26 Czech J. Genet. Plant Breed., 50, 2014 (1): 26–31 Original Paper Expression of Escherichia coli Heat-labile Enterotoxin B Subunit in Transgenic Tomato ( Solanum lycopersicum L. ) Fruit Nguyen Hoang LOC 1 , Dang Thanh LONG 1 , Tae-Geum KIM 2 and Moon-Sik YANG 2, 3 1 Institute of Resources, Environment and Biotechnology, Hue University, Hue, Vietnam; 2 Department of Molecular Biology, Chonbuk National University, Jeonju, Republic of Korea; 3 Jeonju Center, Korea Basic Science Institute, Jeonju, Republic of Korea Abstract Loc N.H., Long D.T., Kim T.-G., Yang M.-S. (2014): Expression of Escherichia coli heat-labile enterotoxin B subu- nit in transgenic tomato (Solanum lycopersicum L.) fruit. Czech J. Genet. Plant Breed., 50: 26–31. We report a feasibility study for expressing the LTB protein ( Escherichia coli heat-labile enterotoxin B subunit) via Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.). We produced five regenerated plants obtained on the selection medium supplemented with an antibiotic. Stable integrations of the LTB gene into the genome of these plants were confirmed by Southern blot hybridization. Western blot analysis showed that only two of the five T 0 transgenic tomato plants expressed the pentameric LTB protein in the fruits. An enzyme-linked immunosorbent assay indicated that these two plants synthesized the LTB protein bound specifi- cally to GM1 ganglioside, suggesting that the LTB subunits formed active pentamers. The LTB protein produced in tomatoes can be a potential candidate for inexpensive, safe, and effective plant-based vaccines. Keywords: B subunit of E. coli heat-labile enterotoxin (LTB); LTB gene; Solanum lycopersicum; plant-based vaccine; tomato fruit; transgenesis Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children in developing coun- tries and in travellers (Chen et al . 2011). The LTB (E. coli heat-labile enterotoxin B subunit) of ETEC is a potent mucosal immunogen and immunoadjuvant for co-administered antigens (Cheng et al . 1999; Ryan et al . 1999; Millar et al . 2001). Many studies have indicated that LTB could be used as a potent adjuvant (Tochikubo & Yasuda 2000). LTB is highly resistant to proteolytic degradation and retains its pentameric quaternary structure in a pH as low as 2.0 (Rez aee et al . 2005). LTB is able to bind to GM1 ganglioside, a glycosphingolipid found ubiquitously on the cell membranes of mammals, and to other related receptors, such as GD1b-ganglioside, asialo-GM1, lactosylceramide and certain galacto- proteins (Williams et al . 1999). The tomato is one of the preferred plants for trans- formation for oral vaccines since its fruits are edible when fresh. Jani et al . (2002, 2004) and Loc et al . (2010b) successfully transferred the CTB (cholera toxin B subunit) gene controlled by the CaMV 35S promoter into tomato plants, and these transgenic plants expressed the CTB subunit in leaves and fruits, which could specifically bind to the GM1-ganglioside receptor, a special receptor for the CTB subunit. The purpose of this work was to determine the ex- pression of recombinant LTB in the fruit of transgenic tomatoes. The gene encoding LTB was introduced into tomato ( Solanum lycopersicum L.) plants, and the LTB protein was found to have a pentameric form with the ability to bind GM1 ganglioside. MATERIAL AND METHODS Plant material. Tomato (Solanum lycopersicum L. Cv. 311) seeds were germinated under sterile condi- tions on 1/2MS medium (Murashige & Skoog 1962) consisting of 30 g/l sucrose and 8 g/l agar. After ten days, cotyledons excised from in vitro-germinated