Function Studies Of Dunaliella Salina Glycerol -3-phosphate Dehydrogenase, Which Has Unique Double Domains

Dunaliella salina is one of most salt-tolerance photosynthetic eukaryotic organism. Glycerol is the key compatible solutes synthesized in salina cell for osmotic balance. NAD+-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is the key enzyme in glycerol metablism. Therefore, function studies of glycerol 3-phosphate dehydrogenase (GPD) is important for plant stress research and glycerol-produce engineering strain construction. Bioinformatics analyses of the full length GPD gene show that the Dunaliella salina Osm-GPD (osmotic isoform) includes two independence domains: glycerol-3-phosphate dehydrogenase (GPD) domain and Ser-phosphatase domain (SGPP). This characteristic is so special and may be related to fast glycerol synthesis in salina cells. We hypothesize that the Osm-GPD possesses both the GPP and GPD enzyme activities. Based on the full length sequence and bioimformatic analysis of Osm-GPD gene, gene function research was carried out.First enzyme assay, the enzyme assay is set up for glycerol 3-phosphate dehydrogenase (GPD) and glycerol-3 phosphatase (GPP) respectively. Total protein was prepared from salina cells grown under different salt concentration and was used for enzyme assay. The results show that GPD activities decreasedcontently with increasing of NaCl concentration and 150mmol/L NaCl in the assay buffer can stimulate GPD activities significantly. GPP activities increase firstly and then decrease and increase and finally remain constant.Second northern blotting, the mRNA level can be significantly induced by NaCl. The results show that the Osm-GPD gene cloned is possible encoded an osmotic isoform.Third subclone, by RT-PCR approaches, the different domains of Osm-GPD (full length) are subcloned into expression vector, named: gpd1.1 (only GPD domain) and gpd1.9 (both GPD and SGPP domain). Base on the references, we hypothesize that this phosphatase domain encodes the very important enzyme in glycerol metabolism: glycerol-3-phosphatase.Forth E. coli expression and purification, E. coli BL21 was transformed with pET-GPD1.1 and pET-GPD1.9. Recombinant protein was purified through Ni column. The enzyme assay results show that GPD1.9 has both GPD and GPP activities, and GPD 1.1 has only GPD activities. This proved our hypothesis.Fifth western blotting, the GPD1.1 and GPD1.9 recombinant protein were injected into rabbits (New Zealand big white rabbits) to prepare muti-antibody. The western blotting results show that the salina Osm-GPD is actually one protein and is not cut into two proteins (GPD and GPP) in salina cells. We conclude the salina osmotic GPD is different from other species; this specific Osm-GPD makes quickly synthesis of glycerol contribute to extreme salt-tolerance.Sixth complementary experiments, the GPD1 defect 5. cerevisiae strain (W303-1A) were transformed with pRS-GPD1.1 and pRS-GPD1.9. The complementary experiments results show that both gpd1.1 and gpd1.9 can restore the phenotype of W303-1A to wild type in some degree. But gpd1.9 seems better than gpd1.1. The results indicate that the double enzyme activities contribute to better salt-tolerance.