scholarly journals Testing Transgenic Aspen Plants with bar Gene for Herbicide Resistance under Semi-natural Conditions

Acta Naturae ◽  
2016 ◽  
Vol 8 (2) ◽  
pp. 92-101 ◽  
Author(s):  
V. G. Lebedev ◽  
V. N. Faskhiev ◽  
N. P. Kovalenko ◽  
K. A. Shestibratov ◽  
A. I. Miroshnikov
Keyword(s):  
Freeze Tolerance ◽  
Pcr Analysis ◽  
Bar Gene ◽  
Forest Trees ◽  
Rt Pcr ◽  
Natural Conditions ◽  
Herbicide Resistant ◽  
Transgenic Aspen ◽  
European Aspen ◽  
Transgenic Lines

Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.

scholarly journals Various effects of the expression of the xyloglucanase gene from Penicillium canescens in transgenic aspen under semi-natural conditions

2020 ◽  
Author(s):  
Elena O. Vidyagina ◽  
Natalia M. Subbotina ◽  
Vladimir A. Belyi ◽  
Vadim G. Lebedev ◽  
Konstantin V. Krutovsky ◽  
...  
Keyword(s):  
Cell Wall ◽  
Decomposition Rate ◽  
Transgenic Line ◽  
Carbohydrate Composition ◽  
Penicillium Canescens ◽  
Natural Conditions ◽  
Biochemical Traits ◽  
Transgenic Aspen ◽  
Transgenic Lines ◽  
Accelerated Growth

Abstract Background: Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to by a number of problems. Therefore, additional research is needed to alleviate them. Results: Results of successful cultivation of the transgenic aspens ( Populus tremula ) carrying the recombinant xyloglucanase gene ( sp-Xeg ) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. Conclusion: A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more markedly were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness and a decrease in the rate of decomposition of wood were observed.

scholarly journals GGR (Geranylgeranyl Reductase) Expression Affects the Allelopathic Response to Arabidopsis Allelochemicals

2018 ◽  
Vol 10 (7) ◽  
pp. 122
Author(s):  
Debora Almeida Alcântara da Silva ◽  
Juliane Laner de Toledo ◽  
Flaviani Gabriela Pierdoná ◽  
Gabriel Sergio Costa Alves ◽  
Michelle de Souza Fayad André ◽  
...  
Keyword(s):  
Growth And Development ◽  
Pcr Analysis ◽  
Enzyme Complex ◽  
Wild Type ◽  
Geranylgeranyl Diphosphate ◽  
Rt Pcr ◽  
Leaf Extracts ◽  
Expression Levels ◽  
Transgenic Lines ◽  
Allelopathic Effects

Allelopathy involves the release of compounds into the environment that affects the growth and development of other organisms. This phenomenon may lead to the production of compounds less harmful to the environment than traditional herbicides used in weed control. In plants, terpenes have been identified as components of allelochemicals and are synthesized by enzymes named as geranylgeranyl diphosphate synthases (GGPPS). There are about 12 GGPPS genes in Arabidopsis, among which is GGR. This work aims to study the association between the expression levels of GGR and the allelopathic response of sesame seedlings to Arabidopsis leaf extracts. Hence, the GGR gene was inserted into Arabidopsis with the purpose to investigate the allelopathic effects of GGR expression levels on sesame seedlings. GGR expression levels were quantified by RT-PCR in both transgenic and non-transgenic [wild-type (WT)] lines. It has been observed that both wild-type and GGR expressing transgenic lines inhibited the growth of sesame seedlings. However, it is noteworthy that the phytotoxicity of extracts from GGR lines were greater than WT extracts. RT-PCR analysis of GGR expression revealed that WT plants had higher levels of GGR expression than GGR transgenic lines, which suggests that a homologous-dependent gene silencing (HDGS) occurred in GGR lines. GGR is part of an enzyme complex that works as a hub that determines the types of terpenes produced in Arabidopsis chloroplasts. The present data indicates that decreases in GGR expression may have favoured the production of terpenes with stronger allelopathic capacity in Arabidopsis leaves.

scholarly journals Various effects of the expression of the xyloglucanase gene from Penicillium canescens in transgenic aspen under semi-natural conditions

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Elena O. Vidyagina ◽  
Natalia M. Subbotina ◽  
Vladimir A. Belyi ◽  
Vadim G. Lebedev ◽  
Konstantin V. Krutovsky ◽  
...  
Keyword(s):  
Cell Wall ◽  
Decomposition Rate ◽  
Transgenic Line ◽  
Carbohydrate Composition ◽  
Penicillium Canescens ◽  
Natural Conditions ◽  
Biochemical Traits ◽  
Transgenic Aspen ◽  
Transgenic Lines ◽  
Accelerated Growth

Abstract Background Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to a number of problems. Therefore, additional research is needed to alleviate them. Results Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. Conclusion A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more pronounced were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness, and a decrease in the rate of decomposition of wood were observed.

scholarly journals Various effects of the expression of the xyloglucanase gene from Penicillium canescens in transgenic aspen under semi-natural conditions

2020 ◽  
Author(s):  
Elena O. Vidyagina ◽  
Natalia M. Subbotina ◽  
Vladimir A. Belyi ◽  
Vadim G. Lebedev ◽  
Konstantin V. Krutovsky ◽  
...  
Keyword(s):  
Cell Wall ◽  
Decomposition Rate ◽  
Transgenic Line ◽  
Carbohydrate Composition ◽  
Penicillium Canescens ◽  
Natural Conditions ◽  
Biochemical Traits ◽  
Transgenic Aspen ◽  
Transgenic Lines ◽  
Accelerated Growth

Abstract Background: Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to by a number of problems. Therefore, additional research is needed to alleviate them. Results: Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. Conclusion: A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more markedly were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness and a decrease in the rate of decomposition of wood were observed.

scholarly journals Protein extract of tobacco expressing StoVe1 gene inhibits Verticillium dahliae proliferation

2013 ◽  
Vol 49 (No. 2) ◽  
pp. 58-64 ◽  
Author(s):  
S.P. Liu ◽  
Y.B. Hong ◽  
Z. Wu ◽  
Y.S. Ma ◽  
D.W. Jue ◽  
...  
Keyword(s):  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Pcr Analysis ◽  
35S Promoter ◽  
Rt Pcr ◽  
Protein Extract ◽  
Solanum Torvum ◽  
Transgenic Lines ◽  
Application Potential ◽  
Anti Fungal

Verticillium dahliae is a principal pathogen causing verticillium wilt in Solanaceae crops. StoVe1 is a gene resisting to verticillium wilt isolated from Solanum torvum. In order to generate resistant tobacco plants, StoVe1 was inserted in the orientation behind CaMV 35S promoter of vector pGS and this construct was introduced into tobacco by Agrobacterium-mediated transformation. A total of 12 kanamycin-resistant plants were generated and 7 independent transgenic lines were identified by PCR analysis. Quantitative RT-PCR analysis showed that the levels of StoVe1 transcript in transgenic lines were up to 2–6 fold higher than in the control. Anti-fungal assay indicated that the protein extract of transgenic tobacco lines showed strong inhibition activity to V. dahliae, 2 fold or higher compared to control plants. This result reveals that StoVe1, as a V. dahliae resistance gene, has an application potential in plant breeding for verticillium wilt resistance.

scholarly journals RT-PCR Analysis and Stress Response Capacity of Transgenic gshI-Poplar Clones (Populus × canescens) in Response to Paraquat Exposure

2006 ◽  
Vol 61 (9-10) ◽  
pp. 699-703 ◽  
Author(s):  
András Bittsánszky ◽  
Gábor Gyulai ◽  
Mervyn Humphreys ◽  
Gábor Gullner ◽  
Zsolt Csintalan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Hybrid Poplar ◽  
Ribosomal Gene ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Gene Expressions ◽  
Response Capacity ◽  
Glutamylcysteine Synthetase ◽  
Transgenic Lines

Abstract Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) of untransformed hybrid poplar, Populus × canescens (P. tremula × P. alba), and two transgenic lines overexpressing γ-ECS (γ-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 × 10-9 to 4.0 × 10-6 m) for 21 days. Significant differences at sublethal (4.0 × 10-7 m) and bleaching (4.0 × 10-6 m) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 × 10-7 m) samples of the transgenic poplar line (cyt-ECS).

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

2021 ◽  
Vol 9 (1) ◽  
pp. 7
Author(s):  
Yusuke Makino ◽  
Kaoru Fujikawa ◽  
Miwako Matsuki-Fukushima ◽  
Satoshi Inoue ◽  
Masanori Nakamura
Keyword(s):  
Bacterial Infections ◽  
Tooth Movement ◽  
Tooth Eruption ◽  
Intercellular Adhesion Molecule ◽  
Enamel Organ ◽  
Pcr Analysis ◽  
Maturation Stage ◽  
Oral Epithelium ◽  
Rt Pcr ◽  
Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

scholarly journals RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

2005 ◽  
Vol 86 (12) ◽  
pp. 3419-3424 ◽  
Author(s):  
Constanze Yue ◽  
Elke Genersch
Keyword(s):  
Varroa Destructor ◽  
Pcr Analysis ◽  
Body Parts ◽  
Rt Pcr ◽  
Larval Food ◽  
Viral Pathogen ◽  
Deformed Wing Virus ◽  
Significant Difference ◽  
Almost All ◽  
Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

scholarly journals Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

2004 ◽  
Vol 183 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Mika Suzuki ◽  
Hiroshi Kobayashi ◽  
Yoshiko Tanaka ◽  
Naohiro Kanayama ◽  
Toshihiko Terao
Keyword(s):  
Real Time ◽  
Trypsin Inhibitor ◽  
Target Genes ◽  
Reproductive Function ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Reproductive Process ◽  
Female Mice ◽  
Retinoid Metabolism ◽  
Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

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