深海白色侧齿霉遗传转化体系建立及渗透压调控相关的EaSHO1基因功能

海洋中具有丰富的动植物及微生物资源，海洋真菌是其重要组成之一。我们前期的研究发现一株深海真菌白色侧齿霉Engyodontium album能产生具有抑菌活性的次级代谢产物engyodontiumin A，该化合物能抑制黑曲霉、金黄色葡萄球菌及创伤弧菌等病原菌的生长，是一种潜在的海洋源抗菌药物。目前，该菌遗传转化体系尚未建立，不利于开展次级代谢产物合成调控机制及其他功能基因研究。本研究成功制备了深海白色侧齿霉菌的原生质体，建立了借助聚乙二醇3350介导的原生质体转化体系，并将pCT74-sGFP载体成功导入白色侧齿霉的原生质体中，结果显示外源GFP能稳定表达。此外，为了明确白色侧齿霉菌是否能够开展基因敲除研究，通过氨基酸序列同源比对，我们选取酵母高渗甘油信号途径中的同源基因EaSHO1进行初步探究。利用同源重组的方法成功将目的基因EaSHO1的开放阅读框(ORF)替换成潮霉素磷酸转移酶基因(HPH)，由此获得EaSHO1基因敲除突变体，并对突变体进行Southern杂交验证及初步的表型分析。结果表明，EaSHO1缺失不影响白色侧齿霉菌的营养生长及对高盐胁迫的响应，亚细胞定位结果显示EaS...

Development of a genetic transformation system for the deep-sea-derived fungus Engyodontium album and loss-of-function analysis of the osmolarity-regulation gene EaSHO1

Ocean is rich in animal, plant and microbial resources, among which marine fungi are an important component part. Our previous study found that Engyodontium album, a deep-sea-derived fungus, can produce a secondary metabolite engyodontiumin A which can inhibit the growth of Aspergillus niger, Staphylococcus aureus, and Vibrio vulnificus, and is a potential marine antibacterial drug. So far, the genetic transformation system of E. album has not been developed, which is disadvantageous to the research on the regulation mechanism of secondary metabolite biosynthesis and other functional genes. In this study, the protoplasts of E. album were prepared, PEG3350-mediated protoplast transformation system was developed, and pCT74-sGFP plasmid was successfully introduced into the protoplast of E. album. Results demonstrated that the exogenous GFP could be stably expressed. To investigate whether the gene deletion strategy could be applicable in E. album, EaSHO1, a homologous gene of the yeast high-osmolarity glycerol pathway related gene identified through homologous comparison of amino acid sequences, was selected for preliminary analysis. The knockout mutants of EaSHO1 gene were obtained by replacing the open reading frame (ORF) of target gene with the hygromycin phosphotransferase (HPH) gene using homologous recombination method, and verified by Southern blot. The phenotypes caused by EaSHO1 deletion were further analyzed. The results showed that the deletion of EaSHO1 did not affect the vegetative growth and response against high salinity stress in E. album. Subcellular localization analysis showed that EaSho1-GFP mainly localized on the hyphal septa. In summary, the genetic transformation system of a deep-sea-derived E. album strain was successfully developed, and the biological function of its gene EaSHO1 was preliminarily analyzed.