Molecular Biology Program, New Mexico State University, Las Cruces, New Mexico, 88003, USA.
J Microbiol Methods. 2010 May;81(2):160-5. doi: 10.1016/j.mimet.2010.02.015. Epub 2010 Mar 6.
Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an alpha-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.
乌地非镰孢（真菌界：子囊菌门；棒孢霉科）是一种生长缓慢的内生真菌，能产生有毒的生物碱苦马豆素。这种内生真菌寄生于疯草，这是一种多年生开花豆科植物。食用这种真菌会导致放牧动物产生一种叫做疯草病的神经紊乱。生物碱苦马豆素是一种α-甘露糖苷酶抑制剂，是导致疯草病田间毒性的罪魁祸首。关于内生真菌中苦马豆素的生物合成途径知之甚少。对内生真菌进行遗传操作对于更好地了解生物碱合成涉及的生化途径很重要，但目前还没有用于研究乌地非镰孢中此类酶的转化系统。在这项研究中，我们报告了乌地非镰孢原生质体和转化系统的开发。用于生成原生质体的真菌菌丝在液体培养基中生长，然后用各种酶进行收获和处理。真菌特异性载体驱动增强型绿色荧光蛋白（EGFP）的表达，将原生质体转化。在整个过程中，监测转化原生质体的质量和转化效率。在所有情况下，都能保持对抗生素潮霉素 B 的抗性。这种操作将为未来的研究开辟途径，以破译真菌的代谢途径。
