High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator

Abstract

In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene's native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 μg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.

Evaluation of Codon Biology in Citrus and Poncirus trifoliata Based on Genomic Features and Frame Corrected Expressed Sequence Tags

“…58 Recently, it has been postulated that codon optimization significantly enhanced MIR gene expression in Solanum lycopersicum cv. Microtom, 59 which potentially depicts the importance and the usage of the optimal codons in gene expression and transgenics. To our knowledge, this is the first time large-scale identification of optimal codons in Citrus species and P. trifoliata , which could serve as a model repertoire to enhance the transformation efficiency.…”

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).…”

Overexpression of a Fungal β-Mannanase from Bispora sp. MEY-1 in Maize Seeds and Enzyme Characterization

“…Using this method, Hiwasa-Tanase et al has successfully achieved high-level expression of a miraculin gene in transgenic tomato [41]. Li et al improved the Bt cry1Ah gene expression in transgenic maize through codon optimization [42].…”