橙色红曲菌AS3.4384转化方法的建立

Transformation of Protoplast from Monascus aurantiacus AS3.4384

本实验优化了橙色红曲菌AS3.4384原生质体制备及再生条件,将孢子接种在MPPY液体培养基中,30℃下,200r/min培养35h,以0.6mol/LMgSO4作为渗透压稳定剂,混合酶解液(0.3%裂解酶+4%纤维素酶+1%蜗牛酶)30℃,酶解3.5h,原生质体可达8×107个/ml,再生率为18%。并建立了PEG8000介导的原生质体转化方法。

In this research, formation and regenaration of protoplast conditions were optimized. The spores suspension up to a final concentration of 1×10^7～4×10^7/ml are inoculated in the liquid medium with MPPY, cultured at 30℃, 200r/min for 35 h. Mycelial masses were resuspended in suitable enzyme solution （1% snaliase ＋ 0.3% lysing enzyme ＋ 4 % cellulase, lytic enzyme dissolved in 0.6 mol/L MgSO4, adjusting pH to 6）, and incubated at 30 ℃ with orbital shaking （60 r/min）. The number of protoplasts were counted by a hemacytometer during the protoplasts formation, and the pesk of 8×10^7/ml after 3.5 h of incubation. The peak of regenerantion frequence is 18%. PEG and CaCl2-mediated protoplast transformation of strain AS3.4384 with pH4, hygromycin B as selective marker is established.