This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see), and thus represents an important step toward understanding the molecular basis governing other subcellular structures. © JoVE 2006-2012. All Rights Reserved.

Fluorescence-microscopy screening and next-generation sequencing: Useful tools for the identification of genes involved in organelle integrity / Stefano G.; Renna L.; Brandizzi F.. - In: JOURNAL OF VISUALIZED EXPERIMENTS. - ISSN 1940-087X. - ELETTRONICO. - (2012), pp. 0-0. [10.3791/3809]

Fluorescence-microscopy screening and next-generation sequencing: Useful tools for the identification of genes involved in organelle integrity

Stefano G.
Writing – Review & Editing
;
Renna L.;
2012

Abstract

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see), and thus represents an important step toward understanding the molecular basis governing other subcellular structures. © JoVE 2006-2012. All Rights Reserved.
2012
0
0
Stefano G.; Renna L.; Brandizzi F.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1230197
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 3
  • ???jsp.display-item.citation.isi??? ND
social impact