The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants

The genomic sequence of a rice (Oryza sativa L.) glycine-rich protein (GRP) gene, designated Osgrp-2, has been previously determined (GenBank U40708). Primer extension analysis indicated that transcription starts 47 bp upstream of the translation start codon. To gain an insight into the transcriptional regulation of this gene, the 2,401-bp promoter sequence and a series of its 5′ deletions were transcriptionally fused to the β-glucuronidase (GUS) gene. GUS activity was subsequently assayed in a transient expression system of tobacco (Nicotiana tabacum L.) protoplasts, which revealed the presence of a positive regulatory region (–2290 to –1406) and two negative regulatory regions (–2401 to –2291 and –1405 to –1022) in the Osgrp-2 promoter for the promoter activity. The positive regulatory region displayed an enhancer-like activity when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter (–89 to +6) to drive GUS expression and assayed on tobacco leaves by the Agrobacterium-mediated transient expression technique (agroinfiltration). Histochemical staining for GUS activity on transgenic tobacco plants has further indicated a preferential expression in vascular tissues of stems and leaves conferred by the positive regulatory region. A 1,023-bp fragment of the Osgrp-2 promoter (–1021 to +2) fused with GUS was transformed into tobacco and proved to be capable of conferring vascular-specific expression. Further 5′ and 3′ deletion analysis of the 1,023-bp promoter revealed that a 99-bp fragment located from –497 to –399 contained cis-elements responsible for vascular-specific expression.