Intron-mediated enhancement of gene expression in transgenic plants using chimeric constructs composed of the Peanut chlorotic streak virus (PClSV) promoter-leader and the antisense orientation of PClSV ORF VII (p7R).

Abstract

The antisense orientation of the Peanut chlorotic streak virus (PClSV) open reading frame (ORF) VII (denoted as p7R), in conjunction with the sense orientation of the PClSV leader sequence, acts as an intron and enhances the expression of a reporter gene, analyzed in protoplasts and transgenic plants of tobacco ( Nicotiana tabacum L.). Correct 5' and 3' splicing sites were determined for intron removal from the chimeric constructs using either beta-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) as a reporter gene. In this splicing process, the active consensus 5' splicing donor site (AG/GTATA) is located at position +283 to +289 from the transcription start site (TSS) of the PClSV full-length transcript (FLt). The 3' splice site (TAG/GATT) is located on the p7R sequence at position +785 to +791 from the TSS. The combination of PClSV FLt leader and p7R enhanced the expression of reporter genes (CAT and GUS) by as much as 2-fold compared to the strong constitutive PClSV FLt promoter without an interfering leader sequence and about 30- to 800-fold compared to constructs containing the sense orientation of PClSV ORF VII (p7) in both protoplast transient-expression experiments and stably transformed transgenic plants. An increased level of mature transcripts accompanied this. This suggests that this combination of elements can mediate the intron-mediated enhancement (IME) phenomenon. We also demonstrated comparative IME with other heterologous promoters from caulimoviruses.