Utilizing modified ubi1 introns to enhance exogenous gene expression in maize (Zea mays L.) and rice (Oryza sativa L.)

Abstract

The phenomenon of intron-mediated enhancement (IME) was discovered in 1990 based on the observation that plant introns can stimulate gene expression, particularly in monocots. However, the intrinsic mechanism of IME remains unclear because many studies have yielded various results depending on the promoter, reporter gene, flanking sequences of the intron, and target cell or tissue. In this study, the effect of the first intron of the maize ubiquitin gene (ubi1 intron) was investigated by changing insertion sites, deleting specific regions and mutating individual motifs in maize (Zea mays L.) and rice (Oryza sativa L.) using ubi1 intron-containing GUS (β-glucuronidase) constructs. In maize callus, the integration of the full-length ubi1 intron into the GUS coding sequence at the +13, +115 and +513 positions by particle bombardment increased GUS activity approximately five-, four- and two-fold, respectively. Eight truncated ubi1 introns in the pSG(13i) N construct significantly influenced GUS gene expression to different degrees in transient assays. Notably, the 3′ region deletions significantly reduced the IME effect, whereas a 142-nt deletion, pSG(13i-P5)N, in the 5′ region caused a 1.5-fold enhancement relative to pSG(13i)N. Furthermore, four site-directed mutageneses were performed in pSG(13i-P5)N; these constructs resulted in the up-regulation of GUS gene expression to different levels. The most effective modified ubi1 intron, pSG(13i-M4)N, was further evaluated and proved in rice using transient experiments. In addition, the sequences flanking the GUS insertion significantly influenced the IME effect of the vectors that were constructed. The modified ubi1 intron had the potential application on crop genetic engineering.