Dunaliella oligosaccharide transferase subunit STT3a Cloning and Functional Analysis

N-linked glycosylation is the most common eukaryotic cells, protein modification, this process is in rough endoplasmic reticulum oligosaccharide transferase enzyme (OST) catalysis. Stt3p catalytic subunit of the OST, it is known OST subunit is the most conservative. Yeast Stt3p found a 78 kDa protein encoded, and almost all eukaryotic genomes are present in yeast Stt3p protein encoded homologue. Mammalian genome encodes two yeast homologs Stt3p STT3a and STT3b, their expression is tissue-specific differences, and can regulate the activity of OST, and contains a STT3a subunit OST OST than subunit containing STT3b completely for selection long-chain oligosaccharides assembly donor (Glac3Man9Glc NAc2-PP-Dol) is more specific. Recent studies have found the OST STT3a Arabidopsis as a transmembrane protein subunits in high salt stress response plays a role. Dunaliella (Dunaliella salina, D. salina) is a highly salt-tolerant unicellular eukaryotic flagellum double green algae, it does not have cell walls, capable of 0.5-5 mol / L NaCl salt to survive, has a strong penetration regulation. In recent years, although people around the salt tolerant algae mechanism for a lot of research, but at present its salt tolerance mechanism is not very clear. In addition, our previous study found that: In high salt conditions, the movement of Dunaliella significantly reduced, and as a salina flagella moving organs directly involved in the movement of Dunaliella. In order to understand the salt-induced transmembrane protein STT3a response in rats and salina salt tolerance and flagellum-mediated motility whether a potential link between, this study Chlamydomonas, Arabidopsis STT3a amino acid highly conserved sequence VCVFTA, DVDYVL to design a pair of degenerate primers, using RT-PCR and RACE methods amplified gene Dunaliella STT3a full-length cDNA sequence. Then Dunaliella STT3a of cDNA was cloned into the prokaryotic expression vector pET-28a () in the recombinant plasmid, by SDS-PAGE method to analyze STT3a fusion protein expression in E. coli BL21. By Primer Premier 5.0 STT3a domain of design in a pair of specific primers, were extracted from different culture conditions and salt flagella regeneration state salina cDNA, by real-time quantitative PCR analysis STT3a in salina salt-induced and flagella regeneration in rats. In addition, the use of improved silver staining flagella and Dunaliella salina algae by silver staining, and according to the formula V = (S2-S1) / t (where V is the relative growth rate, S2 and S1, respectively, for the two adjacent time point average length of the flagellum, t represents time interval), and ultimately find salina flagella regeneration in different periods of relative growth rate. Sequence analysis showed that cloned Dunaliella STT3a full-length cDNA sequence is 2574 bp, including 114 bp of 5'UTR, 279 bp and 2181 bp 3'UTR of an open reading frame (open reading frame, ORF), encoding 727 amino acid residues, and its theoretical isoelectric point of the protein was 9.11, a molecular weight of 80.25 kDa. The resulting amino acid sequence homology analysis, BLAST results show that the resulting cloned sequences Dunaliella STT3a protein gene sequences. SDS-PAGE results showed that: in the 80 kDa occurs around a specific band with the predicted molecular weight size. Real-time PCR results showed that Dunaliella STT3a mRNA levels gradually as the salt concentration increases, the level of 3.5mol / L NaCl concentration than 1.5mol / L NaCl concentration increased 11-fold (P < 0.01). In addition, with no de-flagellated salina compared, STT3a mRNA in flagellar sustained high expression of the regeneration process. Dunaliella flagellum flagellum 15 min before regeneration was significantly increased compared to 420 min when substantially complete flagella regeneration. This study shows that Dunaliella STT3a gene is highly expressed in salina salt adaptation and flagella play an important role in the regeneration process.