The transcriptional properties of artificial promoters are closely related to the type and arrangement position of cis-elements. GWSF (374-bp) was an effective SPIP with four cis-element dimers. There were four pathogen-inducible cis-elements in the GWSF promoter (GST1-boxes, W-boxes, S-boxes, and F-boxes) and a minimal cauliflower mosaic virus 35S promoter. V-element dimers were inserted into the upstream (VGWSF), midstream (GWVSF), and downstream (GWSFV) regions of the original GWSF promoter sequence to examine their affect on the position. The expression activity of promoters was analyzed and estimated using the histochemical staining of leaf discs of eucalyptus with transient expression, an image digitization method to extract the color features, and the induction treatment by a plant pathogenic microorganism/inducer and qPCR assays. The histochemical staining results of the adventitious buds indicated that the promoters had been successfully integrated into the E. urophylla genome and that they drove the expression of the gus gene. There was a noticeable difference in the intensity of color between the adventitious buds on the same callus block, as well as the intensity of color within the same adventitious bud. According to the established two-factor model of blue value, there was a greater difference between the levels of the genotype factor than the promoter factor in eucalyptus leaf discs. Further, the basal and inducible transcriptional levels of the three improved promoters were investigated by qPCR. With the basal transcriptional level of the GWSF promoter normalized to one, the relative basal levels of VGWSF, GWVSF, and GWSFV were 1.40, 1.45, and 4.15, respectively. The qPCR results were consistent with the staining results of GUS histochemical staining. The three improved promoters all had the properties of being induced by salicylic acid, Ralstonia solanacearum, and Phytophthora capsici. The three improved promoters demonstrated a significantly higher TMV induction activity: their induction activity from high to low was GWSFV > GWVSF > VGWSF. The findings will be beneficial to the construction and optimization of artificial promoters for transgenic plants.
Keywords: eucalyptus; expression activity; improved promoter; position effect; tobacco mosaic virus; transgene.