Cloning And Characterization Of Glycerol-3-phosphate Dehydrogenase (EC1.1.1.8) Gene In Dunaliella Salina

Dunaliella salina is the most extreme halotolerat alga in the world. It is a good model organism in the plant halotolerance research. The alga regulates its inside turgor to adapt the outside osmotic change by synthesizing a large amount of glycerol. It is important to study glycerol-3-phosphate dehydrogenase, the key enzyme of the glycerol metabolism to know how the species can sustain the hyperosmotics press by accumulating so much glycerol. Here we constructed a cDNA library using SMART?technique. The recombinant of the library is 100%. The liter of this library is 1.2 × 10⁶cfu/mL which can represent 3.07× 10⁶ mRNA fragments hi the alga cell. A large scale DNA sequencing of the random clone in the library was performed. EST of glycerol-3-phosphate dehydrogenase gene was gained and the full-length cDNA of glycerol-3-phosphate dehydrogenase gene then was achieved as following described. First the 3' coding sequence and 3'UTR glycerol-3-phosphate dehydrogenase gene was sequenced according to subcloning the pGPDH-3 plasmid. The total length of 3' coding sequence and 3'UTR was 1609bp, which included a GT-repeated sequence. Secondly according to the EST sequence the 5' full length of this gene was amplified using RACE technology. The 5' coding sequence and 5'UTR was 1544bp long. At last the Southern analysis was done to identify the cDNA. The result showed that the glycerol-3-phosphate dehydrogenase gene of this species was a single copy gene hi the genome.The gene structure was simply analyzed by PCR method. The partial sequence of this gene was amplified and result showed that it had 8 introns and 9 exons. The splicing site was common to other gene of all the eukaryote. The GC content of intron and exon was greatly different. And the intron had a lot of GT repeated sequence.The DNA and protein sequence of this gene was analyzed using the bioinformatics tools. Two functional domains were found in the protein. One is phosphatase-like domain and the other is glycerol-3-phosphate dehydrogenase domain. The former domain may catalog the dephosphation of the glycerol-3-phosphate and the later presumably synthesize the glycerol-3-phosphate according to similarity searching, domain localization and structure comparison. These should give an evidence for the functional research of this gene.