Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis
Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis
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DOI：
10.1007/s11033-010-0445-z
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发表时间：
2011-06-01
影响因子：
2.8
通讯作者：
Song, Rentao
中科院分区：
文献类型：
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作者：
Meng, Xiangzong;Xu, Zhengkai;Song, Rentao
耐盐盐杜氏藻对高盐环境具有良好的适应性，并具有调节细胞内Na+的有效机制。在植物中，通过H+泵产生的电化学H+梯度驱动Na+螯合到液泡中，并且这种Na+螯合是赋予植物耐盐性的一种机制。为了研究液泡H+泵在杜氏藻耐盐性中的作用，我们从绿色杜氏藻中克隆了液泡质子转运无机焦磷酸酶（V-H+-PPase）的cDNA。DvVP cDNA全长2,984 bp，编码762个氨基酸，具有15个跨膜结构域。DvVP蛋白在其氨基酸序列和其跨膜模式方面与来自其它绿色藻类和高等植物物种的V-H+-PP酶高度相似。系统发育分析表明，杜氏藻与轮藻科绿色藻类和陆生植物的亲缘关系较近。DvVP在酵母突变体G19（Delta ena 1 -4）中的异源表达抑制Na+超敏反应，并且DvVP的GFP融合定位于酵母中的液泡膜，表明DvVP编码功能性V-H+-PPase。北方印迹分析表明，在高盐度下，D.盐胁迫下DvVP的表达与盐诱导的V-H+-PPase的表达不同，表明盐胁迫下DvVP在杜氏藻中的表达可能受到不同机制的调控。本研究不仅丰富了我们对不同生物中V-H+-PPases生物学功能的认识，也加深了我们对杜氏藻耐盐分子机制的理解。
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 (Delta ena1-4) 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.