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RNA Blot Analysis (rna + blot_analysis)
Selected AbstractsMolecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxinFEBS JOURNAL, Issue 14 2001Goro Taguchi Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, ,,20,50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells. [source] Molecular characterization of a human scavenger receptor, human MARCOFEBS JOURNAL, Issue 3 2000Nabil A. Elshourbagy Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria-binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full-length hMARCO clone encoded a 519-amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand-binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low-density lipoprotein (LDL), despite its similarity to the elongated collagen-like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis. [source] Characterization of a Novel RING Finger Gene OsRFP1, which is Induced by Ethylene, Salicylic Acid and Blast Fungus Infection in RiceJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2008Shanyue Zhou Abstract OsRFP1, a C3H2C3 -type zinc finger gene, was isolated through screening a blast-induced rice cDNA library. The full-length cDNA of the OsRFP1 gene is 1393 bp with an open reading frame (ORF) encoding 302 amino acid residues. The deduced amino acid sequence of OsRFP1 contains an N-terminal Pfam:zf-CHY domain and a C-terminal C3H2C3 -type RING signature. OsRFP1 was found localizing in the nucleus based on the fluorescence emitted by OsRFP1-GFP fusion protein expressed in onion epidermal cells. GAL4 DNA-binding vector pBD-containing OsRFP1 could activate expression of the reporter genes of His/Ade/LacZ in yeast strain AH109 indicating that OsRFP1 has the transcriptional activation activity. RNA blot analysis showed that expression of the OsRFP1 gene was significantly induced by ethylene (ET), salicylic acid (SA) and blast fungus infection. Together, these results indicate that the OsRFP1 may function as a transcriptional regulator in ET-dependent signal pathway in plant defense. [source] Identification of potato genes induced during colonization by Phytophthora infestansMOLECULAR PLANT PATHOLOGY, Issue 3 2001Katinka Beyer Summary Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and elevation of transcript levels during P. infestans infection was confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to ,-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillus nidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail. [source] Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA familiesNEW PHYTOLOGIST, Issue 1 2009Guru Jagadeeswaran Summary ,,MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) have emerged as important regulators of gene expression in higher eukaryotes. Recent studies indicate that genomes in higher plants encode lineage-specific and species-specific miRNAs in addition to the well-conserved miRNAs. Leguminous plants are grown throughout the world for food and forage production. To date the lack of genomic sequence data has prevented systematic examination of small RNAs in leguminous plants. Medicago truncatula, a diploid plant with a near-completely sequenced genome has recently emerged as an important model legume. ,,We sequenced a small RNA library generated from M. truncatula to identify not only conserved miRNAs but also novel small RNAs, if any. ,,Eight novel small RNAs were identified, of which four (miR1507, miR2118, miR2119 and miR2199) are annotated as legume-specific miRNAs because these are conserved in related legumes. Three novel transcripts encoding TIR-NBS-LRR proteins are validated as targets for one of the novel miRNA, miR2118. Small RNA sequence analysis coupled with the small RNA blot analysis, confirmed the expression of around 20 conserved miRNA families in M. truncatula. Fifteen transcripts have been validated as targets for conserved miRNAs. We also characterized Tas3-siRNA biogenesis in M. truncatula and validated three auxin response factor (ARF) transcripts that are targeted by tasiRNAs. ,,These findings indicate that M. truncatula and possibly other related legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post-transcriptionally. [source] Over-expression of a Populus peroxisomal ascorbate peroxidase (PpAPX) gene in tobacco plants enhances stress tolerancePLANT BREEDING, Issue 4 2009Y-J. Li Abstract Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. We studied the effect of over-expressing a Populus peroxisomal ascorbate peroxidase (PpAPX) gene under the control of the cauliflower mosaic virus 35S promoter or the rd29 promoter in transgenic tobacco. High levels of PpAPX gene expression were observed in 35S-PpAPX transgenic plants, with a 50% increase in APX activity. The constitutive expression of PpAPX in the tobacco exhibited no morphological abnormalities, while significantly increased root growth was observed in transgenic plants, when compared to control plants. Several independently transformed lines were propagated and evaluated for resistance to methyl viologen (MV), drought and salt stress. Visual assessment of transgenic and control lines exposed to MV (50 or 100 ,mol) confirmed that over-expression of APX minimized leaf damage. APX activity was nearly 80% higher in the leaves of transgenic plants in response to drought or salt stresses. Moreover, the transgenic tobacco also showed significantly improved drought resistance and salt tolerance at the vegetative stage. RNA blot analysis indicated that the PpAPX transcript level was very low under normal growing conditions in rd29Ap-PpAPX plants, but clearly increased under drought stress. Our results show that PpAPX does not play a significant role under normal growing conditions, but did ameliorate oxidative injury under abiotic stress. The Ad29 promoter should be used as an inducible promoter in transgenic works. [source] | ||||||||||