Identification of a rice Actin2 gene regulatory region for high-level expression of transgenes in monocots

We have isolated and characterized the 5' region of the rice actin2 gene (OsAct2), which contains 793 bp of sequence upstream of the OsAct2 transcription initiation site, 58 bp of the first non-coding exon, 1736 bp of the 5' intron and the first 8 bp (non-coding sequence) of the second exon. It was found that the 5' region of OsAct2 is an efficient gene regulatory region for driving the constitutive expression of foreign genes in transgenic rice. In situ histochemical results indicated that OsAct2::GUS (GUS, beta-glucuronidase) gene expression in transgenic rice plants is high in sporophytic and gametophytic tissues. It was demonstrated that a 2.6-kb upstream sequence of the OsAct2 translation initiation codon contains all of the 5' regulatory elements necessary for high-level gus expression in transgenic rice tissues. OsAct2 promoter activity was significantly enhanced by the deletion of a 1590-bp segment from the central region of the first intron. The +96 to +274 region of the intron negatively regulates gus expression in leaves. To identify regulatory elements within the OsAct2 promoter, nested truncations of the promoter region were made and fused to gus. The results showed that the region from -1 to -376 was sufficient for promoter activity. In addition, two OsAct2-based expression vectors for use in monocot transformation were developed to promote the high-level expression of foreign genes.