The synergistic effects of two-intron insertions on heterologous gene expression and advantages of the first intron of a rice [Oryza sativa] gene for phospholipase D

The first intron of a rice gene for phospholipase D (PLD) had an expression-enhancing effect in transient expression assays. This intron also had an enhancing effect in the reverse orientation, and the effect was accompanied by the increase in the steady-state level of transcripts in both orientations. The splicing efficiency of the PLD intron was very low even in the normal orientation, and the intron was not spliced in the reverse orientation. When two different introns were inserted in tandem, synergistic, additive, or negative effect on the reporter gene expression were observed depending on the combination of introns. A synergistic effect was observed for the combinations of PLD and catalase or ubiquitin introns, and sucrose synthase and catalase introns, whereas two PLD introns gave an additive effect. The enhancement of expression in these combinations was accompanied by the increase in the steady-state level of the respective transcripts.