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DIFFERENTIAL SUPPRESSION OF TWO DIFFERENT  ANNEXINS IN CERATOPTERIS USING RNA INTERFERENCE. S. J. Roux, G. B. Clark, S. C. Stout, and S. Archer-Evans.  Sect. of Molecular Cell & Developmental Biology, Univ. of Texas, Austin.

   Annexins, which are calcium-dependent, membrane-binding proteins, have been implicated in a variety of cellular functions in plants and animals, including secretion and calcium channel activity.  We have isolated and sequenced two different full-length cDNAs, AnnCr1 and AnnCr2, that are both expressed in germinating spores of the fern Ceratopteris.  The proteins encoded by these two genes are 78% identical in sequence. To test the possible role of these genes in spore polarity development (which is directed by gravity), we suppressed their expression using RNA interference (RNAi). Gene-specific primers were used to amplify double-stranded (ds) annexin constructs of 200 base pairs from the 3’ regions of AnnCr1 and AnnCr2. Intact spores readily take up dsRNA during their hydration. As judged by sensitive RT-PCR assays, treatment of spores with annexin dsRNA significantly and selectively reduced the level of each annexin mRNA, but not other messages, indicating that the effects of the dsRNA treatments are relatively specific. The effectiveness of these constructs in suppressing the expression of annexin genes was concentration- and sequence-dependent. Sequences of dsRNA based on ORF regions were effective; sequences based on 3’ untranslated regions were not. Suppression was effective for more than
 24 h. Resultant phenotypes from dsRNA treatments ranged from inhibition of germination to delayed and altered development, although initial results indicate these effects occur only when high concentrations of dsRNA are used, and the specificity of RNA suppression at these concentrations is not yet clear. Spores can also take up single-stranded antisense constructs, and these are also effective in selectively depressing annexin mRNA levels, but the sequence dependency of these effects is different from that required for dsRNA effectiveness, suggesting a possibly different mode of action.

(Supported by NAG2-1347 and NAG10-295 to S.J.R.)

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