Characterization of an 18,166 EST dataset for cassava (Manihot esculenta Crantz) enriched for drought-responsive genes.

Cassava (Manihot esculenta Crantz) is a staple food for over 600 million people in the tropics and subtropics and is increasingly used as an industrial crop for starch production. Cassava has a high growth rate under optimal conditions but also performs well in drought-prone areas and on marginal soils. To increase the tools for understanding and manipulating drought tolerance in cassava, we generated expressed sequence tags (ESTs) from normalized cDNA libraries prepared from dehydration-stressed and control well-watered tissues. Analysis of a total of 18,166 ESTs resulted in the identification of 8,577 unique gene clusters (5,383 singletons and 3,194 clusters). Functional categories could be assigned to 63% of the unigenes, while another approximately 11% were homologous to hypothetical genes with unclear functions. The remaining approximately 26% were not significantly homologous to sequences in public databases suggesting that some may be novel and putatively specific to cassava. The dehydration-stressed library uncovered numerous ESTs with recognized roles in drought-responses, including those that encode late-embryogenesis-abundant proteins thought to confer osmoprotective functions during water stress, transcription factors, heat-shock proteins as well as proteins involved in signal transduction and oxidative stress. The unigene clusters were screened for short tandem repeats for further development as microsatellite markers. A total of 592 clusters contained 646 repeats, representing 3.3% of the ESTs queried. The ESTs presented here are the first dehydration stress transcriptome of cassava and can be utilized for the development of microarrays and gene-derived molecular markers to further dissect the molecular basis of drought tolerance in cassava.

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.).

Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing ß-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis.

Highly sensitive northern hybridization using a rapid protocol for downward alkaline blotting of RNA.

A simple and fast RNA gel blot procedure is described that uses 50 mM NaOH to simultaneously transfer and fix RNA to a positively charged nylon membrane. The RNA is transferred in a downward direction, and transfer is routinely completed within 2.5 h. The resulting blots give increased sensitivity over existing methods without affecting RNA integrity and can be used in both radioactive and nonradioactive detection procedures.

Different 3' end regions strongly influence the level of gene expression in plant cells.

We have investigated the functional role of a 3' end region on the expression of a reporter gene in plant cells. In stably transformed plants, expression of the reporter gene without a plant gene 3' end is variable and depends on the fortuitous presence of polyadenylation signals in the downstream sequences. When the reporter gene is flanked by pBR322 DNA, 3'-processing and polyadenylation occurs at (a) cryptic site(s) within these vector sequences. Using a transient gene expression system, we present a deletion analysis of the 3' end of the octopine synthase gene showing that the most proximal polyadenylation signal per se is not sufficient to ensure expression but that a downstream (G)T-rich sequence is also required. Optimal expression of the fusion gene requires more than 98 base pairs and at most 142 base pairs downstream from the most distal polyadenylation site. We analyzed the expression of chimeric genes with 3' end sequences originating from different plant genes. In the transient expression assay, all constructs direct similar neomycin phosphotransferase II activities. However, in stably transformed tissue, the gene constructs displayed characteristic expression levels which varied as much as 60-fold. This result suggests a role for 3' end sequences in post-transcriptional processes such as efficiency of 3'-processing and/or mRNA stability.