Structure, Expression and Promoter Activity of Ubiquitin Genes From Rice (Oryza Sativa L.).

Abstract

Successful transgenic plant production is largely dependent on the use of suitable promoters. In this study, we described isolation of rice ubiquitin genes, and demonstrated the potential utility of the rice ubiquitin promoters in plant transformation systems. Two polyubiquitin genes (RUBQ1 and RUBQ2) and two ubiquitin extension/fusion genes (RUBQ3 and RUBQ4) have been isolated from a rice genomic BAC library. DNA sequence data revealed that both RUBQ1 and RUBQ2 contained an open reading frame encoding a hexameric precursor ubiquitin and an intron immediate upstream of the initiation codon. The two ubiquitin fusion/extension genes RUBQ3 and RUBQ4 contained one repeat of the ubiquitin coding region interrupted by an intron. Northern blot analyses using the 3 '-untranslated region as gene specific probes showed that RUBQ1 and RUBQ2 were actively expressed in all rice plant tissues tested. Deletion analysis of the 5'-upstream region of RUBQ2 revealed a putative 70 bp enhancer region that produced a 2.5-fold stimulatory effect on promoter activity. RUBQ2 promoter activity was dependent on the presence of its own intron since promoter activity was reduced to background levels when the intron was deleted. In transient assays, GUS activity from constructs containing RUBQ1 and RUBQ2 promoters in rice suspension cells was 10 to 15 fold greater than those using the 35S promoter, and 2 to 3 fold greater than constructs with the maize polyubiquitin Ubi1 promoter. In stable transformation experiments, GUS expression levels from constructs containing RUBQ1 or RUBQ2 promoters were 22 and 48-fold greater in stably transformed calli, and 8 to 35-fold greater in transgenic rice plants, respectively, compared to those from a construct containing the 35S promoter. A six fold increase in gene silencing events were observed in R0 transgenic plants containing the GUS gene driven by the 35S promoter (52%) than the rice ubiquitin promoters (0--8%). Southern blot analysis of R0 and R1 transgenic plants showed that 3 to 7 copies of the GUS gene was inserted into the rice genome and inherited as a single locus. In addition, herbicide resistant transgenic rice plants were produced that contained the BAR gene controlled by the RUBQ2 promoter. These results demonstrate the potential usefulness of the rice ubiquitin promoters in rice and other monocot transformation systems.