Cloning and characterization of drought/salinity-responsive genes from sunflower.

摘要

Drought and high salinity are two of the most common stresses that adversely affect plant water status. Cloning and characterization of drought- and/or salinity-responsive genes are very important for our understanding of the mechanisms involved in plant stress response and tolerance.; Differential display reverse transcriptase-PCR was employed in this work because of its simplicity, reproducibility, sensitivity, versatility, ability to detect novel genes and cost-effectiveness. Seventeen putative drought-/salinity-responsive cDNA clones were obtained and characterized. Sequence analysis of these clones identified six with homology to known genes. These included a guanylate kinase (second-messenger signal transduction), LytB (penicillin tolerance), selenium-binding protein (heavy metal stress), AC-like transposable element, polyprotein (reverse transcriptase) and ribosomal protein L41.; The full-length cDNAs corresponding to five clones were isolated using rapid amplification of cDNA ends. These cDNAs encode a guanylate kinase (designated SGK-1), a NAC-domain protein (SNAC-1), a blight-associated protein p12 homolog (SBAP12), ribosomal protein S28 (SRPS28) and an ABA-responsive protein (SABRC5). Also, a simple and efficient PCR-based technique which combines both restriction endonuclease digestion and deoxynucleotide tailing, was developed for cloning unknown genomic sequences (e.g., promoters, introns, 5′ and 3′ untranslated regions) adjacent to a known sequence. Using this method, we cloned the upstream promoter region of SABRC5 . Sequence characterization of the SABRC5 promoter identified three ACGT-containing abscisic acid-responsive elements.; Expression of genes corresponding to thirteen cDNA clones was confirmed to be drought/salinity regulated using quantitative reverse transcriptase PCR. Expression analysis of these genes revealed three different patterns. The first pattern showed that gene expression is either exclusively up- or down-regulated in all organs in response to both stresses. The expression of genes grouped in the second pattern is up- or down-regulated by drought-stress only, and not affected by salinity. The third pattern shows a differential effect between drought- and salinity-stress and between organs. For this pattern, expression is up- or down-regulated depending upon the organ and/or the stress.; Abscisic acid is an important signal that helps modulate a plant's response(s) to environmental stresses (i.e., drought and high salinity). Therefore, gene expression in response to exogenous abscisic acid, was also analyzed for the above clones. Our results showed that expression of genes with pattern I [i.e., clones RSC1-U (SBAP12), RSC5-U (SABRC5), RSG11-U and RSG15-U (SNAC-1)] was up-regulated by exogenous abscisic acid in both seedling roots and shoots, but transcription of genes with patterns II or III [i.e., clones VC2-D (lytB), CAp1-1U (SGK-1) and RSG10-U (polyprotein gene)] was not affected by abscisic acid treatment. These results suggest that plants respond to drought- or salinity-stress in different pathways, and that cross-talk between both stresses is through an abscisic acid-responsive pathway.