A15, A SEED COAT-SPECIFIC PROMOTER FROM ARABIDOPSIS THALIANA

Abstract

The purpose of this project was to characterize a seed-coat specific promoter isolated from Arabidopsis thaliana. The promoter and coding region of the clone A15 was found by screening a silique-specific Arabidopsis thaliana cDNA library followed by Inverse PCR of a genomic Arabidopsis thaliana library. Sequence of the A15 clone was determined and verified by three independent PCRs and used for characterization by molecular and histochemical analysis. The A15 putative promoter region was fused with the B-glucuronidase (GUS) coding region of the uid A gene and cloned into the binary vector pRD400. Transgenic Arabidopsis thaliana plants were produced by Agrobacterium-mediated transformation and used for analysis of temporal and spatial expression. Seed coat-specific expression of GUS occurred in the late stages of silique development. Expression of GUS was localized specifically to the outermost mucilage producing cell layer of the seed coat and expression of GUS in root, stem, bud, flower and leaf was not detected. The specificity of the A15 promoter was tested by genetic ablation using the A-chain of the diphtheriatoxin (DTx-A)as a reporter gene. The expression of even a few DTx-A molecules results in cell death and provides a more sensitive method of detecting A15 promoter-regulated gene activity in other tissues than GUS histochemical analysis. The A15 putative promoter fused to the DTx-A gene was used to produce transgenic plants by Agrobacterium­ mediated transformation and tissues were collected for analysis. Thin section microscopy of transgenic plants revealed a similar pattern of A15 expression with genetic ablation of the mucilage producing cells. The minimal functioning region of the A15 promoter was determined by genetic analysis using 5' deleted promoters of 553, 467, 379, 185bp lengths. These promoters were fused to the gus gene in pRD400 for the production of transgenic arabidopsis plants. Histochemical analysis and fluorometric analysis of the transgenic seed showed that the minimal promoter for gene expression was less that 185bp in length. Genetic analysis of the A15 clone included Southern hybridization and sequence analysis by BLASTn of non-redundant sequence groups. Southern hybridization detected 2 copies of the A15 promoter and 4 copies of the A15 coding region in genomic Arabidopsis thaliana ecotype Columbia DNA. The BLASTn search revealed two overlapping BAC clones on Chromosome 1 in Arabidopsis thaliana that contained the A15 promoter and coding region. Sequence analysis of the BAC clones revealed four copies of the A15 coding region with greater than 90% sequence identity. In addition, two of the copies contained over 90% sequence similarity to the 954bp putative promoter region. The positions of the four A15-like coding regions on the two BAC clones were mapped and found to be clustered in a 50Kb region with two of the coding regions tightly linked within a 5Kb region.