Efficient use of the PMI/mannose selection system in Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum)

Biologia ◽  
2015 ◽  
Vol 70 (4) ◽  
Author(s):  
Ehsasatvatan ◽  
Morad Jafari ◽  
Reza Darvishzadeh
Keyword(s):  
Transgenic Plants ◽  
Leaf Explants ◽  
Basal Medium ◽  
Pcr Analysis ◽  
Environment Friendly ◽  
Reverse Transcription Pcr ◽  
Putative Transformants ◽  
Chlorophenol Red ◽  
Tobacco Genome ◽  
Novel Products

AbstractA safe and environment friendly selectable marker system using phosphomannose isomerase (pmi) gene was employed for Agrobacterium tumefaciens mediated transformation of tobacco (Nicotinia tabacum L.). Sensitivity of tobacco leaf explants to mannose as selective agent was determined before genetic transformation. Twenty mg/L mannose was chosen for selection to suppress the emergence of untransformed shoots from the explants. Leaf explants from 4-week old seedlings of tobacco cv. Gewone Groene were transformed using Agrobacterium harbouring plasmid pCAMBIA3300-PMI carrying the pmi gene. Regenerating transformed shoots were selected on Murashige & Skoog basal medium supplemented with 1.0 mg/L 6-benzylaminopurine, 0.1 mg/L indole-3-butyric acid, and 20 g/L mannose as carbon source. The mannose-resistant shoots were rooted on a plant growth regulator-free Murashige & Skoog basal medium and the potted transgenic plants were acclimatized successfully in the greenhouse, with a survival rate of 100%. Selection efficiency and transformation efficiency based on PCR analysis of individual putative transformants were achieved as high as 94.56% and 21.75%, respectively, with a lower rate of untransformed plant escapes. Southern blot analysis of selected putative transformed plants confirmed the transgene integration into the tobacco genome. The following reverse transcription PCR analysis showed transcriptional activity of the pmi transgene in all T0 transgenic plants analysed with differences in the level of pmi transcripts, and chlorophenol red assay confirmed the activity of PMI in transgenic plants. The PMI/mannose-based transformation system presented here is efficient and reproducible, and can be used for the mass production of transgenic tobacco plants to produce novel products with industrial and medicinal values.

scholarly journals DEVELOPMENT OF TRANSGENIC SWEETPOTATO AND ANALYSIS OF TRANSGENE EXPRESSION

HortScience ◽  
1995 ◽  
Vol 30 (3) ◽  
pp. 441d-441
Author(s):  
C.S. Prakash ◽  
O. Zheng ◽  
A. Porobodessai
Keyword(s):  
Transgenic Plants ◽  
Somatic Embryos ◽  
Transgene Expression ◽  
Fusion Gene ◽  
Leaf Explants ◽  
Organ Specificity ◽  
Pcr Analysis ◽  
Ms Medium ◽  
Gus Assay ◽  
Foreign Genes

Stable, transgenic, sweetpotato plants have been developed using an improved somatic embryogenesis consisting of l) stage I—explants incubated in darkness for 14 days on MS medium with 2,4D (2.5 mg·liter–1) and 6-BAP (0.25 mg·liter–1) and 2) stage II—culture in light for 14 to 28 days on MS medium with ABA (2.5 mg·liter–1). Petiole or leaf explants of the genotype PI318846-3 were co-cultivated with Agrobacterium tumefaciens EHA 101 containing gusA::nptII fusion gene. Transgenic somatic embryos were selected on a kanamycin medium (100 mg·liter–1). The PCR analysis of the transgenic sweetpotato plants showed the presence of foreign genes in the sweetpotato genome. About 100 transgenic plants are being maintained under laboratory and greenhouse conditions. All the transgenic plants showed a strong expression of gusA gene in the histochemical GUS assay but showed quantitative differences in the chemiluminescent assay. The CaMV35S promoter shows a differential expression because there was some degree of tissue- and organ-specificity in the gusA expression. All transgenic plants appear normal with no phenotypic aberrations and are being tested for productivity traits.

scholarly journals MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1202-1212
Author(s):  
Aichun Zhang ◽  
Yangzi Jin
Keyword(s):  
Allergic Rhinitis ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Specific Ige ◽  
Inflammatory Factors ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Pathway Activation ◽  
Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

The effects of quercetin on the expression of SREBP-1c mRNA in high-fat diet-induced NAFLD in mice

2021 ◽  
Vol 32 (4) ◽  
pp. 637-644
Author(s):  
Jamal Nasser Saleh Al-maamari ◽  
Mahardian Rahmadi ◽  
Sisca Melani Panggono ◽  
Devita Ardina Prameswari ◽  
Eka Dewi Pratiwi ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Fatty Liver Disease ◽  
Inhibitory Effect ◽  
Pcr Analysis ◽  
Regulator Gene ◽  
High Fat ◽  
Alcoholic Fatty Liver ◽  
Reverse Transcription Pcr ◽  
Hematoxylin Eosin ◽  
Alcoholic Fatty Liver Disease

Abstract Objectives The study aimed to determine the effect of quercetin on the expression of primary regulator gene involved in lipogenesis and triglycerides synthesis in the liver, and the sterol regulatory binding protein-1c (SREBP-1c) mRNA in non-alcoholic fatty liver disease (NAFLD) with a high-fat diet (HFD) model. Methods Fifty-six Balb/c mice were divided into seven groups: standard feed; HFD; HFD and quercetin 50 mg/kg for 28 days; HFD and quercetin 100 mg/kg BW for 28 days; HFD and quercetin 50 mg/kg for 14 days; HFD and quercetin 100 mg/kg for 14 days; HFD and repaired fed for 14 days. Quercetin was administered intraperitoneally. The animals were sacrificed 24 h after the last treatment; the liver was taken for macroscopic, histopathological staining using hematoxylin–eosin and reverse transcription-PCR analysis sample. Results HFD significantly increased the expression of SREBP-1c mRNA; meanwhile, quercetin and repaired feed significantly reduced the expression of SREBP-1c mRNA in the liver. Quercetin at a dose of 50 mg/kg and 100 mg/kg also improved liver cells’ pathological profile in high-fat diet NAFLD. Conclusions The present study suggests that quercetin has an inhibitory effect on SREBP-1c expression and improved liver pathology in NAFLD mice.

scholarly journals Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

2000 ◽  
Vol 74 (21) ◽  
pp. 10176-10186 ◽  
Author(s):  
T. Yamaguchi ◽  
S. L. Kaplan ◽  
P. Wakenell ◽  
K. A. Schat
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Northern Blotting ◽  
Marek's Disease ◽  
Pcr Analysis ◽  
Marek’S Disease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Herpesvirus Of Turkeys ◽  
Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

2021 ◽  
Author(s):  
Can Chen ◽  
Yi Zong ◽  
Jiaojiao Tang ◽  
Ruisheng Ke ◽  
Lizhi Lv ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

scholarly journals Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

2018 ◽  
Vol 22 ◽  
pp. 222-227
Author(s):  
O. M. Honcharuk ◽  
O. V. Dubrovna
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Bread Wheat ◽  
Binary Vector ◽  
Triticum Aestivum L ◽  
Callus Cultures ◽  
Pcr Analysis ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

scholarly journals Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

2020 ◽  
Author(s):  
Xiya Zuo ◽  
Shixiang Wang ◽  
Wen Xiang ◽  
Huiru Yang ◽  
Muhammad Mobeen Tahir ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Floral Transition ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Transcriptional Responses ◽  
Genome Database ◽  
Reverse Transcription Pcr ◽  
Conserved Core

Abstract Background: Apple (Malus domestica Borkh.) is one of the most popular cultivated fruit crops in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

scholarly journals Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

2020 ◽  
Author(s):  
Xiya Zuo ◽  
Shixiang Wang ◽  
Wen Xiang ◽  
Huiru Yang ◽  
Muhammad Mobeen Tahir ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Economic Value ◽  
Floral Transition ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Transcriptional Responses ◽  
Genome Database ◽  
Reverse Transcription Pcr

Abstract Background: Apple (Malus domestica Borkh.) is a popular cultivated fruit crop with high economic value in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

scholarly journals The Erwinia chrysanthemi 3937 PhoQ Sensor Kinase Regulates Several Virulence Determinants

2006 ◽  
Vol 188 (8) ◽  
pp. 3088-3098 ◽  
Author(s):  
Balakrishnan Venkatesh ◽  
Lavanya Babujee ◽  
Hui Liu ◽  
Pete Hedley ◽  
Takashi Fujikawa ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Regulatory System ◽  
Soft Rot ◽  
Erwinia Chrysanthemi ◽  
Pcr Analysis ◽  
Sensor Kinase ◽  
Virulence Determinants ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Two Component

ABSTRACT The PhoPQ two-component system regulates virulence factors in Erwinia chrysanthemi, a pectinolytic enterobacterium that causes soft rot in several plant species. We characterized the effect of a mutation in phoQ, the gene encoding the sensor kinase PhoQ of the PhoPQ two-component regulatory system, on the global transcriptional profile of E. chrysanthemi using cDNA microarrays and further confirmed our results by quantitative reverse transcription-PCR analysis. Our results indicate that a mutation in phoQ affects transcription of at least 40 genes, even in the absence of inducing conditions. Enhanced expression of several genes involved in iron metabolism was observed in the mutant, including that of the acs operon that is involved in achromobactin biosynthesis and transport. This siderophore is required for full virulence of E. chrysanthemi, and its expression is governed by the global repressor protein Fur. Changes in gene expression were also observed for membrane transporters, stress-related genes, toxins, and transcriptional regulators. Our results indicate that the PhoPQ system governs the expression of several additional virulence factors and may also be involved in interactions with other regulatory systems.

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