限制性内切酶介导的玫烟色拟青霉原生质转化体系构建

Construction of Paecilomyces fumosoroseus protoplast-transforming system by restriction enzyme-mediated integration(REMI)

丝孢类害虫生防真菌的生态适应性和环境抗逆性决定着生防菌剂的田间适用性，兼具多个抗逆性状的菌株一般只能通过相关功能基N的遗传操作和定向选育而获得，但这类真菌遗传操作的可行方法目前相当有限，本文利用限制性内切酶介导插入法将含有草丁膦抗性标记的Bar基因的质粒pAN52-1N-Bar转入玫烟色拟青霉Pfr116菌株的原生质体中，转化效率约为6～13个转化子μg^-1质粒DNA．通过对Pfr116菌株突变转化子的基因组DNA进行PCR产物检测及基因组DNA单酶切片段的Southern杂交分析，证明Bar基因已整合到随机抽取的转化子基因组中，且都在基因组上单位点插入，转化子对草丁膦的抗性具有遗传稳定性，由此构建的原生质体转化体系具有低拷贝、转化率较高，遗传稳定强的特点，不失为一种丝孢类生防真菌遗传操作的有效方法．

Phosphinothricin resistance gene Bar vectored by prepared plasmid pAN52-1N-Bar was transformed into the protoplasts of the entomopathogenic fungus Paecilomyces fumosoroseus Pfr116 using the method of restriction enzyme-mediated integration （REMI）. The protoplast-based transformation resulted in 6-13 mutants per microgram of the plasmid DNA. Subsequently, PCR and Southern Blotting analyses confirmed successful integration of the Bar gene into the genome DNAs of eight mutants randomly taken. The mutants with a single copy of the inserted gene displayed steady resistance to the chemical after three subcultures. The transformation system featured with lower copies, higher efficiency and genetic stable has proven useful for genetic manipulation of the filamentous fungal biocontrol agent.