title: Molecular cloning, characterization, and expression of an alfalfa (Medicago sativa L.) heme oxygenase-1 gene, MsHO1, which is pro-oxidants-regulated.
abstract: It has been documented that plant heme oxygenase-1 (HO-1; EC 1.14.99.3) is both development- and stress-regulated, thus it plays a vital role in light signalling and stress responses. In this study, an alfalfa (Medica sativa L.) HO-1 gene MsHO1 was isolated and sequenced. It contains four exons and three introns within genomic DNA sequence and encodes a polypeptide with 283 amino acids. MsHO1 had a conserved HO signature sequence and showed high similarity to other HOs in plants, especially HO-1 isoform. The MsHO1:GFP fusion protein was localized in the chloroplast. Further biochemical activity analysis of mature MsHO1, which was expressed in Escherichia coli, showed that the Vmax was 48.78 nmol biliverdin-IXα (BV) h⁻¹ nmol⁻¹ protein with an apparent Km value for hemin of 2.33 μM, and the optimum Tm and pH were 37 °C and 7.2, respectively. Results of semi-quantitative RT-PCR and western blot showed that the expressions of MsHO1 were higher in alfalfa stems and leaves than those in germinating seeds and roots. Importantly, MsHO1 gene expression and protein level were induced significantly by some pro-oxidant compounds, including hemin and nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, MsHO1 may play an important role in oxidative responses.
authors.affiliation: College of Life Sciences, Cooperative Demonstration Laboratory of Centrifuge Technique, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Guang-Qing
authors.initials: GQ
authors.lastname: Fu
authors.forename: Sheng
authors.initials: S
authors.lastname: Xu
authors.forename: Yan-Jie
authors.initials: YJ
authors.lastname: Xie
authors.forename: Bin
authors.initials: B
authors.lastname: Han
authors.forename: Li
authors.initials: L
authors.lastname: Nie
authors.forename: Wen-Biao
authors.initials: WB
authors.lastname: Shen
authors.forename: Ren
authors.initials: R
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism*; D006427:Hemin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D018744:DNA, Plant; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D018749:RNA, Plant; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D009599:Nitroprusside; D006427:Hemin; D051547:Heme Oxygenase-1