Thaumatin expression in transgenic cucumber plants

Abstract

Thaumatin is produced in the African plant known Thaumatococcus daniellii as a family of five or more proteins [Higginbotham, Hough, 1977]. Thaumatins are nearly 100000 times sweeter than sucrose on a molar basis [Iyengar et al., 1979]. Taumatins: I and II are the most abundant in fruit, and the cDNA for thaumatin II has been cloned and sequenced [Edens et al., 1982]. So thaumatin is a potentially interesting target for genetic engineering studies and we have recently begun work in this direction developing Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) with thaumatin II gene. We used an efficient plant regeneration procedure described by Burza and Malepszy [1995]. Transformation was carried out using the binary plasmid pRUR528s bearing sense construct of T. daniellii thaumatin II cDNA under the control of constitutive promoter 35S CaMV [Szwacka et al., 1996]. Transformation status was confirmed by PCR amplification of nptII gene. Genomic DNA hybridization experiments proved that independent primary transformants carried one to three copies of the expression cassette. Here, we present data on thaumatin mRNA transcription in independent primary transformants (Fig. 1A). We observed marked differences in the level of thaumatin transcript. Western blot analysis (Fig. 1B) indicated the presence of mature thaumatin (22 kDa) [Vos de, 1985] in five from fifteen analysed primary transformants. In two of them (no. 214 and no. 224) high level of thaumatin mRNA correlated with high level of mature thaumatin (Fig. 1A and 1B).