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

Abstract

Gene dosage and genetic background are factors that influence transgene expression in transgenic plants. In our previous studies, we produced transgenic tomato plants that accumulate miraculin, a taste-modifying protein, in a genetically stable manner. To elucidate the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, we generated hybrid tomato lines between the homozygous transgenic line 56B (background cultivar 'Moneymaker') and the pure cultivars 'Micro-Tom,' 'Moneymaker,' 'Ailsa Craig,' 'M82,' 'Rutgers' and 'Aichi-first' and analyzed them for miraculin mRNA expression and miraculin protein accumulation. The hybrid lines exhibited variation in their fruit structures. Among the hybrid lines heterozygous for the miraculin gene, miraculin accumulation in the fruits varied from 111.0 mg g Ϫ1 fresh weight (FW) to 159.4 mg g Ϫ1 FW. Furthermore, the homozygous line 56B showed higher miraculin accumulation and miraculin mRNA expression than the heterozygous line 56Bϫ'Moneymaker.' These results demonstrate the profound effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits.Key words: Gene dosage, genetic background, miraculin, taste-modifying protein.Plant Biotechnology 27, 333-338 (2010) Original PaperThis article can be found at http://www.jspcmb.jp/ allelic composition and genetic background affect transgene expression and inheritance in white clover. Another study in rice showed that the expression pattern of Xa3/Xa26, which is a rice disease resistance gene, was affected by the genetic background (Cao et al. 2007). The full effects of gene dosage and genetic background on transgene expression and translation remain to be elucidated.In this study, we generated hybrid lines between transgenic line 56B (background cultivar 'Moneymaker'), which is homozygous for the miraculin gene, and the pure line cultivars 'Micro-Tom,' 'Moneymaker,' 'Ailsa Craig,' 'M82,' 'Rutgers' and 'Aichi-first.' Characterization of the hybrids' miraculin mRNA expression and miraculin protein accumulation, revealed the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, and in addition we discuss the significance of combining molecular breeding with cross-breeding to produce transgenic tomatoes that accumulate miraculin. Materials and methods Plant material and growth conditionsThe transgenic tomato line 56B was produced in our previous work (Sun et al. 2007). In brief miraculin encoding 660-bp DNA fragment was inserted into the XbaI/SacI sites of the plant transformation vectors pBI121 (Sun et al. 2006). Line 56B possesses a single copy of the miraculin gene driven by the CaMV 35S promoter.In the present study, the miraculin-accumulating transgenic tomato line 56B (background cultivars 'Moneymaker') was crossed with six other cultivars: 'Micro-Tom (accession number TOMJPF00001), ' 'Moneymaker (accession number TOMJPF00002), ' 'Aichi-first (accession number TOMJPF00003), ' 'Ailsa Cra...

Transgene behavior in Zea mays L. crosses across different genetic backgrounds: Segregation patterns, cry1Ab transgene expression, insecticidal protein concentration and bioactivity against insect pests

“…Ponnala et al [ 77 ] also showed a positive correlation between transcript levels and corresponding proteins in maize, in their case using non-GM varieties. In other GM crops, others have reported a correlation between transgene transcription and Cry1Ab concentrations for transgenic tomato [ 48 , 78 ] and cotton [ 29 , 30 ]. We are led to state, as a working hypothesis, that the crossing of the cry1Ab transgene into heterogeneous genetic contexts differs significantly from crossings within well-defined homogenous backgrounds.…”

High-level expression of sucrose inducible sweet potato sporamin gene promoter: &#x3b2;-glucuronidase fusion gene in transgenic <i>Nicotiana plumbaginifolia</i>

“…In order to overcome degradation of heterologous proteins, previous studies presented some potential solutions, such as to transport heterologous proteins to organelles. For example, the taste-modifying protein, miraculin was able to be accumulated in tomato fruit apoplast at levels over 150 µg/g fresh weight (Hiwasa-Tanase et al 2011;Kim et al 2010). Also, in the case of chloroplast, cry2Aa2 accumulated in tobacco leaf chloroplasts at a level over 70% of TSP (Oey et al 2009).…”

The Effects of Transcription Directions of Transgenes and the gypsy Insulators on the Transcript Levels of Transgenes in Transgenic Arabidopsis

“…This could be the reason why some plants co-overexpressing multiple anti-stress genes don’t perform better than a single gene transgenic plants 22 , 23 . Besides, in some cases, variation of gene expression might even lead to totally unexpected plant phenotypes 24 .…”