Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany.
Appl Environ Microbiol. 2011 May;77(9):2887-97. doi: 10.1128/AEM.02644-10. Epub 2011 Mar 4.
The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.
绿色荧光蛋白（GFP）及其变体已被广泛应用于现代生物学中，作为允许各种活细胞成像技术的报告者。迄今为止，由于荧光强度低，GFP 很少在灰霉菌 Botrytis cinerea 中使用。灰霉菌基因的密码子用法与常用的 GFP 编码基因强烈偏离，并且 GC 含量低于其他真菌。在这项研究中，我们报告了一种经过密码子优化的灰霉菌增强型 GFP（eGFP）编码基因（Bcgfp）的开发和使用，以提高在灰霉菌中的表达。密码子优化以及在较小程度上插入内含子导致更高的 mRNA 水平和增强的荧光。Bcgfp 用于萌发孢子中细胞核的定位以及宿主穿透的可视化。我们进一步证明了启动子-Bcgfp 融合物用于定量评估各种有毒化合物作为编码灰霉菌 ABC 型药物外排转运蛋白的 atrB 基因的诱导剂的用途。此外，构建了一个密码子优化的 mCherry 编码基因，该基因在灰霉菌中产生鲜艳的红色荧光。
