Preliminary Study On Soluble Carbohydrate Composition Of Transgenic Tobacco Plants Containing GalE Or LgtC Gene

galE gene, coming from Escherichia coli, encodes UDP-galactose-4-epimerase (EC 5.1.3.2), which catalyzes the reversible conversation of UDP-Glc to UDP-Gal in regular metabolic pathways. lgtC, which comes from Neisseria Meningitidis, encodesα-1,4-galactosyl-transferase (EC 2.4.1.44). This enzyme mediates the transfer of galactosyl residue from donor to acceptor molecules. In this study, the variation of carbohydrate composition of transgenic tobacco cells containing galE or lgtC were studied respectively, in order to reveal the impact of heterologous expression of galE and lgtC gene on the metabolism and growth of transgenic tobacco plants.Various carbohydrates in transgenic plants were separated and identified by Thin Layer Chromotography (TLC) to determine the constituent and content of soluble carbohydrates in the leaves of transgenic tobacco accurately. Furthermore, the content of sucrose, glucose and fructose were measured with HPLC-RID, and the content of UDP-Glc and UDP-Gal were measured perfectly with RP-HPLC combining with PDAD.Determination of carbohydrate composition and galE enzyme activity in galE transgenic tobacco plantsIn this study, transgenic lines that overexpressed target gene, confirmed by RT-PCR, were chosen for determination of carbohydrate content, including total soluble carbohydrates, monosaccharides, disaccharides and nucleotide sugars. When substrate UDP-Glc were added to the crude protein extracts of transgenic lines, the prospective product of UDP-Gal was detected in the reaction mixture, implying the exist of the reaction catalyzed by galE. The galE activity of transgenic plants was measured under different conditions. The epimerase was active between pH 7and pH9 with optimum at pH 7.5-8, and the optimal reaction temperature of the epimerase was about 20℃. The yield of UDP-Gal in the sense transgenic lines were significantly higher, usually 1.5-1.6 times as that of the antisense transgenic and WT lines.The content of total soluble carbohydrates in sense transgenic lines (E+41, E+54) was significantly higher than that of WT. Accordingly, the levels of glucose, fructose and sucrose in these sense transgenic lines were all increased in considerable degrees. The content of UDP-Gal in sense transgenic lines (E+41, E+54) were extremely (P<0.01) higher than that of WT, while the content of UDP-Glc in the two lines were significantly (P<0.05) higher than that of WT also. However, the content of UDP-sugars, total soluble carbohydrates and other carbohydrate composition in the antisense transgenic line E-23 were not significantly different compared with WT plants.Observation of growth and measurement of photosynthesis of transgenic lines suggested that the heterologous expression of galE from prokaryote activated the growth and photosynthesis of transgenic tobacco plants. The sense transgenic plants had better growth potential than WT plants. They had longer root system in seedling period, much leaf area in vigorous growing period due to longer and wider leaf, and higher plant height in maturation period, most interesting, the initiation of blossom was much earlier than WT plants. Additionally an obvious increase in net photosynthesis rate had been observed in sense transgenic plants, compared with WT plants. On the other hand, the growth potential, initiation of blossom and net photosynthesis rate of the antisense transgenic plants were not significantly different from WT plants.Analysis of carbohydrate composition of lgtC transgenic tobacco plantsTransgenic lines with overexpression of lgtC gene, confirmed by RT-PCR, were chosen for determination of carbohydrate content, to reveal the effect of lgtC from prokaryote on the composition and content of carbohydrate in transgenic tobacco plants.Total soluble carbohydrates of the sense lines (C+27, C+43) were significantly higher than that of WT. The analysis of carbohydrate composition of transgenic tobacco plants indicated that the levels of glucose, fructose and sucrose in these sense lines were increased to some extent. The content of UDP-Gal and UDP-Glc in the two lines were all significantly (P<0.05) lower than that of WT. However, the content of total soluble carbohydrates, as well as glucose, fructose and sucrose in the antisense line C-32 were not significantly different compared with WT plants, while the content of the UDP-Gal and UDP-Glc decreased generally in the antisense line C-32.In this study, the variation of carbohydrate composition in galE or lgtC transgenic tobacco plants were investigated, respectively, and the impact of genetic modification of carbohydrate metabolic pathways on carbohydrate accumulation and growth of transgenic plants were also studied, with an attempt to utilize genes from prokaryote involved in carbohydrate metabolism to plant genetic engineering. This research not only provided some productive and useful lines for further study on the controlling mechanism of carbohydrate metabolism in plant cell, but also shed light on the insight into the cellular carbohydrate metabolism network and the controlling mechanism in plant cells, with great potential in the near future.