Functional characterization of a stilbene synthase gene using a transient expression system in planta

2008 ◽  
Vol 28 (4) ◽  
pp. 589-599 ◽  
Author(s):  
Jose Condori ◽  
Giuliana Medrano ◽  
Ganapathy Sivakumar ◽  
Vipin Nair ◽  
Carole Cramer ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Stilbene Synthase ◽  
In Planta ◽  
Transient Expression System ◽  
Stilbene Synthase Gene

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.

scholarly journals Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

1996 ◽  
Vol 7 (10) ◽  
pp. 1485-1498 ◽  
Author(s):  
M Ramachandra ◽  
S V Ambudkar ◽  
M M Gottesman ◽  
I Pastan ◽  
C A Hrycyna
Keyword(s):  
Drug Resistance ◽  
Atpase Activity ◽  
Cyclosporin A ◽  
Transient Expression ◽  
Expression System ◽  
Rhodamine 123 ◽  
Wild Type ◽  
P Glycoprotein ◽  
Transient Expression System

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

Characterization of transient expression system for retroviral vector production

2006 ◽  
Vol 101 (4) ◽  
pp. 361-368 ◽  
Author(s):  
Akitsu Hotta ◽  
Yoshikazu Saito ◽  
Kenji Kyogoku ◽  
Yoshinori Kawabe ◽  
Ken-ichi Nishijima ◽  
...  
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Retroviral Vector ◽  
Transient Expression System

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 A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yongpeng Li ◽  
Tiantian Chen ◽  
Wei Wang ◽  
Hang Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Transient Expression ◽  
High Throughput ◽  
High Efficiency ◽  
Artemisia Annua ◽  
Expression System ◽  
Functional Characterization ◽  
Cauliflower Mosaic Virus ◽  
Transient Transformation ◽  
Transformation System ◽  
Transient Expression System

Abstract Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

scholarly journals Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

2020 ◽  
Vol 21 (7) ◽  
pp. 2264 ◽  
Author(s):  
Rui Ren ◽  
Jie Gao ◽  
Chuqiao Lu ◽  
Yonglu Wei ◽  
Jianpeng Jin ◽  
...  
Keyword(s):  
Transient Expression ◽  
Transfection Efficiency ◽  
Expression System ◽  
Functional Characterization ◽  
Protoplast Isolation ◽  
Bimolecular Fluorescence Complementation ◽  
Dna Amount ◽  
Time Saving ◽  
Highly Efficient ◽  
Transient Expression System

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana

2012 ◽  
Vol 31 (7) ◽  
pp. 1309-1319 ◽  
Author(s):  
Selvaraju Kanagarajan ◽  
Saraladevi Muthusamy ◽  
Anna Gliszczyńska ◽  
Anneli Lundgren ◽  
Peter E. Brodelius
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Artemisia Annua ◽  
Expression System ◽  
Functional Expression ◽  
Artemisia Annua L ◽  
Transient Expression System

Isolation of Mesophyll Protoplast and Establishment of Gene Transient Expression System in Cotton

2014 ◽  
Vol 40 (2) ◽  
pp. 231 ◽  
Author(s):  
Ni-Na LI ◽  
Lin-Yun DING ◽  
Zhi-Yuan ZHANG ◽  
Wang-Zhen GUO
Keyword(s):  
Transient Expression ◽  
Expression System ◽  
Mesophyll Protoplast ◽  
Transient Expression System
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