Characterization of NtREL1, a novel root-specific gene from tobacco, and upstream promoter activity analysis in homologous and heterologous hosts.

Abstract

A novel root-specific gene and its upstream promoter were cloned and characterized for potential application in root-specific expression of transgenes. The root is an important plant organ subjected to many biotic and abiotic stresses, such as infection by Ralstonia solanacearum. To isolate tobacco root-specific promoters for genetic applications, microarray screening was performed to identify genes highly and specifically expressed in the root. One root-specific gene encoding an extensin-like protein (NtREL1) was isolated, and its expression pattern was further characterized by both microarray analysis and reverse transcription-polymerase chain reaction. NtREL1 was highly expressed only in roots but not in any other organ. NtREL1 expression was affected by hormone treatment (salicylic acid, abscisic acid, and ethephon) as well as low temperature, drought, and R. solanacearum infection. A full-length 849 bp cDNA containing a 657-nucleotide open reading frame was cloned by Rapid Amplification of cDNA Ends. Subsequently, a fragment of 1,574 bp upstream of NtREL1 was isolated by flanking PCR and named pNtREL1. This promoter fragment contains TATA, GATA, and CAAT-boxes, the basic elements of a promoter, and six root-specific expression elements, namely OSE1, OSE2, ROOTMOTIFTAPOX1, SURECOREATSULTR11, P1BS, and WUSATAg. A construct containing the bacterial uidA reporter gene (β-glucuronidase, GUS) driven by the pNtREL1 promoter was transformed into tobacco plants. GUS staining was only detected in the root, but not in leaves and stems. Additionally, transgenic tobacco plants containing peanut resveratrol synthase gene (AhRS) driven by the pNtREL1 promoter produced resveratrol only in the root. Thus, the pNtREL1 promoter can be used to direct root-specific expression of target genes to protect the root from stress or for biological studies.