Microprojectile-mediated DNA delivery to the Salicaceae family

1993 ◽  
Vol 71 (11) ◽  
pp. 1458-1466 ◽  
Author(s):  
Yvonne A. Devantier ◽  
Barbara Moffatt ◽  
Catherine Jones ◽  
Pierre J. Charest
Keyword(s):  
Gene Expression ◽  
Populus Deltoides ◽  
Expression System ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Kanamycin Resistance ◽  
Cell Suspensions ◽  
Neomycin Phosphotransferase ◽  
Salix Alba ◽  
Neomycin Phosphotransferase Ii

A transient gene expression system was developed for the Salicaceae family using microprojectile-mediated DNA delivery (Biolistic™) to cell suspensions. Using Populus nigra × Populus maximowiczii line NM1, 10 variables were optimized. Optimum transient gene expression under the 10 conditions tested was obtained with a 7- to 9-day-old cell suspension. Highest transient levels were observed with samples positioned 13.5 cm from the stopping plate and bombarded with 1.6-μm gold particles coated with 1 μg of DNA precipitated with CaCl2. Assaying for β-glucuronidase gene expression was performed 1 day after bombardment. The fate of the microparticles in the bombarded cells was studied, showing that 58.9% of cells expressing the β-glucuronidase gene had microparticles located in the nucleus or its vicinity and the remaining cells had microparticles in the cytoplasm. Cell suspensions from five different lines (P. nigra × P. maximowiczii lines NM1 and NM6, Populus deltoides × P. nigra line DN106, Populus tremula × Populus alba line 7171-B4, and Salix alba sanguined line SA-2) yielded transient gene expression. The relative strengths of β-glucuronidase expression in the lines tested were NM1 = 7171-B4 > NM6 > DN106 > SA-2. Six plasmid constructions were also tested in line NM1 for transient β-glucuronidase gene expression. The x-glucuronide histochemical assay did not reveal any differences, but the methyl umbelliferone glucuronide fluorescent assay yielded the following relative levels of transient gene expression with the different promoter sequences: 35S-35S-AMV enhancer = 35S-AMV enhancer > 35S-35S = 35S-35S-AMV enhancer with the β-glucuronidase – neomycin phosphotransferase fusion > 35S. Four transgenic cell lines of P. nigra × P. maximowiczii were characterized for kanamycin resistance and neomycin phosphotransferase II gene activity. Polymerase chain reaction and Southern hybridization analyses indicated the presence of the β-glucuronidase and neomycin phosphotransferase II genes in the genome of three of these transgenic lines. Key words: microprojectile, particle bombardment, Salicaceae, poplar, willow.

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 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

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 Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system

2020 ◽  
Vol 37 (1) ◽  
pp. 89-92
Author(s):  
Sakihito Kitajima ◽  
Kenji Miura ◽  
Junko Yasuda
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Gene Expression System ◽  
Radish Sprouts

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

1990 ◽  
Vol 68 (6) ◽  
pp. 983-987
Author(s):  
Chris L. Baszczynski
Keyword(s):  
Brassica Napus ◽  
Bovine Serum Albumin ◽  
Kanamycin Resistance ◽  
Plant Tissues ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Kanamycin Resistance Gene ◽  
Dot Blot ◽  
Immunoprecipitation Assay ◽  
Neomycin Phosphotransferase Ii

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.Key words: anti-kanamycin antibodies, immunoprecipitation, neomycin phosphotransferase II assay, transformation.

An Agrobacterium-mediated transient gene expression system for intact leaves

Plant Science ◽  
1997 ◽  
Vol 122 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Jyoti Kapila ◽  
Riet De Rycke ◽  
Marc Van Montagu ◽  
Geert Angenon
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Gene Expression System ◽  
Intact Leaves
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