红曲霉原生质体的制备、再生及其遗传转化系统

原生质体是研究和建立真菌遗传转化系统的重要工具。为了建立原生质体介导的红曲霉遗传转化系统，考察了各种细胞壁裂解酶和渗透压稳定剂等对红曲霉原生质体形成和再生的影响。将红曲霉分生孢子在铺有玻璃纸的平板上30℃培养30~40 h收获的菌丝体最有利于原生质体的形成和释放。红曲霉菌丝体形成和释放原生质体最适裂解酶和酶解时间分别为：0.3 % lysing enzyme、0.1 % cellulase和1 % snailase的酶组合，30℃作用2.5 h；最适渗透压稳定剂是：1mol /L MgSO4。最适合原生质体再生的培养基为含0.6 mol/L蔗糖的CM培养基。原生质体液涂布单层再生培养基的方法，再生率最高，菌株M34和N18分别为8.5 %和36.4 %。在PEG和CaCl2存在下，以潮霉素B为抗生素选择标记，用质粒pBC-Hygro和pNL1共转化菌株M34原生质体，每微克DNA克获得100个稳定转化子。

Preparation and Regeneration of Protoplasts from Monascus purpureus and Genetic Transformation System

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.