Department of Biotechnology, Indian Institute of Technology, Chennai, India.
J Basic Microbiol. 2014 Jan;54(1):81-7. doi: 10.1002/jobm.201200292. Epub 2013 Apr 2.
Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP-cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and mCherry were fused with RIB1 and RIB3 genes, under their native promoters by PCR-based gene tagging method for their early in vivo expression and cellular localization in A. gossypii. In the integrative transformants, the fusion proteins were expressed as cytoplasmic proteins from the germ bubble stage, in all the cells throughout the hypha. This was evident from the observation that mCherry fusion proteins were seen outside the vacuoles in the cytoplasm. The older matured cells of 14 h hyphae developed large vacuoles which showed green autofluorescence due to riboflavin. It is concluded that RIB1 and RIB3 genes are constitutively expressed in all the cells of this multicellular multinucleate fungus.
阿萨希毛霉是一种核黄素高产丝状真菌。RIB1 和 RIB3 基因分别编码鸟苷三磷酸环水解酶 II（GCH II）和二氢生物蝶呤合酶，这两种酶是核黄素生物合成途径中的两个限速酶。酵母增强型绿色荧光蛋白（yEGFP）和 mCherry 的编码基因通过基于 PCR 的基因标记方法与 RIB1 和 RIB3 基因融合，在其天然启动子下进行早期体内表达和细胞定位在 A. gossypii 中。在整合转化体中，融合蛋白从芽泡阶段开始作为细胞质蛋白表达，在整个菌丝的所有细胞中。这从观察到 mCherry 融合蛋白在细胞质中外层小泡中可以看出。14 h 菌丝的较老成熟细胞形成大的液泡，由于核黄素而显示绿色自发荧光。因此可以得出结论，RIB1 和 RIB3 基因在这种多细胞多核真菌的所有细胞中均持续表达。
