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. 2004 Dec 2;32(21):e171. doi: 10.1093/nar/gnh170

Figure 2.

Figure 2

Preparation of plant 7SL RNA gene promoter-based siRNA expression constructs. A promoter fragment (7SL-P, 289 bp) containing USE and TATA elements (47) and a 3′-UT region (267 bp) of Arabidopsis At7SL4 (AY525344) gene were cloned and ligated in pUC19, from which the 7SL-P::UT construct was excised and cloned into the pGPTV-HPT vector (41) to replace the pAnos-uidA fragment. The resulting vector, pGPSL, contained an hpt selectable marker gene under the control of a nopaline synthase promoter (Pnos)-transcription terminator (pAg7, agropine synthase polyadenylation signal sequence) pair. GUS gene-specific 7SL-GT1, 7SL-GT2 or 7SL-GT3 sequence module, which contained a termination signal of seven thymidines, for the generation of the corresponding hairpin, siRNA was inserted into pGPSL between 7SL-P and 3′-UT. The resulting binary vectors were named pGPSL-HPT-GT1, pGPSL-HPT-GT2 and pGPSL-HPT-GT3, respectively. The binary vector was then mobilized into A.tumefaciens C58 for transforming tobacco. The predicted secondary siRNA structures of 7SL-GT1, 7SL-GT2 and 7SL-GT3 are depicted.