Liang Min, Li Wei, Qi Landa, Chen Guocan, Cai Lei, Yin Wen-Bing
Henan Academy of Science Institute of Biology, Zhengzhou 450008, China.
State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Fungi (Basel). 2021 Feb 14;7(2):138. doi: 10.3390/jof7020138.
Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.
来自独特环境的真菌具有特殊的生理特性和大量生物活性天然产物。然而，顽固的遗传学特性或较低的转化效率阻碍了科学家对其分子生物学机制进行系统研究以及开发其代谢产物。在本研究中，我们以嗜鸟真菌LC5815为研究对象，开发了一种遗传转化系统。我们首先通过对孢子形成和原生质体再生进行条件优化，建立了一种高效的原生质体制备方法。以0.8 M蔗糖作为渗透压稳定剂时，原生质体的再生率高达约34.6%。为了开展遗传转化，我们采用聚乙二醇介导的原生质体转化方法，在菌株LC5815中删除了编码主要协助转运蛋白超家族转运体的测试基因，这证明了该遗传操作体系的可行性。此外，通过使用以5-氟乳清酸作为选择试剂的阳性筛选方案，构建了一个尿苷/尿嘧啶营养缺陷型菌株。最终，在嗜鸟真菌菌株LC5815中成功建立了遗传转化系统，这为分子遗传学研究奠定了基础，并将促进真菌中生物活性次生代谢产物的开发利用。
