摘要

合成细胞外酶降解广谱植物和藻类聚合物底物的能力使得许多真菌与生物技术相关。已经测试了能够植物和藻类降解的陆生嗜热和海洋真菌分离物的抗生素抗性，因为它们可能用于新的遗传转化系统。将编码在花椰菜花叶病毒35S启动子，构巢曲霉的trpC基因启动子和出芽短梗霉TEF基因启动子控制下的潮霉素B磷酸转移酶（hph）的质粒通过电穿孔递送到真菌细胞中。通过在选择培养基上的嗜热热门嗜热菌（以前称为嗜热毁丝霉）的转化和生长以及通过实时PCR分析来比较不同启动子的有效性。通过每1×105个分生孢子使用10μgDNA，在8.5kV / cm的电脉冲下观察到高效转化。尽管所有启动子都能够在Th中表达hph。嗜热菌细胞，trpC启动子提供最高水平的潮霉素抗性。我们进一步成功应用植物二元载体pPZP进行hph基因和增强型绿色荧光蛋白基因的共转化，证实该转化系统可用作基因功能研究和微小菌体中异源蛋白表达的合适工具。

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 μg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

https://www.ncbi.nlm.nih.gov/pubmed/30465791