Cloning And Function Characterization Of Dunaliella Salina NAD-glycerol 3-phosphate Dehydrogenase Isoenzyme Functional Domain

Plants which suffer hyperosmosis, drought or cold are able to produce and accumulate large amount osmolyte to change cell osmotic pressure. This behavior could prevent cell dehydration under hyperosmosis. Dunaliella salina is one of the most salt-tolerance photosynthetic eukaryotic organism, which could survival under 0.1～5.5 mol/L NaCl. As a typical halophytes, composition of osmolyte is its key salt-tolerance strategy. That means it synthesize glycerol for osmotic balance and cell shape recovery. There is a kind of linear correlation between the content of glycerol in Dunaliella salina and the NaCl concentration of the culture medium. Dunaliella salina glycerol metabolism is similar to Saccharomyces cerevisiae. In this metabolic pathway, glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) is the key enzyme, which catalysis the convert from DHAP to glycerol 3-phosphate with the help of NADH and its enzymatic activity is regulated by osmotic pressure of surrouding.Glycerol 3-phosphate dehydrogenase has many kinds of isozyme, which exist in organism widely to perform different function. There are three kinds of glycerol 3-phosphate dehydrogenase isozyme in Saccharomyces cerevisiae and Arabidopsis thaliana. The study of Dunaliella tertiolecta glycerol 3-phosphate dehydrogenase isozyme lay particular emphasis on the separation and purification of the native proteins. Three kinds of GPD isozyme with NAD coenzyme from Dunaliella tertiolecta has been found. Three kinds of Drosophila melanogaster NAD-GPD are encoded by the same structural gene. There are also many kinds of glycerol 3-phosphate dehydrogenase isozyme in Dunaliella salina.During laboratory early days work, the study of glycerol 3-phosphate dehydrogenase isozyme in Dunaliella salina was taken on gene level first time, and got three EST of GPD isozyme named NAD-GPD1,NAD-GPD2 and FAD-GPD respectively. The early work concentrated on the study of NAD-GPD2. The full gene had been cloned and found that it had two independent integrity functional domain by CDD Search, glycerol 3-phosphate dehydrogenase functional domain and glycerol 3-phosphatase functional domain. It was so especial that had been not found in other species. So we suppose that the especial structure may be the key point of the extreme strong ability of glycerol synthesis in Dunaliella salina. That means the reaction from DHAP to glycerol was catalyzed by DsNAD-GPD2 directly. Furthermore, Southern blot, Northern blot, Western blot and the study of recombination protein had been taken to confirm the suppose. Related article was published on . During the following experiments, we found DsNAD-GPD1 had the same structure with DsNAD-GPD2. So what is the cooperation mechanism between the isozyme when Dunaliella salina was suffer hyperosmosis stress? That is the study purpose of this article.Gene and protein study of DsGPD1 was carried out in this article listed as follow.First, clone of DsGPD1 different domain. By RT-PCR approaches, the different domains of DsGPD1 named GPD1.8 and GPD1.1 were subcloned into pMD18-T vector and then were analysised by bioinformatics.Second, prokaryotic expression of DsGPD1 different domain and in vitro enzyme activity determination. Construct recombinant plasmid pET-GPD1.8 and pET-GPD1.1 and then transform E. coli BL21. According to the study of Dunaliella tertiolecta GPD, the effect of NaCl and DTT to recombination protein GPD enzyme activity were determined.And on the other hand, determine whether GPD1.8 recombinant protein have glycerol 3-phosphatase enzyme activitiey.Third, using S. cerevisiae functional complementary experiments verify the function of DsGPD1. S. cerevisiae GPD1△strain were transformed with pYES-GPD1.8 and pYES-GPD1.1. Adding galactose to NaCl culture medium in order to induce the expression of cloned gene and then analyze their salt tolerance extent.Forth, research of DsGPD1 mRNA contrast transcription. DsGPD1 mRNA transcription under salt, oxidative, phosphate and anaerobiosis stress was researched with fluorescent real time PCR. At the same time, the change of total glycerol synthesis of the cells was monitored under the same stress treatments.