Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis
Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis
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DOI：
10.1007/s11103-009-9517-7
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发表时间：
2009-09-01
影响因子：
5.1
通讯作者：
Song, Rentao
中科院分区：
文献类型：
--
作者：
He, Yunxia;Meng, Xiangzong;Song, Rentao
杜氏藻是一种单细胞绿藻，它通过在细胞内积累高浓度的甘油作为相容的溶质，具有在剧烈的渗透胁迫下生存的非凡能力。绿塑性甘油-3-磷酸脱氢酶（GPDH）被认为是杜氏藻中产生渗透调节用甘油的关键酶。在这项研究中，我们从杜氏藻中克隆了两个最突出的GPDH cdna (DvGPDH1和DvGPDH2)，它们分别编码695和701个氨基酸的两个多肽。与高等植物GPDH不同，这两种蛋白在其n端除了c端GPDH结构域外，还含有额外的磷酸丝氨酸磷酸酶（塞族）结构域。这种双结构域GPDH代表了一种新型的GPDH，仅在绿藻谱系中发现。EGFP融合蛋白在烟叶细胞中的瞬时表达表明，DvGPDH1和DvGPDH2都定位于叶绿体中。过表达DvGPDH1或DvGPDH2可以补充酵母GPDH突变体（gpd1 Delta），但不能补充酵母塞族突变体（ser2 Delta）。用纯化的DvGPDH1和DvGPDH2体外实验也显示出明显的GPDH活性，但未检测到塞尔维亚活性。令人惊讶的是，与植物叶绿体GPDHs不同，DvGPDH1和DvGPDH2可以同时利用NADH和NADPH作为辅酶，并且当NADH作为辅酶时，DvGPDH1和DvGPDH2表现出显著更高的GPDH活性。Q-PCR分析显示，这两个基因在高盐休克时都表现出短暂的转录诱导基因表达，然后是基因表达的负反馈。这些结果揭示了盐胁迫下杜氏藻甘油合成的调控。
Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1 Delta), but not a yeast SerB mutant (ser2 Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.