Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

2020 ◽  
Vol 82 (6) ◽  
pp. 74-83
Author(s):  
O. Varchenko ◽  
◽  
M. Kuchuk ◽  
M. Parii ◽  
Y. Symonenko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Water Soluble ◽  
Cauliflower Mosaic Virus ◽  
Histone H4 ◽  
Bisphosphate Carboxylase ◽  
Gfp Gene ◽  
The Difference ◽  
Genetic Constructs

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader

Virus Genes ◽  
1989 ◽  
Vol 3 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Johannes F�tterer ◽  
Karl Gordon ◽  
Pierre Pfeiffer ◽  
H�l�lene Sanfa�on ◽  
Barbara Pisan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Differential Inhibition ◽  
Downstream Gene Expression ◽  
Cauliflower Mosaic Virus 35S

Recognition- and defense-related gene expression at 3 resynthesis stages in lichen symbionts

2015 ◽  
Vol 61 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Sarangi N.P. Athukorala ◽  
Michele D. Piercey-Normore
Keyword(s):  
Gene Expression ◽  
Defense Responses ◽  
Water Soluble ◽  
Physical Contact ◽  
Related Gene ◽  
Defense Related Gene ◽  
The Difference ◽  
Plant Pathogen Interactions ◽  
Cladonia Rangiferina ◽  
Incompatible Interactions

Recognition and defense responses are early events in plant–pathogen interactions and between lichen symbionts. The effect of elicitors on responses between lichen symbionts is not well understood. The objective of this study was to compare the difference in recognition- and defense-related gene expression as a result of culture extracts (containing secreted water-soluble elicitors) from compatible and incompatible interactions at each of 3 resynthesis stages in the symbionts of Cladonia rangiferina. This study investigated gene expression by quantitative PCR in cultures of C. rangiferina and its algal partner, Asterochloris glomerata/irregularis, after incubation with liquid extracts from cultures of compatible and incompatible interactions at 3 early resynthesis stages. Recognition-related genes were significantly upregulated only after physical contact, demonstrating symbiont recognition in later resynthesis stages than expected. One of 3 defense-related genes, chit, showed significant downregulation in early resynthesis stages and upregulation in the third resynthesis stage, demonstrating a need for the absence of chitinase early in thallus formation and a need for its presence in later stages as an algal defense reaction. This study revealed that recognition- and defense-related genes are triggered by components in culture extracts at 3 stages of resynthesis, and some defense-related genes may be induced throughout thallus growth. The parasitic nature of the interaction shows parallels between lichen symbionts and plant pathogenic systems.

scholarly journals Altered Patterns of Gene Expression in Arabidopsis Elicited by Cauliflower Mosaic Virus (CaMV) Infection and by a CaMV Gene VI Transgene

1999 ◽  
Vol 12 (5) ◽  
pp. 377-384 ◽  
Author(s):  
Chiara Geri ◽  
Edi Cecchini ◽  
Maria E. Giannakou ◽  
Simon N. Covey ◽  
Joel J. Milner
Keyword(s):  
Gene Expression ◽  
Transgenic Plants ◽  
Mosaic Virus ◽  
Rna Binding ◽  
Important Determinant ◽  
Blot Hybridization ◽  
Cauliflower Mosaic Virus ◽  
Slot Blot Hybridization ◽  
Pcr Products ◽  
Polymerase Chain

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

Cauliflower mosaic virus as a gene expression vector for plants

1990 ◽  
Vol 79 (1) ◽  
pp. 154-157 ◽  
Author(s):  
J. Futterer ◽  
J. M. Bonneville ◽  
T. Hohn
Keyword(s):  
Gene Expression ◽  
Mosaic Virus ◽  
Expression Vector ◽  
Cauliflower Mosaic Virus

scholarly journals Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery

2016 ◽  
Vol 29 (3) ◽  
pp. 156-164 ◽  
Author(s):  
W. Allen Miller ◽  
Ruizhong Shen ◽  
William Staplin ◽  
Pulkit Kanodia
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Mosaic Virus ◽  
Barley Yellow Dwarf Virus ◽  
Plant Viruses ◽  
Cauliflower Mosaic Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Noncoding Sequences ◽  
Yellow Dwarf Virus

Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like ‘sponges’ that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5′ to 3′ degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host’s RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race.

scholarly journals Genetic constructs creation using Golden Gate cloning method

2019 ◽  
Vol 25 ◽  
pp. 190-196 ◽  
Author(s):  
O. I. Varchenko ◽  
B. M. Krasyuk ◽  
A. A. Fedchunov ◽  
O. V. Zimina ◽  
M. F. Parii ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Golden Gate ◽  
Genes Encoding ◽  
Green Fluorescent ◽  
Genetic Constructs

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

scholarly journals Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

2021 ◽  
Author(s):  
Richard Nelson Hall ◽  
Uri Weill ◽  
Margarita Khariton ◽  
Sergio Leal-Ortiz ◽  
Leonard Drees ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Untranslated Region ◽  
Adult Stem Cells ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
Reporter Expression ◽  
Mrna Transfection ◽  
Genetic Constructs ◽  
Exogenous Gene

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

scholarly journals Studying the effect of tissue-specific expression of the K1 gene encoding LysM-receptor-like kinase on the development of symbiosis in peas

2020 ◽  
Vol 23 ◽  
pp. 03005
Author(s):  
Anna N. Kirienko ◽  
Elena A. Dolgikh
Keyword(s):  
Mosaic Virus ◽  
Infection Process ◽  
Cauliflower Mosaic Virus ◽  
Nodule Formation ◽  
Gene Encoding ◽  
Specific Expression ◽  
Root Cortex ◽  
Tissue Specific ◽  
Receptor Like Kinase ◽  
Genetic Constructs

To study the role of pea LysM receptor-like kinase K1 in the coordination of the infection process, starting in epidermis and nodule organogenesis in the root cortex of plants, during the development of rhizobium-legume symbiosis, the genetic constructs in which K1 gene was cloned under the control of tissue-specific promoter pLeEXT1 of tomato Lycopersicon esculentum extensin gene and the constitutive promoter of cauliflower mosaic virus (CaMV35S, cauliflower mosaic virus 35S) were obtained. During the transformation of the Nod- mutant line, the k1-1, with two types of constructs, the restoration of nodule formation was observed, which indicated the possible participation of K1 in the control not only early, but also later stages of symbiosis development in pea.

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).

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