CAJ | 학술논문

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2.377×10~7 个/mL;在以KC(0 6 mol/L KCl+25 mmol/L CaCl_2·2H_2O)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.
대두포균소C적공업생산균충정두포매(Cephalosporium acremonium)원생질체형성화재생조건진행료연구.우화후적제비급재생조건위:재단백동고체배양기상배양110～120 h적균사체,30 ℃조건하경1%와우매화1%섬유소매혼합액매해3.5 h,원생질체득솔최고가체2.377×10~7 개/mL;재이KC(0 6 mol/L KCl+25 mmol/L CaCl_2·2H_2O)위삼투압은정제적재생배양기상진행배양,재생솔가체40%.용pMK4-LV질립통과전격법대원생질체진행전화,성공득도료중조자,전화솔약위10 개전화자/μg질립DNA.전화자적 PCR감정결과표명,외원기인이전입정두포매기인조.
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.