scholarly journals The influence of posttranscription silensing protein-suppressor P19 on the transient gfp gene expression level in aztec tobacco plants (Nicotiana rustica L.)

2020 ◽  
Vol 26 ◽  
pp. 169-175
Author(s):  
O. I. Varchenko ◽  
M. S. Dzuh ◽  
M. F. Parii ◽  
Yu. V. Symonenko
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gene Expression Level ◽  
Expression Level ◽  
Nicotiana Rustica ◽  
Gfp Gene ◽  
Green Fluorescent ◽  
Genetic Constructs ◽  
Spectrofluorimetric Analysis

Aim. Genetic constructs creation for studying the influence effect of the viral posttranscriptional silencing protein suppressor p19 on transient reporter green fluorescent protein (GFP) expression and accumulation. Methods. The Golden Gate molecular cloning method was used to create the genetic constructs; the leafy tissues of the Aztec tobacco plants (Nicotiana rustica L.) were infiltrated with a suspension of Agrobacterium tumefaciens L.; the gfp gene expression level was determined by spectrofluorometric and quantitative protein (Bradford method) assays. Results. As a result of the work, the pSPV2324 genetic construct was created, which contained the reporter gene for the green fluorescent protein gfp and the gene for the synthesis of the viral posttranscriptional silencing protein suppressor p19 and its effect on the accumulation of the recombinant GFP protein was determined. A comparative analysis of the gfp gene expression level without and with the suppressor protein synthesis gene in the genetic vector showed that the fluorescence level of GFP protein in Aztec tobacco tissues was 1.3 times higher during spectrofluorimetric analysis using the p19 suppressor gene construct. Conclusions. The positive effect of the viral suppressor silencing P19 gene on the accumulation of recombinant GFP protein in tissues plants of N. rustica L. was shown for the first time. The increase in GFP protein fluorescence when using the p19 suppressor protein construct in spectrofluorimetric analysis coincides with an increase in the total concentration of total water-soluble proteins and the level fluorescence of GFP protein in their native electrophoretic separation. Keywords: cloning, genetic constructs, transient expression, silencing protein suppressor p19, green fluorescent protein (GFP).

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

scholarly journals Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Liyue Wang ◽  
Kao Zhang ◽  
Hongyu Lin ◽  
Wenyan Li ◽  
Jiexia Wen ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
North American ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Type Ii ◽  
Nsp2 Gene ◽  
Gfp Gene ◽  
Overlap Pcr ◽  
Green Fluorescent ◽  
North American Type

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

scholarly journals The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in Phanerochaete chrysosporium

2001 ◽  
Vol 67 (2) ◽  
pp. 948-955 ◽  
Author(s):  
Biao Ma ◽  
Mary B. Mayfield ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Phanerochaete Chrysosporium ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Extracellular Fluid ◽  
Gene Promoter ◽  
Green Fluorescent Protein Gene ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly withegfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of theegfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of theegfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studyingcis-acting elements in the mnp1 gene promoter.

Sign in / Sign up

Export Citation Format

Share Document