Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis
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The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na+. In plants, sequestration of Na+ into the vacuole is driven by the electrochemical H+ gradient generated by H+ pumps, and this Na+ sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H+ pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V–H+-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V–H+-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V–H+-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na+ hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V–H+-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V–H+-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V–H+-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.
KeywordsDunaliella viridis H+-pyrophosphatase Cloning Functional characterization Expression analysis Salt tolerance
Open reading frame
Expressed sequence tag
We are grateful to Dr. Alonso Rodriguez-Navarro (Universidad Politécnica de Madrid, Spain) for providing the yeast G19 mutant, Dr. Erin K. O’Shea (University of California, San Francisco) for providing the plasmid EB0666 and Dr. Anu Saloheimo (VTT Biotechnology, Finland) for providing the pAJ401 vector. This work was supported by National Natural Sciences Foundation of China (30871278, 30970242), Ministry of Agriculture of China (2008ZX08003-001, 2008ZX08003-005) and the Research Foundation from Shanghai Municipal Education Commission (09DZ2271800).
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