Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2)
Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2)
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DOI：
10.1099/00221287-145-9-2221
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发表时间：
1999-09-01
期刊：
影响因子：
--
通讯作者：
Bibb, MJ
中科院分区：
文献类型：
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作者：
Sun, JH;Kelemen, GH;Bibb, MJ
增强型绿色荧光蛋白(EGFP)基因是水母Aequorea Victoria的绿色荧光蛋白基因的修饰版本，其密码子使用与许多富含GC的链霉菌基因中的密码子用法一致。在这里，使用绿色荧光蛋白作为分析天蓝色链霉菌A3(2)中空间和时间调控基因表达的报告。将绿色荧光蛋白基因插入到可在大肠杆菌中复制的载体中，极大地促进了绿色荧光蛋白基因融合的构建。通过接合，两个质粒(pIJ8630和pIJ8660)整合到温带噬菌体fC31的染色体附着点。这些载体被用来分析编码孢子成熟所需的S因子的sigf和合成十一烷基灵芝菌素的途径特异性调控基因redd的时空表达，十一烷基灵芝菌素是天蓝色5种抗生素之一。Sigf的转录仅限于发育中和成熟的孢子链，而Redo的转录仅在老化的底物菌丝体中发生。另一个缺乏fC31连接位点的质粒衍生物(PIJ8668)被制造出来，使EGFP基因在转录上与感兴趣的基因在其天然染色体位置上融合。
The enhanced green fluorescent protein (EGFP) gene is a modified version of the green fluorescent protein gene of the jellyfish Aequorea victoria with a codon usage that corresponds well to that found in many GC-rich streptomycete genes. Here the use of EGFP as a reporter for the analysis of spatially and temporally regulated gene expression in Streptomyces coelicolor A3(2) is demonstrated. The EGFP gene was inserted into plasmids that can replicate in Escherichia coli, greatly facilitating the construction of EGFP gene fusions. The plasmids can be transferred readily to S. coelicolor by conjugation, whereupon two of them (pIJ8630 and pIJ8660) integrate at the chromosomal attachment site for the temperate phage fC31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a s factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by 5 coelicolor. While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redo occurred only in ageing substrate mycelium. A further plasmid derivative (pIJ8668) was made that lacks the fC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations.