Functional characterization of full-length and 5′ deletion fragments of Citrus sinensis-derived constitutive promoters in Nicotiana benthamiana

Abstract

Full-length and 5′ deletion fragments of three constitutively expressed gene promoters identified from the Citrus sinensis L. genome (cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1)) were evaluated for their ability to express the uidA gene in leaf, stem, and root tissues of transgenic N. benthamiana plants. According to the fluorometric GUS assays, the CsGAPC2 promoter activity was significantly reduced in the leaves when the sequence between position − 1090 and − 497 was removed, while the activity remained unchanged in the roots. The CsCYP promoter activity was not affected by the different deletions, and even the smallest evaluated fragment was sufficient to maintain the expression of the uidA gene in N. benthamiana leaves and roots. Truncated promoter fragments of CsEF1 conferred higher GUS activity in leaves compared with the full-length promoter, while GUS activity was not affected in the roots. The removal of the intron in the 5′ UTR of the CsEF1 promoter reduced gene expression in the roots, although it did not cause any reduction in the gene expression level in the leaves. These results indicate that any of the three deletions of the CsCYP promoter can be utilized for efficient gene expression. In addition, the full-length CsGAPC2 promoter and two of the truncated CsEF1 promoter fragments were sufficient for efficient uidA gene expression.