Cloning, Expression Of GLDH Gene Of Tobacco And Analyzing The Contribution Of GLDH To AsA Biosynthesis In Cells

Ascorbic acid (AsA) has been found to play important roles in many physiological processes of higher plants including resistance to abiotic stresses and pathogen attack. In plants AsA is biosynthesized in mitochondria with the last step (L-galactono-γ-lactone → AsA) being catalyzed by L-galactono-y-lactone dehydrogenase (GLDH EC 1.3.2.3). Some refernces reported that AsA biosynthesis in plants could be modulated by mitochondrial respiratory chains. But the deep modulating mechanisms are still not quite clear. This thesis is aimed at ecucidating the modulating mechanisms of mitochondrial respiratory chains on AsA biosynthesis in plants.At first, an 805bp fragment of tobacco GLDH encoding gene was successfully amplified by RT-PCR. This fragment was further cloned into a prokaryotic expression vector, pET-42a. Results of restriction enzyme digestion and DNA sequencing showed that the insertion direction was correct and no frameshift existed. This recombinant prokaryotic expression vector (pET-GLDH) was used to transform E. coli. SDS-PAGE results of the proteins from the transformed E. coli showed a specifically expressed band with the apparent molecular weight of about 66kD, which suggests that the fragment of tobacco GLDH encoding gene was expressed in the transformed E. coli as a part of a fusion protein. This fusion protein was found to acquire a better expression under the induction of 0.75mM IPTG for 3hr at 27℃. The above research will help to get the antibody against GLDH which is necessary for the study on the modulating effects of mitochondrial respiratory chains on GLDH expression in plants.Secondly, HPLC (high performance liquid chromatography) was succefully used to detect the AsA biosynthesis activity in plant. The contributions of GLDH and respiration complex I to the AsA biosynthesis activity in suspension cultured tobacco cells were determined. It was found that during high temperature stress (40℃) AsA biosynthesis activity in suspension cultured tobacco cells decreased. The contribution of GLDH to AsA biosynthesis activity under this stress condition obviously decreased, but the contribution of respiration complex I obviously increased.