title: Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.
abstract: 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.
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China.
authors.forename: Yumei
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Luo
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China.
authors.forename: Mengru
authors.initials: M
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Gong
authors.affiliation: School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Yunnan Normal University, Kunming, 650500, Yunnan, China. zzr09@sina.com.
authors.forename: Zhurong
authors.initials: Z
authors.lastname: Zou
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D003001:Cloning, Molecular; D055864:Droughts; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D043564:Inorganic Pyrophosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D031286:Jatropha / Q000235:genetics; D058977:Molecular Sequence Annotation; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D055049:Salt Tolerance / Q000235:genetics*; D012965:Sodium Chloride / Q000633:toxicity; D013312:Stress, Physiological / Q000235:genetics*
chemical_list: D012965:Sodium Chloride; D043564:Inorganic Pyrophosphatase
keywords: Gene cloning; H+-PPase; Heterologous expression; Jatropha curcas; Salt tolerance