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.

Download the abstract

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…

The impacts of frozen material-other-than-grapes (MOG) on aroma compounds of red wine varieties

An undesirable note called “floral taint” has been observed in red wines by winemakers in the Niagara region caused by large volumes of frozen leaves and petioles [materials-other-than-grapes (MOG)] introduced during mechanical harvest and subsequent winemaking late in the season. The volatiles, which we hypothesized are responsible, are primarily terpenes, norisoprenoids, and specific esters in frozen leaves and petioles. The purpose of this study was to investigate the volatile compounds which may cause the floral taint problem and explore how much of them (thresholds) may lead to the problem. Also, the glycosidic precursors of some of these compounds were analyzed to see the changes happening during frost events.

Read More

Using 1H-NMR combined with chemometrics to discriminate the effect of different cuts and toasting of woods used for grape pomace distillate ageing

The purpose of this research study is to consider new solutions for distillate ageing, in alternative to conventional oak chips or barrels in particular sliced wood and peeled wood were compared to oak cubes, normally employed during both wine and distillate ageing.

Read More

Biochemical characterization of grape skin cell wall during ripening in relation to Botrytis cinerea susceptibility of two Champagne cultivars

Pectins or pectic polysaccharides are one of the major components in grape skin cell wall, they contribute to physiological processes which determine the integrity and rigidity of grape skin tissue

Read More

SSR analysis of some Vitis sylvestris (GMEL.) accessions of the Szigetköz and Fertő-hanság national park, Hungary

The evolution of cultivated plants played important role in the ascent of humanity. Research of their origin and evolution started at the beginning of the20th century, but till nowadays a lot of questions remain open. A large number of theories exist about the evolution of the European grapevine (Vitis vinifera L.). The Vitis sylvestris GMEL. in Hungary is a protected species.

Read More

Precipitation variability in a temperate coastal region and how it affects Tannat and Albariño cultivars 

Climate is one of the main components that defines the development and behavior of the plant, conditioning the health status and the final quality of the grapes. In temperate coastal climates such as in Uruguay (latitude 35° S, longitude 55° O), precipitations during the growing season present high interannual variability, with a average of 100 mm per month. This variability means that plants must adapt to conditions from one year to the next.

Read More