Plant Genome IV Conference Plant Genome IV Conference
Coffea arabica (coffee) is a widely cultivated tetraploid tropical tree species. Promoters in this plant provide long term, constitutive or inducible expression of the genes under their control. A potential application of such promoters is the control of new transgenes introduced into the plant. Since few DNA sequences are known from Coffea spp., selected homologous genes of interest from different plants were compared for similarity among their promoters, finding it to be very low. Therefore, PCR primers were designed from consensus sequences in the coding regions. For the constitutive alpha-tubulin gene, a 350 bp fragment was amplified in C. arabica var. Caturra and Nicotiana tabacum var. Havana (control), corresponding to the first intron and parts of exons 1 and 2. Similarly, primers for the inducible defense gene phenylalanine ammonia lyase (PAL) produced a 250 bp fragment from exon 1. To amplify unknown upstream regions, 2 reverse primers designed from the cloned sequences (Specific primers) were used along with 12 non-specific forward primers (Walking primers), under combinations of low and high stringency annealing PCR conditions. Products were probed with alpha-tubulin or PAL digoxigenin labeled clones. 16 primer pairs amplified products ranging from 600bp to 1Kb, which hybridized with the probes (putative promoters). Sequencing and engineering of these products is under way to evaluate transient expression of GUS in microprojectile bombarded tissue. These results indicate that this system could be useful for cloning promoters of homologous genes and may be an alternative to methods involving screening of genomic libraries.