粗壮脉纹孢菌原生质体的制备、再生及转化的条件

Protoplast Production, Regeneration and Transformation of Neurospora crassa

为建立原生质体介导的粗壮脉纹孢菌遗传转化系统,本研究主要考察了复合酶解液、菌龄及酶解时间对粗壮脉纹胞菌原生质体生成及再生的影响。结果表明,粗壮脉纹胞菌孢子悬液接种在马铃薯葡萄糖琼脂(potato dextrose agar,PDA)液体培养基中,30℃条件下摇床培养10 h的菌丝,采用0.5 g/100 m L蜗牛酶+0.5 g/100 m L溶壁酶+1 g/100 m L纤维素酶的复合酶解液、30℃条件下酶解10 h,粗壮脉纹胞菌原生质体生成及再生最为适合。在此条件下,原生质体生成数可达4.53×106个,38#再生培养基中的再生数可达1.41×106个,再生率达31.71%,聚乙二醇介导PAKHB载体转化NC原生质体,每微克质粒可获得8个以上的转化子。

This study aimed to establish protoplast-mediated genetic transformation system of Neurospora crassa. Several major factors influencing the production and regeneration of N. crassa protoplast, such as mycelium incubation time, macerozyme and digestion time, were investigated. The results showed that mycelium incubation in liquid PDA medium at 30 ℃ for 10 h, and enzymatic hydrolysis at 30 ℃ for 10 h with a macerozyme solution containing 0.5 g/100 mL snailase, 0.5 g/100 mL lyticase and 1.0 g/100 mL cellulase were optimal for the production and regeneration of N. crassa protoplast. Under these conditions, the number of production and regeneration in 38# regeneration medium of Neurospora crassa protoplast were 4.53 ×10^6 and 1.41 × 10^6, respectively, and the regeneration rate was 31.71%. Transformation efficiency with PAKHB plasmid by PEG-mediated was more than 8 transformants per μg of plasmid DNA.