Cloning And Preliminary Functional Study Of The NAD~+-dependtent Sorbitol Dehydrogenase (NAD-SDH) In Tobacco

Sorbitol plays a very important role in the plant carbohydrate metabolism, it is the primary product of photosynthesis and the major translocation form of photosynthate in the Rosaceae family. It was found that sorbitol accumulation could improve plant stress tolerance. However, transformants that accumulate overfull sorbitol often show growth inhibition and symptom. NAD+-dependent sorbitol dehydrogenase (SDH) is a key enzyme in the metabolism of sorbitol, it catalyses the oxidation of sorbitol to fructose. The results of our research are as follows:1. We first cloned the full-length cDNA of tobacco (Nicotiana tabacum L.cv Xanthi NN) NAD+-dependent sorbitol dehydrogenase (Named NAD-SDH) by RT-PCR. It contains 1119 base pairs, and encodes a polypeptide consisting of 311 amino acid residues, with 94% and 87% identity to tomato and grape NAD-SDH gene respectively.2. NAD-SDH was cloned into pMAL-C2X vector and transformed into the E.coli TB1strain in order to express NAD-SDH. It is useful for protein purification and further study on it.3. NAD-SDH was cloned into novelty transient expression vector E3025-GFP. The reconstructed plasmid, E3025-GFP+ NAD-SDH, was bombarded into onion (Allium cepa) epidermal cell. It was found that NAD-SDH localized in the cell membrane.4. We obtained Arabidopsis NAD-SDH gene T-DNA knockout mutation, pCAMBIA1302+ NAD-SDH and pCAMBIA1305.1+ NAD-SDH transformed Arabidopsis. The preliminary comparative experiment in the drought tolerance showed that the drought tolerance ability of Arabidopsis T-DNA mutant was enhanced to some extent, compared with wild-type. It was also found that the drought tolerance ability of transgenic was depressed, compared with wild-type Arabidopsis. In a word, we first cloned the full-length cDNA of tobacco NAD-SDH and found that NAD-SDH mainly distributed in the membrane. This research provided an experimental and theoretical basis to the further study on molecular mechanism of tobacco sorbitol dehydrogenase, and the further study on the sorbitol function in plant.