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

ZHOU Li-Hong,LI Guo-Qin,WANG Zheng-Xiang,ZHUGE Jian (Research Center of Industial Microbiology The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036,China)  
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 30℃. Mycelia obtained through cellophane-mediated culture for 30~40 h 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 MgSO_4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5 h. 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 CaCl_2- 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.
【CateGory Index】: TS201.3
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