Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits

Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.