/L CaCl2.2H2O)为渗透压稳定剂的再生培养基上进行培养,再生率可达40%。用pMK4-LV质粒通过电击法对原生质体进行转化,成功得到了重组子,转化率约为10个转化子/μg质粒DNA。转化子的PCR鉴定结果表明,外源基因已转入顶头孢霉基因组。

Study on preparation and regeneration condition of protoplasts of Cephalosporium acremonium and transformation of plasmid DNA with vgb gene

The formation and regeneration conditions of Cephalosporium acremonium protoplast were studied. The optimum conditions of protoplast preparation were as follow: After Cephalosporium acremonium mycelium has been cultivated for 110 -120 h on peptone medium, using enzyme 1%snailase+1% cellulase("Onozoka R - 10") to digest the cell wall for 3. 5 h at 30 ℃ , the number of protoplast reaches about 2. 377 × 10~7 per milliliter. Meanwhile the regeneration was tested on solid media containing KC (0. 6 mol/L KCl+25 mmol/L CaCL_2 · 2H_2O) as osmotic stabilizer, the regeneration ratio is about 40%. Then the constructed shuttle plasmid pMK4 - LV was transformed into protoplasts by using electroporation method. The recombi-nant was obtained. Successfully. The transfer ratio is about 10 recombinants/μg DNA. The vgb gene was testified to have been recombined into the Cephalosporium acremonium genome through the PCR amplification.