|Joung, Young hee|
|Kamo, Kathryn - Kathy|
Submitted to: Plant Cell Reports
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/3/2006
Publication Date: 12/16/2006
Citation: Joung, Y.H. and Kamo, K. 2006. Expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Reports. 25:1081-1088. Interpretive Summary: Genetic engineering of floral crops for disease resistance requires the use of a promoter that directs high levels of the resistance gene. It has been difficult finding a promoter that expresses well in Gladiolus. This report is the isolation of a promoter from Gladiolus that results in high levels of gene expression. High levels of gene expression were demonstrated in rose cells making it possible to use this promoter in other floral crops.
Technical Abstract: A polyubiquitin promoter (GUBQ1) was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using currently available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5’ and 3’ intron splicing sites for the 1.228 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86% respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that levels of relative GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus, and this level of expression was comparable to that of the CaMV 35S promoter. The relative GUS activity was lower for the GUBQ1 promoter in cannas as compared to the CaMV 35S promoter. The full length GUBQ1 promoter including 5’UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5’UTR and 398 bp of the full length 1.228 kb intron was 41%. A 3’UTR region specific to the GUBQ1 coding region was identified by PCR using an oligodT primer set and used as a probe in Northern hybridization to show polyubiquitin expression in roots, leaves, and callus of Gladiolus. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia where high levels of gene expression are important.