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…

Unveiling the bioactive potential of aglianco grape pomace: oleanolic acid as a promising natural product

The winemaking industry generates a substantial amount of byproducts, including grape pomace, which is often discarded as waste. However, this seemingly useless material holds a wealth of bioactive compounds with potential health benefits. Recognizing the value of circular economy principles, this study delves into the comprehensive chemical analysis of aglianco grape pomace, aiming to transform this byproduct into a valuable resource.

Aspect juridiques des terroirs

Le “terroir” est dans tous les discours, les articles, les étiquettes et les publicités. Le voca­ble est en situation d’utilisation euphorique. Indiscutablement l’emploi historique est agri­cole, puis viticole, mais il n’est jamais juridique.

Red wine astringency: correlations between chemical and sensory features

Astringency is a crucial sensory attribute typically described as the drying and/or puckering sensation occurring after the consumption of tannin-rich foods and beverages. In this study, thirty-seven red wines from different varieties, origins and styles were evaluated, analyzing both chemical and sensory features. Principal Component Analysis was used for dimensionality-reduction and for correlating selected chemical parameters against astringency. The results showed that tannin content was the most important chemical parameter influencing overall astringency but more clearly the dryness sub-quality, followed by pH, titratable acidity and alcohol content.

EUGENOL AS QUALITY MARKER OF WINES AND SPIRITS FROM HYBRID VINES: IMPACT OF DIFFERENT WINEMAKING AND DISTILLATION PROCESSES

Eugenol, widely spread in various plants notably cloves, basil and bay, was identified too in wines from hybrid grapes without contact with oak wood. This aromatic molecule presents a strong spicy note of clove and also antifongic properties. Eugenol was described as an endogenous compound of Baco blanc, from the grapes to the spirits of Armagnac area. Moreover, this compound is a chemical marker of Baco blanc products quality.
Influences of harvest time and different winemaking processes (settling, use of enzymatic preparations, lees content and stock time before distillation) on Baco blanc wine eugenol contents were explored using a two-levels full factorial Design of Experiments (DoEs).

Combined use of leaf removal and natural shading to delay grape ripening in Manto negro (Vitis vinifera L.) under deficit irrigation 

The increasingly frequent heat waves during grape ripening pose challenges for premium wine grape production. This makes the development of irrigation and canopy management techniques of great importance to maximize yield and grape quality. A field experiment was carried out during 2021 and 2022 using Manto negro wine grapes to study the effect of two irrigation strategies and different light exposure levels on grape quality.