目的:制备致病性烟曲霉菌原生质体,并观察原生质体从细胞壁剥离到恢复为完整形态这一时间窗内的生物学性状。方法:1mol/L山梨醇为渗透压稳定剂配制浓度为1g/dL蜗牛酶、1g/dL纤维素酶及0.1g/dL溶壁酶复合裂解酶液,与浓度为5×109个/L的烟曲霉菌分生孢子液按体积比1∶1配比,30℃下以80r/min酶解2h,使用3-氨基-7-甲氨基-2-甲基吩嗪盐酸盐溶液检测原生质体活力。结果:烟曲霉菌分生孢子原生质体的平均生成量为2.84×109个/L,完成酶解后0,6,12,18,24h活力分别为86.6%,77.4%,73.9%,70.6%,66.2%,原生质体最早在完成酶解18h后出现再生现象,36～48h内有活性的原生质体基本完成再生。结论:采用复合酶法可以获得生成率达56.8%的烟曲霉菌原生质体,恢复为完整形态前的可干扰时间窗至少为18h,原生质体可以在24h内保持较高活力。

AIM: To product protoplasts of pathogenic Aspergillus fumigatus,and to observe biological properties of protoplasts from stripped the cell wall to return to full form·METHODS: 1mol/L sorbitol was used as the osmotic stabilizer to product complex enzyme including 1g/dL snailase,1g/dL cellulase and 0.1g/dL lysing enzyme.Complex enzyme was confected with 5×109/L Aspergillus fumigatus conidia liquid by volume ratio of 1∶1 to digest 2 hours in gas bath thermostatic oscillator at 30℃ and 80r/min.The activity of protoplasts was detected by the neutral red solution·RESULTS: The average generation capacity of Aspergillus fumigatus conidia protoplasts was 2.84×109/L.After digestion the activity of protoplasts at 0,6,12,18,24 hours were 86.6%,77.4%,73.9%,70.6%,66.2%.The regeneration phenomenon of protoplasts began at 18 hours after the digestion,and basically completed within 36-48 hours·CONCLUSION: The generation rate of Aspergillus fumigatus protoplasts was 56.8% with complex enzyme.The time window that we can interfere was at least 18 hours.Protoplasts could maintain a high activity in 24 hours.

中国国家自然科学基金资助项目(No.81170825);中国青岛市科技发展计划资助项目[No.11-2-3-1-(5)-nsh]