| Mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH), a nuclear-encoded mitochondrial protein, is located in the inner mitochondrial membrane with its catalytic site facing toward the cytosol. It oxidizes glycerol-3-phosphate (G-3-P) and transfers reducing equivalents to the respiratory chain, combined action of this enzyme with the Cytosolic NAD~+-linked isoform of glycerol-3-phosphate dehydrogenase (NAD~+-GPDH) forms the glycerol-phosphate shuttle, In this shuttle, Cytosolic NAD~+-glycerol-3-phosphate dehydrogenase oxidizes Cytosolic NADH to NAD~+, and catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G-3-P. Subsequently G-3-P passes the outer mitochondrial membrane and is oxidized to DHAP by FAD-GPDH, simultaneously delivers its electrons to the respiratory chain. The DHAP then returns to the cytosol for next cycle or enters EMP pathway. In fact the role of glycerol-phosphate shuttle is transferring reducing equivalents from cytosol into the mitochondrial respiratory chain and maintaining redox balance in cytosol.The glycerol-phosphate shuttle universally exists in eukaryote, has been detailed in yeast and animal systems. However, until recently it has been demonstrated that a glycerol-3-phosphate shuttle in Arabidopsis thaliana. In addition to this, there are no other reports about glycerol-3-phosphate shuttle in plants.Dunaliella salina is a unicellular green alga that can adapt to an extremely wide range of salinities, from 0.1 to 5.5 M NaCl. However, it lacks a rigid cell wall, and responds to osmotic changes by modulating its cell volume through expanding or shrinking plasmalemma, and regulating intracellular concentration of glycerol. Previous studies show that G-3-P, the substrate of mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, is an obligatory precursor for the biosynthesis of glycerol and all glycerolipids, including membrane and storage lipids. It may uncover the mechanism how Dunaliella salina adapts to high osmotic conditions to investigate the metabolism of G-3-P. The cellular NADH/NAD~+ ratio will be disturbed when cells encounter stress conditions. Therefore, the study of Dunaliella salina FAD-GPDH will improve our knowledge about glycerol-phosphate shuttle and its role in maintaining cellular redox balance. This paper reports cloning of the full length cDNA of Dunaliella salina FAD-GPDH, sequence analysis of Dunaliella salina FAD-GPDH ORF, Expression profile of Dunaliella salina FAD-GPDH, prokaryotic expression of Dunaliella salina FAD-GPDH and its enzyme assay. The following are details:.1. Clone the full length cDNA of Dunaliella salina FAD-GPDH. 3'RACE and 5'RACE were carried out based on Dunaliella salina FAD-GPDH EST from cDNA library. A full length cDNA was obtained by assembling 3'RACE and 5'RACE fragments.2. Analyze the deduced amino acid sequence. Signal peptide analysis indicates that the protein has an apparent mitochondrial targeting sequence in its N-terminal. Membrane-spanning helices predicted results show that Dunaliella salina FAD-GPDH has three strong transmembrane helices. It is easy to identify the putative FAD-binding site and G-3-P binding domains when aligned Dunaliella salina FAD-GPDH with other FAD-GPDHs.3. Assay the enzyme activity of Dunaliella salina FAD-GPDH expressing in E.coli in vitro. The ORF of Dunaliella salina FAD-GPDH is constructed into prokaryotic expression vector pET-32a, and induced to express in E.coli BL21. Target protein is purified with affinity chromatograph. Its activity is assayed in vitro. The results show that recombination protein can oxidize glycerol-3-phosphate, exhibits the FAD-GPDH activity. 4. Analyze the expression profile of Dunaliella salina FAD-GPDH under stress conditions. The results of Real-Time Quantitative PCR and enzyme assay show that expression of Dunaliella salina FAD-GPDH is enhanced at first by salt treatment, and repressed by oxygen deficiency and cold stress.
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