非模式真菌雷斯青霉遗传操作系统的建立  

Establishment of a genetic transformation system in non-model fungus Penicillium raistrickii

作　　者：张姝 陈登辉 陈国参[3] 徐新然 尹文兵[2] ZHANG Shu;CHEN Denghui;CHEN Guocan;XU Xinran;YIN Wenbing(College of Biomedical Sciences,Shandong First Medical University,Jinan 250117,Shandong,China;State Key Laboratory of Mycobiology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;Institute of Biology Co.,Ltd.,Henan Academy of Sciences,Zhengzhou 450008,Henan,China)

机构地区：[1]山东第一医科大学生物医学科学学院,山东济南250117 [2]中国科学院微生物研究所真菌学国家重点实验室,北京100101 [3]河南省科学院生物研究所有限责任公司,河南郑州450008

出　　处：《菌物学报》2024年第6期86-95,共10页Mycosystema

基　　金：中国科学院战略生物资源计划(KFJ-BRP-009-005);中国科学院基础前沿科学研究计划从0到1原始创新项目(ZDBS-LY-SM016);河南省科学院省级特聘研究员项目(230505002)。

摘　　要：雷斯青霉Penicillium raistrickii能够产生丰富的次级代谢产物,然而,对其生物合成途径和沉默基因簇的产物研究鲜有报道,这是由于高效遗传操作系统的缺乏严重阻碍了其分子生物学机制研究和天然产物的开发利用。本研究建立了未作为特定对象广泛研究的非模式真菌雷斯青霉CGMCC 3.1066的遗传操作系统。我们逐步确定了孢子萌发培养条件、抗生素筛选浓度、原生质体制备条件和转化DNA片段浓度,利用PEG介导的原生质体转化技术,成功敲除了5个长度分布在1-10kb的基因,平均敲除阳性率为17.5%。此外,我们敲除了雷斯青霉中黑色素前体1,8-dihydroxynaphthalene(DHN)-melanin的合成基因。结果表明,该基因的缺失会导致雷斯青霉孢子颜色变为白色,但不影响正常生长,这使得该位点可作为以雷斯青霉为宿主的异源表达整合位点,可快速可视化辨别整合外源基因的转化子。雷斯青霉遗传操作系统的建立,将为其基因功能鉴定、化合物生物合成途径解析等研究奠定基础,同时为其他非模式丝状真菌转化体系的建立提供重要参考。Penicillium raistrickii can yield plenty of secondary metabolites.However,there are few reports about its synthetic pathways and unexpressed biosynthetic gene clusters because of the lack of a genetic transformation system,as a result,the study of its biosynthesis mechanism and natural products is seriously hampered.An efficient genetic transformation system for P.raistrickii CGMCC 3.1066 was built in this study.The conditions for spore germination,the antibiotic concentration,the optimum condition for preparing protoplasts,and the concentration of DNA fragments have been determined.Five genes ranging in length from 1–10 kb were knocked out by polyethylene glycol-mediated protoplast transformation,and the average positive rate of gene knockout was 17.5%.In addition,the gene for synthesizing 1,8-dihydroxy naphthalene(DHN)-melanin has been knocked out.The results show that the lack of this gene can cause spore discoloration from green to white,but the growth of the strain is not affected,making this site to act as a fixed insertion site because the transformants can be identified quickly through observation.The genetic transformation system successfully established in P.raistrickii CGMCC 3.1066 will facilitate the identification of gene function,analysis of biosynthetic pathways of compounds,and other studies,and this study can provide a reference for building the transformation system of other non-model fungi.

关 键 词：雷斯青霉 遗传操作系统 基因敲除 潮霉素B 原生质体 

分 类 号：Q78[生物学—分子生物学]