Biomaterials and Biocatalysts Group, Temasek Life sciences Laboratory, Research Link, National University of Singapore, Singapore 117604, Singapore.
Fungal Genet Biol. 2010 Apr;47(4):279-87. doi: 10.1016/j.fgb.2010.01.002. Epub 2010 Jan 15.
Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1x10(4) Hygromycin B resistant colony forming units (CFU) have been achieved on each 90mm agar plate using 10(6) sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5kb from the coding sequences at the 5' UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.
玉米黑粉菌是一种重要的模式真菌生物，可用于多种研究。自 20 多年前报道的原生质体 PEG 介导转染以来，其转化方法几乎没有得到改进。我们构建了携带潮霉素和新霉素抗性基因盒的二元 T-DNA 载体，并基于根癌农杆菌介导的转化（ATMT）开发了一种高效的转化方法。通过一系列优化，使用 10^6 分生孢子，在每个 90mm 琼脂平板上至少获得了 1x10^4 潮霉素 B 抗性菌落形成单位（CFU）。ATMT 的最佳 pH 值约为 5.6。约 96%的潮霉素 B 抗性转化体含有一个单拷贝 T-DNA 插入核基因组。对 204 个 T-DNA 侧翼序列的分析表明，其中 15.2%位于编码序列中，另有 37.25%位于编码序列 5'UTR 或启动子区域的 0.5kb 范围内。此外，还开发了一种转化就绪的 T-DNA 供体和受体细胞的制备和保存方法，允许按需进行基因标记实验。对 5000 个突变体的初步筛选确定了一个假定的法呢基转移酶β亚基和 PRE6 同源物作为玉米黑粉菌性交配的新参与者。
