terclim by ICS banner
IVES 9 IVES Conference Series 9 Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Abstract

Cis-acting regulatory elements are DNA sequences that can be bound by transcription factors to regulate the expression of genes in a condition-dependent and tissue-specific way. It is nowadays possible to search for DNA motives and sequences that a given transcription factor is binding or at least can, but it is still hard to have a glance at all the transcription factors that are contemporaneously located at the same locus. Inspired by an existing technique that uses the CRISPR-Cas system in mammal cells, we are trying to develop a protocol to study such regulation in Vitis vinifera. Using the highly sequence-specific binding capacity of a catalytically inactive Cas9 protein (dCas9), our idea is to set up a system to target a desired sequence and precipitate all the crosslinked proteins and distantly interacting chromatin at this locus and analyze them. After conducting preliminary assays on protoplast system, we got introduced to the CRISPR-FISH technique, that uses dCas9 and a fluorescent guide to label telomeres on nuclei directly isolated from a small quantity of fixed leaves with a very simple procedure. We used part of such technique to easily obtain the chromatin that was needed for our trials and eventually performed the pull-down of the targeted DNA sequences directly on these nuclei after transforming them with the dCas9 complex. Sequencing the obtained fragments allows to verify the specificity of the tool. Far from having eliminated the idea of using protoplasts as model system, we proceed developing both approaches in parallel.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Aurélien Devillars1*, Silvia Farinati1, Edoardo Bertini2, Chiara Fattorini2, Sara Lissandrini2, Adriana Fernanda Soria Garcia1, Bhanu Prakash Potlapalli3, Andreas Houben3, Sara Zenoni2 and Alessandro Vannozzi1

1 Department of Agriculture Food Natural Resources Animals and Environment, University of Padova, Agripolis, Legnaro, Italy
2 Department of Biotechnology, University of Verona, Verona, Italy
3 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany

Contact the author*

Keywords

Molecular Biology, Grapevine, Gene Regulation, CRISPR-Cas9, Protoplasts

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

New satellite-based sampling protocols for grapevine nutrient monitoring

Extension specialists often recommend nutrient monitoring through leaf blade or petiole sampling twice a season for each vineyard block. However, due to the time and labor required to collect a large, random sample, many growers complete the task infrequently or incorrectly. Readily available remote sensing images capture the vineyard variability at both spatial and temporal scales, which can capture canopy and soil variability and be used to guide growers to representative sampling locations.

Colloids in red wines: new insights from recent research

Despite their significant impact on wine quality and stability, colloids in red wine remain relatively under-researched. A series of studies, developed in the context of the d-wines project, aimed to provide a comprehensive understanding of the structure, composition, and formation mechanisms of red wine colloids by studying monovarietal wines from 10 of the most significant Italian red grape varieties. Starting from the idea that proteins, polysaccharides, and tannins should be involved in colloid formation, 110 monovarietal red wines were analysed for these components, revealing high inter- and intra-varietal diversity [1].

From local classification to regional zoning. The use of a geographic information system (GIS) in Franconia / Germany. Part 3: classification of soil parameters in vineyards

La conservation de la fertilité du sol est un aspect primordial dans la viticulture durable. Différents paramètres, comme par exemple la topographie, la composition du sol, les conditions climatiques, influencent la fertilité du sol des surfaces viticoes.

la caratterizzazione dell’areale viticolo “terre alte di brisighella”: aspetti metodologici e primi risultati

La zonazione viticola rappresenta un importante strumento di indagine per valutare e interpretare le potenzialità produttive e qualitative di un territorio. Con l’obiettivo di studiare come l’ambiente influisca sulla qualità dell’uva nell’areale di Brisighella, sono stati monitorati, nelle annate 2007, 2008 e 2009, 14 vigneti per la varietà Albana e 38 per la varietà Sangiovese, rappresentativi di una area vitata di circa 1000 ha.

SENSORY EVALUATION OF WINE AROMA: SHOULD COLOR-DRIVEN DESCRIPTORS BE USED?

The vocabulary used to describe wine aroma is commonly organized according to color, raising the question of whether they reflect the reality of olfactory perception. Previous studies have assumed this convention of color-aroma matching, and have investigated color’s influence on the perception of aroma only in dyed white wine or in red wine from particular places of origin. Here 48 white and red varietal wines from around the world were evaluated in black glasses then in clear glasses by a panel of wine experts, who gave intensity ratings for aroma attributes commonly used by wine professionals. In black glasses, aromas conventionally associated with white wine were perceived in the red wines, and vice versa.