Leaf and Root-Specific Sorghum Promoters Whose Transcripts Are Also Tissue-Specific in Maize and Sugarcane

Future crop developments in the GM arena will undoubtedly require functional promoters with expressions restricted to specific tissues. We targeted tissue-specific transcripts by querying the Sugarcane Affymetrix® microarrays with leaf, root and stem cDNAs from sorghum and maize. The results provided a set of sugarcane sequences whose homologous counterparts in sorghum and maize are tissue-specifically expressed. A further selection of sequences was based on possible identity (or lack of) and ascertaining similar gene expression pattern in other plant species (BLAST and UniGene). The validation and quantification of expression of the selected sequences were carried out by qPCR on sugarcane, maize and sorghum tissue-specific cDNAs. In addition, a preliminary search was done to assess the Freedom to Operate of potential promoters associated with these transcript sequences (NCBI patent sequences database). Several candidates for promoter recovery were chosen. We assessed the expression of the different alleles by PCR amplification and cloning of a portion of the coding sequences. By sequencing a range of cloned amplicons and identifying SNPs within these sequences, we were able to target the strongest functional sorghum promoters corresponding to each chosen tissue-specific transcripts. Primers were designed from the sorghum genome sequence and the promoters were PCR-amplified. After cloning and sequencing, these promoters were introduced into GUS reporter-gene cassettes as transcriptional fusions. We will consider different avenues for validating in planta their true functionality and tissue-specificity.