PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9404544

Factors affecting transient expression of vector constructs in wheat protoplasts.

K Z Ahmed·S Omirulleh·F Sági·D Dudits
View original source ↗Export metadata ↓

Abstract

Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

Explore related subjects

Gene ExpressionGenes, ReporterGenetic VectorsGlucuronidasePlants, Genetically ModifiedPlasmidsPolyethylene GlycolsProtoplastsTransformation, GeneticTriticumJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

K Z Ahmed, S Omirulleh, F Sági, D Dudits. 1997. Factors affecting transient expression of vector constructs in wheat protoplasts.. https://pubmed.ncbi.nlm.nih.gov/9404544/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Protocol to improve isoform-level quantification of low-abundance transcripts via STALARD pre-amplification.

STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 μg input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.

Gene Expression↗
PubMed2007

Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform.

Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.

Gene Expression↗
PubMed2007

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants.

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H(+)-ATPases. We investigated expression of Lycopersicon esculentum Mill. H(+)-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H(+)-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.

Gene Expression↗