THE GENE FOR THE MAJOR CUTICULAR WAX-ASSOCIATED PROTEIN AND 3 HOMOLOGOUS GENES FROM BROCCOLI (BRASSICA-OLERACEA) AND THEIR EXPRESSION PATTERNS

The gene for the major protein (WAX9) found in surface wax of broccoli , designated wax9D, and three homologous genes (wax9A, B and C) were i solated from a genomic library using the previously isolated cDNA enco ding the WAX9 protein as the probe; all four genes were sequenced. Gen omic Southern blot analysis using the WAX9 cDNA as a probe showed the presence of at least four homologous genes in broccoli genome. The seq uence of the originally isolated WAX9 cDNA matched with that of gene D . All four genes have an intron two codons before the stop codon. The putative promoter regions of the four genes, beyond the first 200 bp i mmediately 5' to the translation start sites, are quite different. Ess ential elements such as TATA and CAAT boxes and several regions homolo gous to the promoter regions of other plant Itp genes were identified. The expression patterns of the genes were determined by RT-PCR with g ene-specific primers and sequencing of the PCR products. All the genes were expressed in leaves and flower buds. While genes A, B and D also were expressed in stems and open flowers, expression of gene C was no t detected in these organs. None of them were expressed in roots. The 972 bp 5'-flanking region of wax9D when fused to beta-glucuronidase (G US) gene, directed tissue-specific GUS expression in transgenic tobacc o plants; GUS expression was found in the epidermis of leaves, stems a nd flower petals, sepals, trichomes, and ovules but not in roots.