Genetic Transformation Of SusA, GalE And LgtC Into Tobacco And Analysis Of Soluble Carbohydrates In Transgenic Lines

Compared to other biological reactors, like microbes and animal cell expression systems, plants have many advantages, including: 1) post-translational modification to produce biologically active products; 2) simpler isolation and purification procedure than that of other systems; 3) more abundant substrate and thus lower cost; 4) no ethical concerns, nor viral contaminations, compared with transgenic animals. Therefore, transgenic plants as a new type of biological reactor posses great potentials. Using plant to produce biologically active products, including higher value proteins, carbohydrates and lipids for clinical, food and industrial purpose has been widely reported. In plants carbohydrates are produced via photosynthesis in leaves and transferred to root, stem and seeds to be stored. Modification of the carbohydrate metabolic pathways using genetic approaches could transform plants into biological reactors to produce higher value oligo-, poly-saccharide and glycoconjugates.Globotriose is an oligosaccharide with many important application. In E.coli., three genes were needed for its synthesis: 1) susA, encodes Sucrose synthase (EC2.4.1.13): 2) galE, encodes UDP-galactose 4-epimerase (EC 5.1.3.2) and 3) lgtC, encodes α-1,4-galactosyltransferase (EC 2.4.1.x). We intend to transform the above genes into tabacco to produce globotriose. First we cloned these genes into a plant expression vector pCAMBIA1300-35S-Tnos-als individually in sense or antisense direction. Six constructs were obtained: plgtC (+/-), pgalE (+/-) and psusA (+/-). Then Agrobacterium tumefaciens strain LBA4404 carrying the above constructs were used to transform the tobacco leaf discs. Putative transgenic plantlets were selected on selecting medium and analyzed by PCR, and some of transgenic plantlets were confirmed by Northern blotting. Expression of target genes were confirmed in the transgenic tobaccos.Concentration of soluble carbohydrate (CSC) was examined in transgenic tobacco lines and compared with control. Out of the 4 lines transformed with psusA (+), CSC was comparable and significantly higher (P≤0.05) in 3 lines but not significantly different in one line. Out of the 4 lines transformed with psusA (-), CSC was extremely higher (P≤0.01) in 3 lines but not significantly different in the other one.