Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

Ma Cai,Li-Hong He,Tu-Yuan Yu,Sue Cotterill

doi:10.1371/journal.pone.0062287

Abstract

Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.

Highlights

Dunaliella salina, one member of the genus Dunaliella (Chlorophyceae, Volvocales), is an extremely halotolerant, unicellular, green, and motile algae
glycerol3-phosphate dehydrogenase (G3PDH) catalyze dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate, which is converted to glycerol by glycerol-3-phosphate phosphatase [18,19]
When using total RNA from D. salina cells as Reverse transcription (RT) substrate, an expected 583 bp fragment amplified with primers Dsgdph1-F/R was cloned and sequenced (Fig. 1a)

Summary

Introduction

Dunaliella salina, one member of the genus Dunaliella (Chlorophyceae, Volvocales), is an extremely halotolerant, unicellular, green, and motile algae. The genus Dunaliella is unique in its remarkable ability to survive in the media with a wide range of NaCl concentrations, from about 0.05 M to saturation (around 5.5 M), while maintaining a relatively low intracellular sodium concentration [1,2]. This remarkable osmotic adaptability is mediated primarily by the massive de novo synthesis of the compatible solute, the glycerol, following salt stress [3]. G3PDH catalyze dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate, which is converted to glycerol by glycerol-3-phosphate phosphatase [18,19]

Methods

Results

Conclusion