Spatial and Developmental Profiling of Miraculin Accumulation in Transgenic Tomato Fruits Expressing the Miraculin Gene Constitutively

Abstract

We previously developed a transgenic tomato that expresses the miraculin gene using a constitutive promoter. In this study, we profiled the developmental and spatial accumulation of the miraculin protein and mRNA in transgenic tomato fruits. Miraculin mRNA expression was almost constant up to orange stage, and then the expression increased at red stage. The miraculin protein accumulated gradually during fruit development and reached its highest level at the overripe stage. At the red stage of fruit, miraculin protein was accumulated at the highest level in the exocarp, and similar in other fruit tissues: mesocarp, dissepiment, upper placenta, lower placenta and jelly. Moreover, the pattern of miraculin accumulation in fruit tissues was the same regardless of genetic background and position at which the miraculin gene was inserted in the genome. We also discuss suitable tomato types expressing miraculin for their commercial use.

Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits

“…The mesocarp, dissepiment, upper placenta, lower placenta, and seeds showed almost the same accumulation levels. The jelly showed the lowest accumulation levels, which corroborates our previous report (Kim et al 2010). Despite the different genetic backgrounds, the accumulation pattern of miraculin in the hybrid lines was similar to that of line 56B.…”

“…Once elite transgenic line is obtained, the elite line can be improved by crossing with other lines conferring superior traits. Previously, we reported the ratio of individual tissue weights to whole fruit weight in pure cultivar lines (Kim et al 2010). The fruits weights of pure cultivar lines, 'Aichi-first' and 'Micro-Tom' used in this study were 263.4 and 3.66 g FW, respectively.…”

“…Ubiquitin3 has been described previously (Hoffman et al 1991) and was used as a control (Hackel et al 2006;Leclercq et al 2005). The primer sequences have also been described previously (Kim et al 2010). …”

“…The extracts were centrifuged at 15,000ϫg for 20 min at 4°C. The resulting supernatant was used for immunoblot analysis and ELISA, which were performed according to Sun et al (2007) and Kim et al (2010), respectively. …”

“…We developed transgenic tomato plants that accumulate significant levels of miraculin protein (Sun et al 2007), and the protein produced had almost same level of tastemodifying activity as native miraculin from miracle fruits. Furthermore, we characterized the miraculin accumulation pattern in the transgenic tomato fruits in detail and found that miraculin accumulation was highest in the exocarp (Kim et al 2010). The exocarp is composed of small cells that allow high levels of miraculin protein to accumulate.…”