Abstract:

The 1 216 bp 5'' upstream r-egion of the gene encoding the class ⒒ chitinase VCH3 was isolated from grapevine (Vitis amurensis Rubr.) (Genbank accession number AF441123) and two inverse salicylic acid (SA) responsive cis -acting motifs (TGACG) were found at -1 181 bp and -293 bp upstream of the transcriptional start site, respectively. To characterize the VCH3 promoter, four chimeric constructs varied in the length of promoter fragments from -1 187 bp, -892 bp, -589 bp and -276 bp to +7 bp relative to the transcriptional start site were placed to the upstream of the 円-glucuronidase (GUS) coding region and transferred to Nicotiana tobacum L. cv. NC89 by Agrobacterium tumefaciens-mediated leaf discs transformation. The functional properties of each promoter fragment were examined by fluorometric and histochemical analysis of GUS activity in the transgenic tobacco root treated with SA. The VCH3 (-276) GUS construct, containing only the TATA and CAAT boxes was shown to have little inducibility upon treatment with SA. However, the similarly higher level of GUS expression was observed in the VCH3 (-589) GUS or VCH3 (-892) GUS transgenic plants with only one cis -acting motif, while the most abundance of GUS expression was found in the full-length promoter (-1187 bp to +7 bp) with two cis -acting motifs. These results indicated that the two cis -acting motifs were required for the maximal expression of the GUS reporter gene by SA induction. In addition, the histochemical analysis of GUS activity showed that the four VCH3 promoter fragments were more active in vascular tissue than that in outer and inner cortexes of the transgenic tobacco roots treated by SA, suggesting that the region involved in vascular tissue-specific expression of VCH3 promoter upon SA inducibility appears to be located between positions -276 bp and +7 bp relative to the transcriptional start site. In general, these results indicate a potential use for the SA induction of VCH3 promoter in genetic engineering.

Key words: salicylic acid (SA), induction, VCH3 promoter, cis-acting motif, vascular tissue-specific expression