Expression of thymosin α1 concatemer in transgenic tomato (Solanum lycopersicum) fruits.

Tα1 (thymosin α1), 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. Tα1 is one of two thymosin proteins that have potential future clinical applications. In order to express Tα1 protein in plants, we designed and synthesized the Tα1 gene according to the plant codon usage bias and created a novel 4×Tα1 concatemer (four copies of the Tα1 gene arranged end-to-end in tandem, designated 4×Tα1). Subsequently, a plant binary expression vector, PG-pRD12-4×Tα1, 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×Tα1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4×Tα1 protein reached a maximum of 6.098 μg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4×Tα1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4×Tα1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Tα1 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 Tα1 in plants, and also it will provide the basis for further development of the plant system to produce Tα1. [ABSTRACT FROM AUTHOR]