Bos C J, Slakhorst S M
Can J Microbiol. 1981 Apr;27(4):400-7. doi: 10.1139/m81-061.
Protoplasts were prepared from conidiospores of Aspergillus nidulans. The mononucleated conidia gave protoplasts of a uniform size, approximately 5-micron diameter, depending on the strain and the stabilizing medium used. Conidia were preincubated with 2-deoxy-D-glucose in a minimal medium at 37 degrees C for 3 h. The swollen conidia were collected, resuspended in a buffer containing 0.4 M (NH4)2SO4 as stabilizer, and incubated with Oerskovia lytic enzymes at 30 degrees C for 3 or 4 h. Approximately 80% of the conidia were converted into protoplasts. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 30% sucrose. This isolation procedure gives a suspension of mononucleated or binucleated protoplasts suitable for recombination experiments and other studies for which a homogenous protoplast suspension is required. The procedure was also successful for Aspergillus niger.
原生质体是由构巢曲霉的分生孢子制备而来。单核分生孢子产生大小均匀的原生质体，直径约5微米，这取决于所使用的菌株和稳定培养基。分生孢子在含有2-脱氧-D-葡萄糖的基本培养基中于37℃预孵育3小时。收集肿胀的分生孢子，重悬于含有0.4M硫酸铵作为稳定剂的缓冲液中，并在30℃下与溶壁奥氏杆菌酶孵育3或4小时。约80%的分生孢子转化为原生质体。通过在30%蔗糖上离心，将原生质体与细胞壁碎片和完整的分生孢子分离。该分离程序得到单核或双核原生质体的悬浮液，适用于重组实验和其他需要均匀原生质体悬浮液的研究。该程序对黑曲霉也成功。
