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…

Etude préliminaire des influences pédoclimatiques sur les caractéristiques quali-quantitatives du cépage aglianico dans une zone de la province de benevento-ltalie

The need to classify the vineyards of an area according to the quality of its wines is not recent, but it is only in the last ten years that studies on the suitability of different areas for the cultivation of vineyard take on an integrated and interdisciplinary character (Boselli, 1991). The definition of the suitability of the environment is thus obtained by making the climatic, pedological, topographical and cultural information interact with the vegetative, productive and qualitative expression of the grape varieties.

Analyses of a long-term soil temperature record for the prediction of climate change induced soil carbon changes and greenhouse gas emissions in vineyards

The evaluation of the current and future impact of climate change on viticulture requires an integrated view on a complex interacting system within the soil-plant-atmospheric continuum under continuous change. Aside of the globally observed increase in temperature in almost all viticulture regions for at least four decades, we observe several clear trends at the regional level in the ratio of precipitation to potential evapotranspiration. Additionally the recently published 6th assessment report of the ipcc (the physical science basis) shows case-dependent further expected shifts in climate patterns which will have substantial impacts on the way we will conduct viticulture in the decades to come.

Polyphenolic profile and dietary fiber content of skins and seeds from unfermented and fermented grape pomace

The valorization of winemaking byproducts is subordinated to the knowledge of their chemical characteristics. This work concerned the determination of the polyphenolic profile and the dietary fiber content of skins and seeds from unfermented and fermented pomace of different cultivars (Moscato bianco, Cortese, Arneis, Pinot Noir, Barbera, Grignolino, Nebbiolo), sampled from some wineries in the Piedmont area (Italy) during the 2020 harvest.

Sustainability as system innovation: sustainability as system innovation: a returnable system for glass wine bottles

Introduction increasing sustainability is essential and a societal challenge, requiring fundamental changes in behaviour and attitudes. This applies to both producers and consumers. For the wine industry in particular, such a change is a major challenge. An eip-agri research project is evaluating the introduction of a returnable glass system in the german wine industry as a key solution for increasing sustainability. Given the need for change associated with a returnable system, the project is theoretically grounded in systems innovation, as this approach provides solutions for complex, transformative change.

Efficient irrigation strategies and water use reduction in the high quality production regions of Priorat and Montsant (Spain)

Priorat and Montsant Appellations of Origin are located in the south of Catalonia (North‐East Spain), under severe Mediterranean climatic conditions