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Procedures of Mitochondria Purification and Gene Expression to Study Alternative Respiratory and Uncoupling Pathways in Fruits

  • Jurandi Gonçalves de OliveiraEmail author
  • Luis Miguel Mazorra Morales
  • Gláucia Michelle Cosme Silva
  • Kátia Daniella da Cruz Saraiva
  • Diederson Bortolino Santana
  • Clesivan Pereira dos Santos
  • Marcos Goes Oliveira
  • José Hélio Costa
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1670)

Abstract

We describe detailed procedures to get intact and well-coupled mitochondria from a variety of fruit species such as papaya (Carica papaya), guava (Psidium guajava), tomato (Solanum lycopersicum), and strawberry (Fragaria x ananassa) as well as the protocols to assess the capacities of AOX and UCP pathways in intact fruit mitochondria. The procedures presented here were tested for the species mentioned above; their use with other types of fruits must be tested for yield of intact and active mitochondria. This is possible from individual adjustments. Strict care during extraction, including the use of osmotic protectants (i.e., mannitol/sucrose) and antioxidants (i.e., cysteine, ascorbate) at defined concentrations, are critical factors to ensure mitochondrial integrity and to obtain higher yields. The mitochondria purified using the discontinuous Percoll gradients described here can be used for the analysis of the capacity of alternative respiration and uncoupling pathways in fruits. In addition, protocols for quantitative and semiquantitative PCR applicable for the analysis of AOX and UCP gene expression in fruits are shown. Microarray and RNA-seq data from public databases are also valuable for the analysis of AOX and UCP genes. In both cases having the sequences of genes or cDNAs to be used in primer design or probe identification is necessary.

Key words

Gene identification Mitochondria isolation Respiratory control ratio RNA extraction Semiquantitative RT-PCR 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jurandi Gonçalves de Oliveira
    • 1
    Email author
  • Luis Miguel Mazorra Morales
    • 1
  • Gláucia Michelle Cosme Silva
    • 1
  • Kátia Daniella da Cruz Saraiva
    • 2
  • Diederson Bortolino Santana
    • 1
  • Clesivan Pereira dos Santos
    • 2
  • Marcos Goes Oliveira
    • 3
  • José Hélio Costa
    • 2
  1. 1.Universidade Estadual do Norte Fluminense Darcy RibeiroCampos dos GoytacazesBrazil
  2. 2.Functional Genomics and Bioinformatics, Department of Biochemistry and Molecular BiologyFederal University of CearaFortalezaBrazil
  3. 3.Universidade Federal do Espirito SantoSão MateusBrazil

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