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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Deciphering Precise Gene Transcriptional Expression Using gwINTACT in Tomato.

Yiyang Chu1, Jiachen Gong1, Peiwen Wu1

  • 1College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.

Frontiers in Plant Science
|May 2, 2022
PubMed
Summary

A new tomato nuclear purification method, gwINTACT, effectively isolates nuclei from leaves, flowers, and fruits, removing over 95% of chloroplasts. This technique enhances the study of gene transcription in various plant tissues.

Keywords:
gwINTACTnuclear RNAnuclei purificationtomatotranscriptional expression

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Area of Science:

  • Plant molecular biology
  • Gene expression analysis

Background:

  • Nuclear gene transcription is crucial for plant physiology.
  • The Isolation of Nuclei Tagged in Specific cell type (INTACT) technique purifies nuclei for studying gene expression dynamics.
  • Traditional INTACT methods are limited in plants due to chloroplast contamination, especially in non-root tissues.

Purpose of the Study:

  • To develop a simplified and efficient INTACT method for nuclear purification in tomato (Solanum lycopersicum) leaves, flowers, and fruits.
  • To overcome chloroplast pollution issues in plant nuclear isolation.

Main Methods:

  • A novel gwINTACT method was designed using mas-enhanced GFP (eGFP)-SlWIP2.
  • The gwINTACT method was applied to various tomato tissues (leaves, flowers, fruits).
  • Nuclear yield and purity were compared to traditional INTACT procedures.

Main Results:

  • The gwINTACT method successfully purified nuclei from tomato leaves, flowers, and fruits.
  • Nuclear yield from tomato leaves using gwINTACT was doubled compared to traditional INTACT.
  • Over 95% of chloroplasts were removed using the gwINTACT method.
  • Re-evaluation of ethylene-related gene expression in gwINTACT leaves revealed distinct results compared to total RNA-based assays.

Conclusions:

  • The established gwINTACT system enables precise deciphering of transcriptional status in diverse tomato tissues.
  • This method provides a foundation for advanced studies like ChIP-seq and ATAC-seq to investigate nucleus-related molecular regulation, particularly in fruit ripening.