Mycobiology ( IF 1.9 ) Pub Date : 2021-08-19 , DOI: 10.1080/12298093.2021.1961431 Min-Hye Jeong 1 , Jung A Kim 2 , Seogchan Kang 3 , Eu Ddeum Choi 1 , Youngmin Kim 1 , Yerim Lee 1 , Mi Jin Jeon 2 , Nan Hee Yu 4 , Ae Ran Park 4 , Jin-Cheol Kim 4 , Soonok Kim 2 , Sook-Young Park 1
Abstract
An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.
中文翻译:
根癌农杆菌介导的 Xylaria grammica EL000614 转化的优化,一种产生革兰霉素的内生真菌
摘要
内生真菌Xylaria grammica EL000614 产生克霉素,这是一种有效的杀线虫吡喃酮衍生物,可作为根结线虫的新控制选择。我们针对X. grammica优化了根癌农杆菌介导的转化 (ATMT) 方案以支持遗传研究。将X. grammica的匀浆幼菌丝体与A. tumefaciens菌株 AGL-1共培养后成功生成转化子,该菌株携带包含细菌潮霉素 B 磷酸转移酶 ( hph ) 基因和eGFP的双元载体T-DNA 中的基因。所得转化体有丝分裂稳定,PCR 分析显示转化体基因组中的两个基因均已整合。通过荧光显微镜确认 eGFP 的表达。Southern 分析显示 166 个转化体中有 131 个 (78.9%) 包含单个 T-DNA 插入。主要生产单一 T-DNA 转化体的关键因素包括 48 小时的共同培养、在共同培养之前用乙酰丁香酮预处理农杆菌细胞,以及使用新鲜制备的菌丝体。已建立的 ATMT 协议为研究X. grammica的生物学和生态学提供了随机插入诱变和基因转移的有效工具。