Mora-Lugo Rodrigo, Zimmermann Judith, Rizk Amira M, Fernandez-Lahore Marcelo
BMC Microbiol. 2014 Sep 25;14:247. doi: 10.1186/s12866-014-0247-x.
Aspergillus sojae has been an important filamentous fungus in Biotechnology due to its use in diverse fermentative processes for the production of various food products. Furthermore, this fungus is a common expression system for the production of enzymes and other metabolites. The availability of molecular genetic tools to explore its biology is thus of big interest. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system for A. sojae was developed and its applicability evaluated.
The donor plasmid named pRM-eGFP was constructed for ATMT of A. sojae. This plasmid contains the ble and egfp genes in its transfer DNA element (T-DNA) to confer phleomycin resistance and express the enhanced green fluorescent protein (EGFP) in A. sojae, respectively. Agrobacterium tumefaciens (LBA4404) harboring the donor plasmid and A. sojae (ATCC 20235) were co-cultured under diverse conditions to achieve ATMT. The maximum number of transformed fungi was obtained after three days of co-culturing at 28°C, and selection with 50 μg/ml phleomycin. Polymerase chain reaction (PCR), fluorescence microscopy and Western Blot analysis for EGFP expression confirmed successful genomic integration of the T-DNA element in A. sojae. The T-DNA was mitotically stable in approximately 40% of the fungal transformants after four generations of sub-culturing under phleomycin pressure.
We successfully established a new ATMT protocol for A. sojae. This transformation system should enable further protein expression studies on this filamentous fungus.
米曲霉因其在多种食品发酵生产的不同发酵过程中的应用，一直是生物技术领域重要的丝状真菌。此外，这种真菌还是生产酶和其他代谢产物的常用表达系统。因此，开发用于探索其生物学特性的分子遗传工具备受关注。在本研究中，我们开发了一种用于米曲霉的根癌农杆菌介导转化（ATMT）系统，并评估了其适用性。
构建了名为pRM-eGFP的供体质粒用于米曲霉的ATMT。该质粒在其转移DNA元件（T-DNA）中包含ble和egfp基因，分别赋予对潮霉素的抗性并在米曲霉中表达增强型绿色荧光蛋白（EGFP）。携带供体质粒的根癌农杆菌（LBA4404）与米曲霉（ATCC 20235）在不同条件下共培养以实现ATMT。在28°C共培养三天并用50μg/ml潮霉素筛选后，获得了最大数量的转化真菌。通过聚合酶链反应（PCR）、荧光显微镜和针对EGFP表达的蛋白质免疫印迹分析证实了T-DNA元件在米曲霉中成功进行了基因组整合。在潮霉素压力下继代培养四代后，约40%的真菌转化体中T-DNA在有丝分裂过程中保持稳定。
我们成功建立了一种新的米曲霉ATMT方案。该转化系统应能推动对这种丝状真菌的进一步蛋白质表达研究。
