ABSTRACT
Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that are able to make cells to uptake heavy metals from the environment. Molecular and functional characterization of this gene family improves understanding of the mechanisms underlying heavy metal tolerance in higher organisms. In this study, a cDNA clone, encoding 74-a.a. metallothionein type 1 protein (ZjMT), was isolated from the cDNA library of Ziziphus jujuba. At the N- and C-terminals of the deduced amino acid sequence of ZjMT, six cysteine residues were arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC structure, respectively, indicating that ZjMT is a type 1 MT. Quantitative PCR analysis of plants subjected to cadmium stress showed enhanced expression of ZjMT gene in Z. jujuba within 24 h upon Cd exposure. Escherichia coli cells expressing ZjMT exhibited enhanced metal tolerance and higher accumulation of metal ions compared with control cells. The results indicate that ZjMT contributes to the detoxification of metal ions and provides marked tolerance against metal stresses. Therefore, ZjMT may be a potential candidate for tolerance enhancement in vulnerable plants to heavy metal stress and E. coli cells containing the ZjMT gene may be applied to adsorb heavy metals in polluted wastewater.
Subject(s)
Metallothionein/metabolism , Metals, Heavy/metabolism , Ziziphus/metabolism , Amino Acid Sequence , Cadmium/metabolism , Cadmium/toxicity , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Escherichia coli/metabolism , Metallothionein/chemistry , Metallothionein/genetics , Metals, Heavy/toxicity , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sequence Alignment , Ziziphus/drug effectsABSTRACT
Plant metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, and metal-binding proteins, which play an important role in the detoxification of heavy metal ions, osmotic stresses, and hormone treatment. Sequence analysis revealed that the open-reading frame (ORF) of ZjMT was 225 bp, which encodes a protein composed of 75 amino acid residues with a calculated molecular mass of 7.376 kDa and a predicated isoelectric point (pI) of 4.83. ZjMT belongs to the type I MT, which consists of two highly conserved cysteine-rich terminal domains linked by a cysteine free region. Our studies showed that ZjMT was primarily localized in the cytoplasm and the nucleus of cells and ZjMT expression was up-regulated by NaCl, CdCl2 and polyethylene glycol (PEG) treatments. Constitutive expression of ZjMT in wild type Arabidopsis plants enhanced their tolerance to NaCl stress during the germination stage. Compared with the wild type, transgenic plants accumulate more Cd2+ in root, but less in leaf, suggesting that ZjMT may have a function in Cd2+ retension in roots and, therefore, decrease the toxicity of Cd2+.
Subject(s)
Adaptation, Physiological/genetics , Genes, Plant , Metallothionein/genetics , Plant Proteins/genetics , Stress, Physiological/genetics , Ziziphus/genetics , Ziziphus/physiology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/physiology , Cadmium/metabolism , Gene Expression Regulation, Plant , Metallothionein/chemistry , Metallothionein/metabolism , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants, Genetically Modified , Protein Structure, Tertiary , Protein Transport , Salinity , Salt Tolerance/genetics , Subcellular Fractions/metabolismABSTRACT
Chinese jujube (Ziziphus jujube Mill.) is important economically for its fruit and also bears attractive medical value. Its flower development concurs with the growth of current fruit bearing shoots (FBSs). However, events involved in current FBSs, including FBS differentiation, flower development, and reliable housekeeping gene are still unknown. In this study, the morphology of FBS development and floral formation were examined and expression profiles of several potential internal control genes was determined. These included genes encoding proteins involved in protein translation (translation elongation factor 1alpha, ZjEF1), protein folding (cyclophilin, ZjCyP) or degradation (ubiquitin extension protein, ZjUBQ), and proteins involved in the structure of the cytoskeleton (beta-actin, ZjACT) or nucleosome (histone3, ZjH3). Our results showed that the floral development in early growing FBSs (less than 20 mm in length) or shoot apices was not complete. Among ZjACT, ZjEF1, ZjCyP, ZjUBQ, and ZjH3, ZjH3 was the most suitable housekeeping gene to evaluate FBS development, based on their expression in early growing FBSs, shoot apices, and different organs. These results will be useful for further molecular mechanism study about FBS development in Chinese jujube.
Subject(s)
Genes, Plant , Ziziphus/physiology , Fruit/genetics , Fruit/growth & development , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Histocytochemistry , Plant Shoots/genetics , Plant Shoots/growth & development , RNA, Messenger/analysis , RNA, Plant/analysis , Reverse Transcriptase Polymerase Chain Reaction , Ziziphus/genetics , Ziziphus/growth & development , Ziziphus/metabolismABSTRACT
Two orthologues of FLORICAULA/LEAFY AFL1 and AFL2 (apple FLO/LFY), were isolated from the floral buds of apple trees. Their expression was detected in various tissues and during differentiation of the floral buds. Furthermore, the flowering effectiveness of each gene was assessed with transgenic Arabidopsis. Both AFL1 and AFL2 showed high homology to each other (90%) and a high degree of similarity to PTLF and PEAFLO (70%), which are homologues of FLO/LFY from poplar and pea, respectively. RNA blot analysis showed that AFL1 was expressed only in the floral bud during the transition from vegetative to reproductive growth, whereas AFL2 was expressed in vegetative shoot apex, floral buds, floral organs and root. Genomic Southern analysis showed that apple had other homologues in addition to AFL1 and AFL2. The transgenic Arabidopsis with over-expressed AFL2 showed accelerated flowering and gave rise to several solitary flowers from rosette axils directly. AFL1 had similar effects, but the phenotypes of the transgenic Arabidopsis with AFL1 were weaker than those with AFL2. These results suggest that both genes are involved in flower differentiation in apple.
