An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin
The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.
Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.
KeywordsExpression vector E8 promoter HSP terminator Miraculin Transgenic tomato plants
We thank the members of the Ezura laboratory for their helpful discussions. This study was supported by the project ‘‘Development of Fundamental Technologies for the Production of High-value Materials Using Transgenic Plants’’ by the Ministry of Economy, Trade, and Industry of Japan (to H.E.). The Micro-Tom seeds (TOMJPF00001) were provided by the National BioResource Project (NBRP) of the MEXT, Japan.
- Bhattacharyya J, Chowdhury AH, Ray S, Jha JK, Das S, Gayen S, Chakraborty A, Mitra J, Maiti MK, Basu A, Sen SK (2012) Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants. Plant Cell Rep 31:271–279. doi: 10.1007/s00299-011-1161-4 PubMedCrossRefGoogle Scholar
- Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS (2004) Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic Res 13:541–549. doi: 10.1007/s11248-004-2737-3 PubMedCrossRefGoogle Scholar
- Hiwasa-Tanase K, Nyarubona M, Hirai T, Kato K, Ichikawa T, Ezura H (2011) 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. Plant Cell Rep 30:113–124. doi: 10.1007/s00299-010-0949-y PubMedCrossRefGoogle Scholar
- Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedCrossRefGoogle Scholar
- Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD, Buetow DE (2000) Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res 9:127–135. doi: 10.1023/a:1008979525909 PubMedCrossRefGoogle Scholar