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Reporter Genes02:11

Reporter Genes

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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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Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions
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Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Jimmy Villalta-Villalobos1,2, Luiz Filipe Pereira3,4, Andrés Gatica-Arias5,6

  • 1School of Biology, University of Costa Rica, San José, Costa Rica.

Methods in Molecular Biology (Clifton, N.J.)
|April 24, 2024
PubMed
Summary

Researchers developed a new method for genetically modifying coffee plants to resist the coffee berry borer (CBB). This involves using a gene from Bacillus thuringiensis in a cell suspension system for faster development of resistant coffee varieties.

Keywords:
Agrobacterium tumefaciensBacillus thuringiensisEmbryogenic cell suspension culturesGenetic transformationcyt1Aa, barCoffee

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Area of Science:

  • Agricultural Science
  • Molecular Biology
  • Entomology

Background:

  • Coffea arabica L. is genetically uniform, increasing vulnerability to pests like the coffee berry borer (CBB).
  • Current CBB control methods are limited as the pest spends most of its life cycle inside coffee beans.
  • Bacillus thuringiensis cyt1Aa gene offers potential as a biological insecticide.

Purpose of the Study:

  • To establish a transient expression system for validating insecticidal genes in Coffea arabica.
  • To develop a method for genetically engineering coffee cell suspensions to express the cyt1Aa gene.
  • To lay the groundwork for creating coffee varieties resistant to CBB and other stresses.

Main Methods:

  • Utilized embryogenic cell suspensions of Coffea arabica L. var. Catuaí.
  • Employed Agrobacterium tumefaciens (strain GV3101::pMP90) for genetic transformation, introducing the bar and cyt1Aa genes.
  • Verified cyt1Aa gene integration and expression using PCR at 2, 5, and 7 weeks post-infection.

Main Results:

  • Successfully demonstrated the transient expression of the cyt1Aa gene in coffee cell suspensions.
  • Confirmed the presence of the cyt1Aa gene via PCR analysis over several weeks.
  • Established a functional protocol for genetic modification of coffee cell cultures.

Conclusions:

  • The developed cell suspension system is an efficient platform for testing candidate genes for pest resistance in coffee.
  • This method facilitates the development of genetically improved coffee varieties resistant to CBB.
  • The protocol supports future applications in developing resistance to various biotic/abiotic stresses and genome editing.