title: Cloning of PEPC-1 from a C4 halophyte Suaeda aralocaspica without Kranz anatomy and its recombinant enzymatic activity in responses to abiotic stresses.
abstract: Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthetic pathway and plays an important biochemical role in higher plants and micro organisms. To gain understanding of the role of PEPC in stress adaptation in plant, we cloned PEPC gene from Suaeda aralocaspica, a C4 species without Kranz anatomy, and performed a series of experiments with PEPC gene expressed in Escherichia coli under various abiotic stresses. Results showed that, based on the homology cloning and 5'-RACE technique, the full-length cDNA sequence of PEPC (2901 bp) from S. aralocaspica was obtained, which shares the typical conserved domains to documented PEPCs and was identified as PEPC-1 in accord to the reported partial sequence (ppc-1) in S. aralocaspica. qRT-PCR analysis revealed the expression patterns of PEPC-1 and PEPC-2 (known as ppc-2, another plant type of PEPC) in S. aralocaspica, suggesting that PEPC-1 was up-regulated during seed germination and under NaCl stress, and presented higher level in chlorenchyma than other tissues, which were significantly different with PEPC-2. Afterwards, PEPC-1 was recombinant in E. coli (pET-28a-PEPC) and expressed as an approximate 110 kDa protein. Under various abiotic stresses, the recombinant E. coli strain harboring with PEPC-1 showed significant advantage in growth at 400-800 mmol L(-1) NaCl, 10-20% PEG6000, 25 and 30 °C lower temperature, 50-200 μmol L(-1) methyl viologen, and pH 5.0 and 9.0 condition, compared to control. Further analysis of the enzymatic characteristics of the recombinant PEPC-1 suggests that it was the higher enzyme activity of PEPC-1 which might confer the stress tolerance to E. coli. We speculate that over expression of PEPC-1 is probably related to regulation of oxaloacetate (OAA) in tricarboxylic acid (TCA) cycle in E. coli, which may contribute to further understanding of the physiological function of PEPC in S. aralocaspica.
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. Electronic address: enjoy123fly@163.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. Electronic address: 1259008920@qq.com.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. Electronic address: lanhaiyan@xju.edu.cn.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Lan
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017343:Genes, Plant; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D010788:Photosynthesis; D010802:Phylogeny; D010940:Plant Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055051:Salt-Tolerant Plants / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013312:Stress, Physiological
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D010730:Phosphoenolpyruvate Carboxylase
keywords: Abiotic stresses; Phosphoenolpyruvate carboxylase; Recombinant enzyme activity; Single-cell C4 halophyte; Suaeda aralocaspica