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…

Accumulation of deleterious mutations in grapevine and its relationship with traits of interest for wine production and resilience

Deleterious mutations that severely reduce population fitness are rapidly removed from the gene pool by purifying selection. However, evolutionary drivers such as genetic drift brought about by demographic bottlenecks may comprise its efficacy by allowing deleterious mutations to accumulate, thereby limiting the adaptive potential of populations. Moreover, positive selection can hitchhike mildly deleterious mutations due to linkage caused by lack of recombination. Similarly, in the context of species domestication, artificial selection mimics these evolutionary processes, which can have undesirable consequences for production and resilience. In this study, we evaluated the extent of the accumulation of deleterious mutations and the magnitude of their effects (also known as genetic load) at the whole-genome scale for ca.

A multidisciplinary approach to grapevine zoning G.I.S. technology based: an example of thermal data elaboration

Un grand nombre d’études ont été consacrées à l’évaluation quantitative des effets de climat sur la qualité des vignes, dans différents contextes climatiques. Généralement, la vocation viticole d’un terroire peut être étudiée par des approches mono ou multidisciplinaires.

La Région Délimitée du Douro et le Vin de Porto — un terroir historique —

The viticulture of the Douro Delimited Region, one of the heirs of ancestral viticulture, traditionally empirical and of quality, while integrating modernity and contemporary tools, respects and has always present the principles on which it was developed.

INFLUENCE OF WINEMAKING VARIABLES AND VINEYARD LOCATIONS ON CHEMICAL AND SENSORY PROFILES OF SOUTH TYROLEAN PINOT BLANC

Pinot Blanc, an important grape variety grown in some mountain areas of Northern Italy such as South Tyrol over the last decades, with its cultivation covering 10.3% of the total vineyards, has compatible climatic conditions (e.g. heat requirements) which are normally found in the geographical areas of the mountain viticulture [1,2,3,4]. Climatic changes are hastening the growth of this variety at higher elevations, particularly for the production of high quality wine.

Organoleptic and analytical impacts of the color of glass of the bottles on Chasselas wine

This study was performed on Chasselas wine to assess the impact of exposure to wine light according to several glass color of bottles. The aim was to highlight any differences whether from an organoleptic or analytical point of view depending on the color. For this, four different shades were compared, dead leaf, green, cinnamon and transparent. A control, not treated with light, was also included in the study…