【摘要】 为了建立原生质体介导的黑曲霉转化系统,研究了菌龄、酶系统、渗透压稳定剂、酶解时间对葡萄糖淀粉酶生产菌株黑曲霉CICIM F0410原生质体形成与再生的影响。结果表明,培养4 d的幼嫩菌丝体最适于制备原生质体;综合考虑原生质体形成与再生的情况,作者选用1 mol/L山梨醇为最适渗透压稳定剂,1 g/dL蜗牛酶-1 g/dL纤维素酶-0.1 g/dL溶壁酶为最适裂解酶组合,30℃酶解2.5~3 h,最适合的原生质体的再生培养基为含0.6 mol/L MgSO4的TZ培养基。在PEG和CaCl2存在条件下,以潮霉素B为选择性标记,质粒pBC-Hygro转化原生质体,每微克DNA可获得4~5个转化子。更多还原

【Abstract】 To establish protoplast-mediated genetic transformation system of Aspergillus niger,the conditions for its protoplast preparation and regeneration of the mycelium incubation time,various enzymes and osmotic stabilizers were examined.The results showed that the mycelia incubated for 4 days were most suitable for protoplast release.1 mol/L sorbitol was the most effective osmotic stabilizer and the mixture solution of 1 % snailase/1% cellulose/0.1% lysing enzyme was most favorable for protoplast preparation.The suitable incubation temperature and time for the release of protoplast was 30℃ and 2.5～3 h.For the regeneration of the protoplast prepared freshly,the TZ medium containing 0.6 mol/L MgSO₄was the best.PEG and CaCl₂ mediated protoplast transformation of A.niger with pBC-Hygro was performed and 4⁵ stable transformats per microgram DNA were obtained.更多还原