Procedures of Mitochondria Purification and Gene Expression to Study Alternative Respiratory and Uncoupling Pathways in Fruits
| Autor: | Clesivan Pereira dos Santos, José Hélio Costa, Marcos Góes Oliveira, Diederson Bortolino Santana, Luis Miguel Mazorra Morales, Jurandi Gonçalves de Oliveira, Kátia Daniella da Cruz Saraiva, Gláucia Michelle Cosme Silva |
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| Rok vydání: | 2017 |
| Předmět: |
0106 biological sciences
0301 basic medicine Alternative respiration biology food and beverages Mitochondrion biology.organism_classification 01 natural sciences Cell biology 03 medical and health sciences 030104 developmental biology Gene expression medicine RNA extraction Mannitol Carica Solanum Gene 010606 plant biology & botany medicine.drug |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493972913 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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