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ABSTRACT: Northern blot analysis showed that a metallothionein gene,ricMT, is expressed strongly in the stem of rice with an expression level that could be more than 100-fold stronger than in leaf
blades. The results suggest that the 5′ upstream region flanking the coding sequence of thericMT may contain a fairly strong promoter. To elucidate its regulation and promoter structure, the genomic clones ofricMT were screened out from a rice genomic library and a fragment of about 4 084 bp was sequenced. The fragment included a 5′
upstream region of ca. 2 970 bp, a transcription region of ca. 690 bp and a 3′ downstream region of ca. 420 bp. Computer analysis
of the sequence homology showed that the 5′ upstream region included a putative TATA box, a putative CAAT box, and a typical
metal-responsive element TGCGCGCG. The results will promote further understanding of the mechanisms of gene regulation and
metal response of plant metallothionein proteins.
Chinese Science Bulletin 04/2012; 45(2):153-156. · 1.32 Impact Factor
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ABSTRACT: The transcription factors DREB1s/CBFs play important roles in the regulation of plant resistance to environmental stresses and are quite useful for generating transgenic plants tolerant to these stresses. In the present work, a cDNA encoding DREB1/CBF-like protein (GhDREB1L) from cotton was isolated, and its sequence features, DNA binding preference, and expression patterns of the transcripts were also characterized. GhDREB1L contained one conserved AP2/ERF domain and its amino acid sequence was similar to the DREB1/CBF group of the DREB family from other plants. The DNA-binding domain of GhDREB1L was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Electrophoretic mobility shift assay revealed that the purified GhDREB1L fusion protein had a specific binding activity with the previously characterized DRE element (core sequence, ACCGAC) and also with the DRE-like sequence (core sequence, GCCGAC) in the promoter of the dehydration-responsive late embryogenesis-abundant gene LEA D113. Semi-quantitative RT-PCR showed that GhDREB1L was induced in the cotton cotyledons by low temperature, as well as drought and NaCl treatments. These results suggested that the novel cotton GhDREB1L might play an important role in response to low temperature as well as drought and high salinity through binding to the DRE cis-element.
Science in China Series C Life Sciences 03/2007; 50(1):7-14. · 1.61 Impact Factor
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ABSTRACT: Beta-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) beta-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the beta-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, sequence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.
Science in China Series C Life Sciences 05/2006; 49(2):105-14. · 1.61 Impact Factor
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ABSTRACT: The metal ion binding properties of a phytochelatin (PC) analogue, (Glu-Cys)4-Gly (named as EC4), have been studied by a divalent metal ion binding assay monitored by UV-visible spectroscopy, circular dichroism and NMR spectroscopy. Spectro- photometric titration with different divalent metal ions have revealed that the stiochoimetry of metal-bound EC4 was 1:1, and its metal binding affinities with different divalent metal ions in the order of Cd(II)>Cu(II)>Zn(II)>Pb(II)>Ni(II)>Co(II). UV-visible spectroscopic analysis of metal complexes indicated that four sulfur atoms in cysteine residues are attributable to ligand-to-metal charge transfer (LMCT) between divalent metal ions and EC4, and further confirmed by 1D H1 NMR study and Circular Dichroism. In addition, Circular Dichroism spectra of both free and metal-bound forms of EC4 revealed that metal coordination drives the nonapeptide chain to fold into a turned conformation. The comprehensive analysis of spectroscopic properties of the nonapeptide complexed with metal ions not only provides a fundamental description of the metal ion binding properties of PC analogue, but also shows a correlation between metal binding affinity of PC analogue and the induction activity of metal ions.
Journal of Inorganic Biochemistry 11/2005; 99(10):1952-62. · 3.35 Impact Factor
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ABSTRACT: Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.
PROTEOMICS 09/2005; 5(12):3162-72. · 4.51 Impact Factor
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ABSTRACT: Tabtoxin resistance protein (TTR) is an enzyme that catalyzes the acetylation of tabtoxin rendering tabtoxin-producing pathogens tolerant to their own phytotoxins. According to the structure based detoxification mechanism of TTR, three site-directed mutants Y141F, D130N and Y141F-D130N were constructed and overexpressed in E. coli. The products were then purified and their properties were analyzed by CD and DLS. Crystallization trials of two mutants Y141F andY141F-D130N were preformed.
Protein and Peptide Letters 07/2003; 10(3):255-63. · 1.94 Impact Factor
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Hongzhen He,
Yi Ding,
Mark Bartlam,
Fei Sun,
Yi Le,
Xincheng Qin,
Hong Tang,
Rongguang Zhang,
Andrzej Joachimiak, Jinyuan Liu,
Nanming Zhao,
Zihe Rao
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ABSTRACT: Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.
Journal of Molecular Biology 02/2003; 325(5):1019-30. · 4.00 Impact Factor
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ABSTRACT: One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.
Protein Expression and Purification 05/2002; 24(3):439-44. · 1.59 Impact Factor
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ABSTRACT: The molecular techniques including Northern blot, dot blot,in situ hybridization, etc. have been successfully used to estimate semi-quantitatively mRNA levels in plant samples. In this study,
we employed a real-time reverse transcription-PCR (RT-PCR) assay using SYBR Green I fluorescence methodology to evaluate accurate
quantitation and sequence specific detection ofAux/IAA mRNA levels inArabidopsis. Results obtained indicate a linear dynamic range of 102–106
Aux/IAA mRNA copies with standard deviations of generally less than 15%. As a model experiment, the outcome of analysis of expression
patterns of fiveAux/IAA genes inArabidopsis under various chemical and temperature treatments is presented. The method presented here provides a sensitive and rapid
technique to evaluate plantAux/IAA mRNA expression levels in nanogram order.
Chinese Science Bulletin 09/2001; 46(19):1642-1645. · 1.32 Impact Factor
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ABSTRACT: Kiwifruit metallothionein, kiwi503, is a typical plant metallothionein protein. It has 63 amino acid residues in two cysteine-rich
regions and one spacer region of about 32 residues. In this note, the two cysteine-rich regions and the spacer region have
been modeted separatety by the distance geometry and the homology method. The three parts are then connected to generate a
three-dimensional structural modet of kiwifruit metallothionein kiwi503. The result shows that there is no structural or energy
barrier preventing either cysteine rich domain from independently forming a metal-sulfur chetating cluster. The method can
also be applied to predicting the structures of the same type of other proteins.
Chinese Science Bulletin 07/2000; 45(15):1413-1417. · 1.32 Impact Factor
