PEG介导的黑附球菌遗传转化体系的建立

以生防真菌黑附球菌原生质体为受体、潮霉素抗性基因（ hph）为筛选标记,应用PEG介导法进行了遗传转化体系的研究。潮霉素敏感性测试结果表明,黑附球菌对潮霉素的耐受浓度为50mg/L。PEG介导的黑附球菌原生质体最佳遗传转化体系为：30℃下用2%裂解酶酶解黑附球菌菌丝2h,过滤收集原生质体,经MTC清洗重悬后将质粒pYF11-hph转化黑附球菌原生质体,在RM培养基中混匀再生;经潮霉素药剂筛选和PCR验证,表明外源基因 hph已转入到黑附球菌中并获得稳定表达。本实验成功地建立了稳定的PEG介导的黑附球菌遗传转化体系,为研究其代谢途径、生防机制及构建工程菌株提供了技术保障。

Establishment of genetic transformation system of Epicoccum nigrum using PEG-mediated method

The PEG mediated method for genetic transformation of Epicoccum nigrum was carried out. The sensitivity assessment of E. nigrum to hygromycin B showed that the lowest selection concentration was 50mg/L. The optimum condition for preparing protoplasts and transformation was set up by using 2% lysing enzyme in 0.7mol/L NaCl to digest the fresh mycelia at 30°C for 2h to produce an efficient yield of protoplasts. After washing by MTC buffer twice, the protoplasts were incubated with expression plasmid pYF11-hph and mixed with RM medium. Verifications of the putative transformants by hygromycin B seletion and PCR analysis indicated that the hph gene has successfully transformed into the protoplast of E. nigrum. The stable PEG-mediated genetic transformation system provides an essential technology for studying the metabolic pathways, molecular mechanisms, and constructing engineering strains.