Role of miniature inverted repeat transposable elements inserted into the promoter region of a carrot phenylalanine ammonia-lyase gene and its gene expression

Abstract

The expression of the phenylalanine ammonia-lyase gene (PAL) was induced in anthocynanin-producing suspension-cultured carrot cells. In our previous study, we isolated two PAL genes, DcPAL3 and DcPAL4, from a carrot genomic library, the nucleotide sequences of which are highly similar. Here, the complete nucleotide sequence of DcPAL4 was determined and compared with that of DcPAL3, revealing two miniature inverted repeat transposable elements (MITEs), here designated MITE1 and MITE2, inserted into the proximal promoter region of DcPAL3, but not into that of DcPAL4. The expression of DcPAL4 was not detectable by RT–PCR in cultured carrot cells or the tissues of carrot plants, whereas DcPAL3 expression was detected. Transient expression experiment in carrot protoplasts showed that the DcPAL4 promoter actively produces transcripts, as does the DcPAL3 promoter. To characterize the roles of MITE1 and MITE2 (MITE1/2) in the transgenes of stable transformants, they were inserted into a plant transformation vector containing the 35S–β-glucuronidase (GUS) reporter gene and selectable antibiotic-resistance marker genes, and then introduced into tobacco BY-2 cells using an Agrobacterium method. More regenerated calli carrying the MITE1/2 construct survived on medium containing kanamycin than calli lacking MITE1/2. Regenerated calli containing MITE1/2 were more numerous having stronger GUS activity than those without MITE1/2. These results suggest that MITE1/2 enhances the expression of adjacent transgenes introduced into stable transformants and possibly into the endogenous DcPAL3 gene.