S-Adenosyl Homocysteine Hydrolase (SAHHase) Gene Is Involved In The Regulation Of Flagellar Length In Dunaliella Salina

Dunaliella salina, a unicellular eukaryotic green alga, is easy to culture and has a pair of equal-length flagella. The flagellum of Dunaliella salina has a typical structure that was generally acknowledged as "9+2". Flagellar resorption and regeneration occur after treated with some chemical agents such as colchicine, so Dunaliella salina is much fitted to be a model organism for reaserch on flagella.The flagellum/cilium is an organelle projecting from the surface of cells and plays key roles in cell division, motion and signal transduction. The dysfunction of flagellum/cilium is associated with several human diseases, such as polycystic kidney disease, Kartagener syndrome and so on. But the exact regulation mechanism of flagellum/cilium is poorly understood. Flagellar/cilia assembly is a bidirectional processes, containing anterograde and retrograde processes. Intraflagellar transport (IFT) particles are a large protein complexs that transport along the axonemal out doublet microtubles from the base of the flagella to its distal tip by kinesin motor and then from the tip back to the cell body by cytoplasmic dynein. When the anterograde process is predominant, the flagella will regenerate; on the contrary, flagella will tend to resorption. There is a definite link among flagella regeneration, resorption and cell devision, and it has been reported that abnormality of flagellar results many human diseases.S-Adenosyl homocysteine hydrolase (SAHHase) is unique enzyme to degrade S-adenosyl homocysteine, a byproduct of methionine synthase (MetE)-catalyzing methylation reactions. So inhibiting activity of SAHHase can interfere with methylation pathway. Schneider et al. reported that protein methylation pathway was active during flagellar resorption. Although Pazour et al. have demonstrated S-Adenosyl homocysteine hydrolase are components of the membrane-matrix fraction in Chlamydomonas, its role in the regulation of flagella length has been poorly understood so far.In order to investigate whether SAHHase is involved in the regulation of flagellar length, a pair of degenerate primers was designed to obtain a fragment of SAHHase cDNA, and the RACE method was used to amplify the full length of SAHHase cDNA. The results of PCR and RACE showed that the full length of SAHHase cDNA cloned in this study was 1926 bp, consisting of ORF 1458 bp,3' UTR 61 bp and 5'UTR 407 bp. The Real-time fluorescence quantitative PCR was employed to monitor the transcriptional level of the SAHHase gene from D. salina during flagella regeneration and resorption. The result of real-time fluorescence quantitative PCR indicated that abundance of SAHHase mRNA was induced during the process of the flagella regeneration.SAHHase is a key enzyme in methylation pathway. Our study shows SAHHase involved in the regulation of flagellar length. Some studies suggested that methylation pathway may play a role in the process of flagellar length regulation, our study also provide evidences for this hypothesis.Materials and Methods1 D.salina algae stranis, bacteria stranins and vectors D. salina algae strain UTEX-LB-1644 was purchased from the University of Taxas, USA. The alga were cultured in the modified PKS liquid medium, at 26℃and 4500 Lux for light/dark culture of 12 h each. E.coli DH5a, E.coli BL21 and pET28a (+) were conserved by our laboratory, and pMD 18T-vector was purchased from TaKaRa.2 Cloning of SAHHase cDNA from D. salinaA pair of degenerate primers was designed according to conserved homologous amino acid residues of SAHHase. Trizol reagent was used to extract total RNA of D.salina, and then the total RNA was reversely transcripted to cDNA. RT-PCR and RACE were used to obtain the full lengtn of SAHHase cDNA.3 Relationship between SAHHase and regulation of flagellar length at transcription level in D.salinaDeflagella was carried out through grinding the cell of D.salina in glass pestle, and then the cells cultured for the flagella regeneration; Additionally, D.salina cells was cultured after adding colchicine to medium for the flagellar resorption. Total RNA of different samples was extracted for preparing cDNA, and then Real time-PCR was used to investigate whether SAHHase had a role in the regulation of flagellar length in D.salina.4 Heterologous expression of SAHHaseA recombinant plasmid pET 28a(+)-SAHHase was constructed and then to transformed toE.coli BL21. IPTG was used for inducing the expression of exogenous proteins. 5 Purification of SAHHase and antibody preparationSAHHase was purified by His-Bind Purification Kit (Novagen), and then animals were immunized with the purified protein for preparing antibody.Results1 Cloning of SAHHase cDNA from D. salinaThe full-length SAHHase cDNA of 1926 bp from D. salina was obtained, which consists of a 61-bp 5'UTR, a 407-bp 3'UTR and a 1458-bp open reading frame (ORF) (Genbank No. HM180944). Sequence analysis by BLASTX program in NCBI database revealed that the putative SAHHase amino acids shared high identities with the known SAHHase, such as Chlamydomonas reinhardtii (83%).2 Relationship between SAHHase and the regulation of flagellar length at transcript level in D. salinaThe results of Real-time Q-PCR demonstrated that the expression level of the SAHHase gene from D. salina was increased during both flagellar regeneration and flagellar resorption after treated with colchicine.3 Heterologous expression of SAHHaseThe result of SDS-PAGE proved that SAHHase of D.salina was expressed successfully, and the molecular weight of SAHHase was about 52 kDa. 4 Purification of SAHHase and antibody preparationThe result of SDS-PAGE showed that the purity of SAHHase was larger than 90%. The antibody titcr was reached to 1:1,280,000.Conclusion1. A 1926 bp full-length SAHHase cDNA from D. salina was obtained, which consists a 61-bp 5'UTR, a 407-bp 3'UTR and a 1458-bp open reading frame.2. SAHHase play a role in the regulation of flagellar length at transcript level.3. SAHHase of D. salina is about 52 kDa.