School of Biological Sciences, UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (East), Mumbai, 400098, India.
Sci Rep. 2020 Feb 11;10(1):2323. doi: 10.1038/s41598-020-59159-0.
Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5' ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.
双向启动子（BDP）是一种调控 DNA 序列（长约 1000bp），它位于两条反向排列的基因之间，其 5'端彼此靠近。这些基因通常是共表达的；但也有观察到反相关和独立转录的情况。在真菌系统中，BDP 通过组装和调节共同代谢途径的不同基因的转录，提供了一种改进的遗传电路。我们从香蕉根病原菌尖孢镰刀菌（Foc）的基因组中鉴定出一个基因间区（1063bp）。该基因间区调控一对分解半纤维素所需基因的表达。为了进行特征分析，它被克隆到 pCSN44 载体骨架之间的两个报告基因，即β-葡糖苷酸酶（GUS）和增强型绿色荧光蛋白（EGFP）之间。新形成的载体被转化到 Foc 中，并测试其双向表达活性。通过组织化学染色和荧光显微镜，分别在不同的生长条件下测试了 GUS 和 EGFP 表达的动力学。活性受到木聚糖、阿拉伯半乳聚糖和果胶等诱导剂的差异调控。这是首次从尖孢镰刀菌中分离出具有诱导性双向启动子活性的基因间区的报告。此类 BDP 的特性研究将在利用真菌系统进行遗传工程、代谢工程和合成生物学方面得到应用。
