Introns and their positions affect the translational activity of mRNA in plant cells

Abstract

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.

Fig. 1. Chimeric luciferase constructs. The maize ubiquitin promoter (black box) plus leader intron (grey line) is fused to the Luc⁺ reporter gene (grey box) in presence of the Nos terminator (black squares). White boxes represent maize introns and their corresponding number is indicated. Restriction sites used to introduce the different introns in the luciferase cDNA are indicated as well as their position in the coding region. Arrowheads indicate primers used for the PCR amplifications.

Fig. 2. Luciferase activity in transformed maize BMS cells and protoplasts. Average luciferase activities from 11 bombardments of BMS cells (in grey) and three electroporations of BMS protoplasts (in black) are given as the ratio of luciferase units to GUS units. Luciferase activity of the plasmid pAL25 was set to one.

Fig. 3. PCR amplification of the spliced products from mRNA isolated from bombarded maize BMS cells. Lane 1, plasmid amplicons; lane 2, RT–PCR amplicons; lane 3, mRNA controls from RT–PCR. The size of the expected spliced product (1.5 kb) is indicated with arrows.

Fig. 4. Quantification of the luciferase mRNA in maize BMS protoplasts. (A) Example of two cDNA amplifications using GUS and both LUC probes. Only the PCR cycles in which the reporter dye emission crosses the default threshold (horizontal line) are shown, corresponding Ct values for both cDNA with each probe (GUS in blue, 5′LUC in red and 3′LUC in yellow) are indicated. (B) Comparison of mRNA ratios between GUS and 5′Luc, GUS and 3′Luc, and 5′Luc and 3′Luc. Ratios are set to pAL25 values.

Fig. 4. Quantification of the luciferase mRNA in maize BMS protoplasts. (A) Example of two cDNA amplifications using GUS and both LUC probes. Only the PCR cycles in which the reporter dye emission crosses the default threshold (horizontal line) are shown, corresponding Ct values for both cDNA with each probe (GUS in blue, 5′LUC in red and 3′LUC in yellow) are indicated. (B) Comparison of mRNA ratios between GUS and 5′Luc, GUS and 3′Luc, and 5′Luc and 3′Luc. Ratios are set to pAL25 values.