Cloning Of NAD-dependent Sorbitol Dehydrogenase (NAD-SDH) From Apple (Malus Pumila) Fruit And The Construction Of Its Antisense Expression Plasmid

As an alditol, sorbitol existing widely in most of the Rosaceae plants is the primary photosynthetic product, the major translocated sugar, and the main storage carbohydrate, playing a very important role in the carbohydrate metabolism of the Rosaceae plants. In addition, sorbitol is a kind of osmotic matter with small molecular weight, and it can change the osmotic potential of the cell; therefore, sorbitol can help plants out of many difficult environment, such as water stress, salt, chilling, and Boron efficient. The enzymes related with sorbitol metabolism are 6-phosphate sorbitol dehydrogenase, sorbitol dehydrogenase and sorbitol oxidase, which play a key role in the sorbitol metabolism; thereinto, sobitol dehydrogenase catalyzes the oxidation of sorbitol to fructose. In this dissertation, a partial fragment of NAD-SDH from apple fruit was cloned, the NAD-SDH antisense expression plasmid was constructed, and the antisense expression has been doing; thus, the role of NAD-SDH in the sobitol metabolism could be clarified in detail via researching the antisense expression of the NAD-SDH; otherwise, the influence of sorbitol on the environmental tolerance of plants and its taste could be studied. The following were the main experimental results: 1. The method of isolation of total RNA from apple fruit tissue which rich in polysaccharides and phenol was optimized. Isopropanol with the same volume is better than LiCl for the precipitation of total RNA, and the oxidation of phenol could be inhibited by adding 8% PVP and 1%β-mercaptoethanol simultaneously. 2. A new number of NAD-SDH gene family was separated form the apple fruit, and the Genbank accession number was AY707458. The sequence showed that the fragment is 377bp, encoding 125 amino acid, cDNA sequence similarity searching against GenBank by using BLAST program revealed that the fragment had high identity to other NAD-SDHs (the highest is 99%). The fragment was named as NAD-SDH8, which contained a characteristic amino acid sequence G–H–E-X(2)–G-X(5)-[G/A]-X(2)-[I/V/S/A/C], the amino acid residues for catalytic and structural zinc binding site, the NAD binding pocket, and the zinc-containing alcohol dehydrogenase signature sequence,then the NAD-SDH8 belonged to the alcohol dehydrogenase family. According to the phylogenetic tree of NAD-SDH from Rosaceae plants, NAD-SDHs had two branches, and NAD-SDH8 had the shortest phylogenetic distant to NAD-SDH from loquat. 3. The antisense expression plasmid of NAD-SDH8 was constructed and has been directly transformed to Agrobacterium (strain EHA105), which had been testified by the PCR and sequencing.