Cloning And Characterization Of STT3a Gene Of Oligosaccharyltransferase Subunit From Dunaliella Salina

N-linked glycosylation is one of the most common protein modification reactions in eukaryotic cells, and the process is catalyzed by oligosaccharide transferase enzyme (OST) in the rough endoplasmic. STT3p is a highly conserved catalytic subunit in the OST complex, which encodes a 78 kDa protein in yeast cells. The homologues of yeast STT3p protein almost exist in all eukaryotes cells, which have two homologues STT3a and STT3b in mammalian genome. The expression of STT3a and STT3b has tissue-specific property, which can regulate the activity of OST. OST complexes with a STT3a subunit display a higher specificity for selecting the fully assembled dolichol-linked oligosaccharide donor (Glc3Man9GlcNAc2-PP-Dol) than OST complexes with a STT3b subunit. Recent studies showed that the STT3a subunit of the Arabidopsis oligosaccharyltransferase controlled adaptive responses to salt/osmotic stress.Dunaliella salina (D. salina) is a unicellular, highly halotolerant and biflagellate eukaryotic alga. It does not have cell wall and is able to live in a variety of salt concentrations ranging from 0.05 to 5 M solution of sodium chloride. Although the halotolerant mechanism of D. salina has been highly studied during the past decade, it is not very clear now. Our observation result that D. salina swam slowly in high salt concentrations and the knowledge that the flagellae are directly involved in the movement of D. salina cells encourage us to explore the potential link between salt stress and flagella-mediated motility by cloning and function analysis of transmembrane protein STT3a gene in D. salina.In this study, a pair of degenerate primers was designed according to conserved homologous amino acid sequences of "VCVFTA" and "DVDYVL" of STT3a from Chlamydomonas, Arabidopsis thaliana and other organisms. The full-length cDNA sequence was amplified from D. salina by RT-PCR and Rapid Amplification of cDNA Ends (RACE) and inserted into the NcoI-EcoRI sites of the pET-28a(+) vector, generating the recombinant vector pET28a(+)-STT3a. The expression of recombinant STT3a protein was analyzed by SDS-PAGE. To confirm whether the STT3a gene could response to salt stress and flagellar regeneration, D. salina cells grown in different media containing 0.75,1,1.5,2,3,3.5 mol/L NaCl and different stages of flagellar regeneration were respectively collected. A pair of specific primers in the functional domain of STT3a was designed by Primer Premier 5.0. Real-time fluorescence quantitative PCR (real-time Q-PCR) was employed to analyze the change of STT3a mRNA levels in salt adaption and flagellar regeneration. D. salina cells and flagellae in different stages of flagellar regeneration were stained by the improved silver staining method. The length of the D. salina flagellae in different stages of flagellar regeneration with the method of thread fitting flagellae. The relative growth rate of D. salina in different times of flagellar regeneration was accounted by formula V=(S2—S1)/t (V represents the relative growth rate of D. salina, S2—S1 represents the average length of two adjacent time points. T represents the time of two adjacent time points).Sequence analysis showed that the cloned STT3a cDNA possessed a 114 bp 5'UTR, a 279 bp 3'UTR and an 2181 bp open reading frame (ORF) encoding 727 amino acid residues. The theoretical molecular weight is 80.25 kDa and the pI value is 9.11. Sequence analysis by BLAST in NCBI database confirmed that the cloned sequence was D. salina STT3a amino acid sequences. The result of SDS-PAGE showed that the recombinant STT3a proteins were about 80.0 kDa, which was consistent with the expected molecular weight of D.salina STT3a. Real-time Q-PCR demonstrated that the STT3a mRNAs from D. salina were induced by increased concentrations of NaCl, and increased to 11-fold higher by 3.5 mol/L NaCl than that by 1.5 mol/L NaCl (P＜0.01). Also, STT3a mRNA of D. salina maintained at a higher level in the process of flagellar regeneration with than without experiencing deflagellar treatment. The flagellae of D. salina grew quickly at the initial stage of flagellar regeneration and the flagellar regeneration of D. salina almost finished in 420 min after deflagellar treatment. In conclusion, the high expression of the STT3a gene plays important roles in the process of salt adaptation and flagellar regeneration of D. salina.