Transient expression of foreign chimeric genes in the gymnosperm hybrid larch following electroporation

1991 ◽  
Vol 69 (8) ◽  
pp. 1731-1736 ◽  
Author(s):  
Pierre J. Charest ◽  
Yvonne Devantier ◽  
Christine Ward ◽  
Catherine Jones ◽  
Ulriche Schaffer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Inducible Promoter ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Hybrid Larch ◽  
Naked Plasmid ◽  
Angiosperm Species

A transient gene expression system using electroporation and naked plasmid DNA has been developed for hybrid larch (Larix × eurolepis). The β-glucuronidase, neomycin phosphotransferase II, and chloramphenicol acetyltransferase genes were used effectively, but the latter was found to be the most useful. Electroporation conditions were comparable with protocols developed for other conifer and angiosperm species. Of the parameters tested the optimum conditions were 300 V, 150 μF, and 300 μg/mL pCaMVCN DNA. The 35S promoter of cauliflower mosaic virus yielded a stronger level of transient expression than the nopaline synthase promoter, which is consistent with other studies. A construct with the wound-inducible promoter of the potato proteinase IIK gene and the chloramphenicol acetyl transferase reporter coding sequence did not yield to any transient gene expression, even after induction with acetylsalicylic acid and exposure to ultraviolet radiation. Key words: larch, Larix × eurolepis, electroporation, transient gene expression.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Grapevine Protoplasts as a Transient Expression System for Comparison of Stilbene Synthase Genes Containing cGMP-Responsive Promoter Elements

1999 ◽  
Vol 54 (3-4) ◽  
pp. 220-229 ◽  
Author(s):  
Ilka Brehm ◽  
Regina Preisig-Müller ◽  
Helmut Kindl
Keyword(s):  
Cell Wall ◽  
Transient Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Stilbene Synthase ◽  
Fungal Cell Wall ◽  
Cauliflower Mosaic Virus ◽  
Fungal Cell ◽  
Stilbene Synthase Gene ◽  
Rna Promoter

Abstract A method for preparing elicitor-responsive protoplasts from grapevine cells kept in suspen­sion culture was established. The protoplasts were employed in order to perform transient gene expression experiments produced by externally added plasmids. Using the gene coding for bacterial β-glucuronidase as the reporter gene, the transient expression under the control of various promoters of stilbene synthase genes were analyzed. The elicitor-responsiveness of promoters from grapevine genes and heterologous promoters were assayed: the grapevine stilbene synthase gene VST-1 and pine stilbene synthase genes PST-1, PST-2 and PST-3. Compared to the expression effected by the cauliflower mosaic virus 35S RNA-promoter, the stilbene synthase promoters caused a 2-5-fold increase in GUS-activity. Incubation of transformed protoplasts with fungal cell wall further stimulated the stilbene synthase promoters but not the 35S RNA-promoter. An even more pronounced differentiation between the promoters was observed when cGMP was included in the transient expression assays. Instead of treating transformed protoplasts with fungal cell wall we administered simultaneously cGMP and the plasmid to be tested. The cGMP-responsive increase was (a) specific concerning the nucleotide applied, (b) characteristic of grapevine protoplasts, and (c) not seen with shortened promoter-GUS constructs or GUS under the control of the 35S RNA-promoter. The highest cGMP-dependent reponse to stress was shown by the promoter of the grapevine stilbene synthase gene VST-1.

scholarly journals Factors Affecting Transient Gene Expression in Apple Cotyledons following Particle Bombardment

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 617a-617
Author(s):  
Hong Y. Yang ◽  
Schuyler S. Korban
Keyword(s):  
Gene Transfer ◽  
Transient Expression ◽  
Particle Bombardment ◽  
Transient Gene Expression ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Tungsten Particle ◽  
Factors Affecting ◽  
Efficient Gene

Developing an efficient gene transfer system for apple (Malus ×domestica L.) remains a major objective in genetic engineering efforts of this fruit crop. Transient expression of the uidA gene coding for β-glucuronidase (GUS) and driven by the cauliflower mosaic virus 35S promoter (CaMV35S) has been induced in apple cotyledonary explants of mature seeds by tungsten particle bombardment using the Particle Inflow Gun (PIG). Several factors that affect transient expression of the GUS gene in apple cotyledons were investigated. The gene transfer efficiency was monitored by recording the number of blue spots observed on explants two days following bombardment. Precultivation of cotyledons for 18 hours before bombardment significantly increased the number of blue foci. Of the three different precipitation methods tested including water, 25% PEG, and 60% glycerol, the latter was the most effective for coating DNA onto tungsten particles. Washing DNA-coated tungsten particles with 70% ethanol and resuspending in 100% ethanol significantly enhanced gene delivery to cotyledons. The amount of particles used for each bombardment also influenced GUS expression. About 0.5 mg of particles per shot resulted in the highest number of blue foci. Using larger quantity of particles (i.e., 2 mg) drastically decreased GUS expression probably due to the toxicity of tungsten particles.

scholarly journals Transient Gene Expression System Using Protoplasts from Developing Soybean Cotyledons.

1993 ◽  
Vol 43 (1) ◽  
pp. 53-60
Author(s):  
Mayuko ISHIKAWA ◽  
Kyuya HARADA ◽  
Fukumi SAKAI ◽  
Yuko OHASHI ◽  
Tadao NAITO
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Gene Expression System

Human growth hormone as a reporter gene in regulation studies employing transient gene expression

1986 ◽  
Vol 6 (9) ◽  
pp. 3173-3179 ◽  
Author(s):  
R F Selden ◽  
K B Howie ◽  
M E Rowe ◽  
H M Goodman ◽  
D D Moore
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Transient Expression ◽  
Human Growth Hormone ◽  
Expression System ◽  
Simian Virus 40 ◽  
Human Growth ◽  
Assay System ◽  
Transient Assay ◽  
Pituitary Cells

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.

scholarly journals Molecular Rewiring of the Jasmonate Signaling Pathway to Control Auxin-Responsive Gene Expression

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 641
Author(s):  
Ning Li ◽  
Linggai Cao ◽  
Wenzhuo Miu ◽  
Ruibin Cao ◽  
Mingbo Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathway ◽  
Developmental Process ◽  
Expression System ◽  
Transient Gene Expression ◽  
Transcriptional Repressor ◽  
Auxin Signaling ◽  
General Strategy ◽  
Auxin Signaling Pathway ◽  
Responsive Gene

The plant hormone jasmonic acid (JA) has an important role in many aspects of plant defense response and developmental process. JA triggers interaction between the F-box protein COI1 and the transcriptional repressors of the JAZ family that leads the later to proteasomal degradation. The Jas-motif of JAZs is critical for mediating the COI1 and JAZs interaction in the presence of JA. Here, by using the protoplast transient gene expression system we reported that the Jas-motif of JAZ1 was necessary and sufficient to target a foreign reporter protein for COI1-facilitated degradation. We fused the Jas-motif to the SHY2 transcriptional repressor of auxin signaling pathway to create a chimeric protein JaSHY. Interestingly, JaSHY retained the transcriptional repressor function while become degradable by the JA coreceptor COI1 in a JA-dependent fashion. Moreover, the JA-induced and COI1-facilitated degradation of JaSHY led to activation of a synthetic auxin-responsive promoter activity. These results showed that the modular components of JA signal transduction pathway can be artificially redirected to regulate auxin signaling pathway and control auxin-responsive gene expression. Our work provides a general strategy for using synthetic biology approaches to explore and design cell signaling networks to generate new cellular functions in plant systems.

scholarly journals Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

1999 ◽  
Vol 42 (3) ◽  
pp. 299-302 ◽  
Author(s):  
Romulo Marino Llamoca-Zárate ◽  
Luiz Ferreira Aguiar Ponte ◽  
Joerg Landsmann ◽  
Francisco de Assis Paiva Campos
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Prickly Pear ◽  
Target Tissue ◽  
Gus Gene ◽  
Transformation System ◽  
Opuntia Ficus Indica ◽  
Apical Meristems

We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transformation system can be used to express a transgene in a member of the Cactaceae.

scholarly journals Human growth hormone as a reporter gene in regulation studies employing transient gene expression.

1986 ◽  
Vol 6 (9) ◽  
pp. 3173-3179 ◽  
Author(s):  
R F Selden ◽  
K B Howie ◽  
M E Rowe ◽  
H M Goodman ◽  
D D Moore
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Transient Expression ◽  
Human Growth Hormone ◽  
Expression System ◽  
Simian Virus 40 ◽  
Human Growth ◽  
Assay System ◽  
Transient Assay ◽  
Pituitary Cells

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.

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