Molecular Cloning, Expression Analysis, and Functional Characterization of the H+-Pyrophosphatase from Jatropha curcas
Molecular Cloning, Expression Analysis, and Functional Characterization of the H+-Pyrophosphatase from Jatropha curcas
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DOI：
10.1007/s12010-015-1944-0
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发表时间：
2016-04
影响因子：
3
通讯作者：
Yumei Yang;Zhu Luo;Mengru Zhang;Chang Liu;M. Gong;Z. Zou
中科院分区：
文献类型：
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作者：
Yumei Yang;Zhu Luo;Mengru Zhang;Chang Liu;M. Gong;Z. Zou
H(+)-焦磷酸酶(H(+)-PPase)是一种主要的焦磷酸(PPI)供能的质子泵，可产生电化学H(+)梯度，用于ATP的产生和跨膜物质的转移。它在逆境适应中发挥着重要作用，大量过量表达H(+)-PPase的转基因植物证实了这一点，但缺乏针对精英能源植物麻疯树的相关研究。在此，我们根据与巴西橡胶树H(+)-PPase高度匹配的EST序列，采用逆转录-聚合酶链式反应(RT-PCR)技术，克隆了巴西橡胶树H(+)-PPase(JcVP1)互补DNA的全长。该基因编码一个由765个氨基酸组成的多肽，被预测为K(+)依赖的H(+)-PPase，在进化上与其他大戟科植物的H(+)-PPase最接近。许多与环境胁迫、分子信号或组织特异性相关的顺式调控元件通过在JcVP1编码序列上游1.5kb区域的启动子预测而被鉴定。同时，JcVP1的表达对几种常见的非生物胁迫(盐、干旱、热、冷)的响应特征与魔芋的内在抗逆性有很好的一致性。此外，我们还发现JcVP1的异源表达可以显著提高重组大肠杆菌和酿酒酵母的耐盐性，并且通过在酵母中添加克氏锥虫H(+)-PPase的液泡靶向信号肽，这种作用可以在酵母中进一步增强。
H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.