In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Existem quase 260.000 clones independentes, seqüenciados a partir da extremidade 5’, no banco de dados do SUCEST (Sugarcane Expressed Sequence Tag), os quais foram obtidos a partir de 37 bibliotecas de cDNA preparadas de diferentes tecidos. Este grande número de etiquetas de sequências expressas (ESTs) fornece uma oportunidade, sem precedentes em plantas, de realizar um ‘digital differential screening’ em bibliotecas de cDNA selecionadas. Geralmente, a frequência de um determinado EST está correlacionada ao acúmulo de transcritos nos tecidos dos quais as bibliotecas de cDNA foram construídas, e desta forma, é possível comparar o transcriptoma completo de diferentes tecidos, usando uma análise computacional de um banco de dados de ESTs. Em nossa pesquisa, analisamos os ESTs de cana-de-açúcar de acordo com sua expressão tecidual e identificamos mais de 1.000 putativos genes específicos de flor. O fato de que usando esta técnica fomos capazes de identificar homológos em cana-de-açúcar, de vários genes previamente descritos como específicos de pólen, sustenta este método de estimar especificidade tecidual. Além disto, ESTs com similaridade a genes específicos de órgãos reprodutivos foram revelados, como por exemplo, o gene que codifica uma proteína meiótica essencial para a montagem do complexo sinaptonêmico e sinapse normal. Esta abordagem também permitiu a identificação de muitas sequências anônimas, específicas de flor, que são boas candidatas para novos genes envolvidos com a reprodução de plantas. Este trabalho descreve a análise dos níveis de expressão gênica de 24 clusters de ESTs, durante o desenvolvimento floral, usando um ‘northern blot digital’ construído a partir da contagem direta dos ESTs das bibliotecas não-normalizadas de cDNAs de cana-de-açúcar.

There are almost 260,000 independent clones sequenced from the 5’ end in the Sugarcane Expressed Sequence Tag (SUCEST) database, which have been obtained from 37 cDNA libraries prepared from different tissues. This large number of expressed sequence tags (ESTs) provides an opportunity, unprecedented in plants, to perform ‘digital differential screening’ on selected cDNA libraries. In general, the frequency of a particular EST correlates with transcript accumulation in the tissues from which the cDNA libraries were constructed, so it is possible to compare the whole transcriptome from different tissues using computer-assisted analysis of an EST database. In our research we analyzed sugarcane ESTs according to tissue expression and identified more than 1,000 putative flower-specific genes. The fact that using this technique we were able to identify sugarcane homologues of several genes previously described as pollen-specific justifies this method of assessing tissue specificity. In addition, ESTs similar to genes specific to reproductive organs were detected e.g. a sugarcane gene encoding a meiotic protein essential for assembly of the synaptonemal complex and normal synapsis. This approach also allowed the identification of many flower-specific anonymous sequences that are good candidates for being novel genes involved in plant reproduction. This paper describes the analysis of the gene expression levels of 24 EST clusters during flower development using a ‘digital northern blot’ constructed from direct EST counts made on the non-normalized sugarcane cDNA libraries.