Role of miniature inverted repeat transposable elements inserted into the promoter region of a carrot phenylalanine ammonia-lyase gene and its gene expression
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The expression of the phenylalanine ammonia-lyase gene (<i>PAL</i>) was induced in anthocynanin-producing suspension-cultured carrot cells. In our previous study, we isolated two <i>PAL</i> genes, <i>DcPAL3</i> and <i>DcPAL4</i>, from a carrot genomic library, the nucleotide sequences of which are highly similar. Here, the complete nucleotide sequence of <i>DcPAL4</i> was determined and compared with that of <i>DcPAL3</i>, revealing two miniature inverted repeat transposable elements (MITEs), here designated MITE1 and MITE2, inserted into the proximal promoter region of <i>DcPAL3</i>, but not into that of <i>DcPAL4</i>. The expression of <i>DcPAL4</i> was not detectable by RT–PCR in cultured carrot cells or the tissues of carrot plants, whereas <i>DcPAL3</i> expression was detected. Transient expression experiment in carrot protoplasts showed that the <i>DcPAL4</i> promoter actively produces transcripts, as does the <i>DcPAL3</i> 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 (<i>GUS</i>) reporter gene and selectable antibiotic-resistance marker genes, and then introduced into tobacco BY-2 cells using an <i>Agrobacterium</i> 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 <i>DcPAL3</i> gene.
- Plant tissue culture letters
Plant tissue culture letters 25(5), 473-481, 2008-12-01
Japanese Society for Plant Cell and Molecular Biology