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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Optimized methodology for obtention of high-yield and-quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri

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Author(s):
Nobrega, Bianca B. [1, 2] ; Soares, Douglas M. M. [2] ; Zamuner, Caio K. [2] ; Stevani, V, Cassius
Total Authors: 4
Affiliation:
[1] Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo - Brazil
[2] V, Univ Sao Paulo, Inst Quim, Dept Quim Fundamental, Sao Paulo - Brazil
Total Affiliations: 2
Document type: Journal article
Source: Journal of Microbiological Methods; v. 191, DEC 2021.
Web of Science Citations: 0
Abstract

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna like vegetation) in Northeast Brazil, especially in Piaui State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and-quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy (R) kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of similar to 130% in comparison to the RNeasy (R) method and of similar to 40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy (R) method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and-yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes. (AU)

FAPESP's process: 20/16000-3 - Functional characterization of candidate genes for the biological clock of Neonothopanus gardneri and its relationship with bioluminescence
Grantee:Bianca de Barros Nóbrega
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 19/12605-0 - Bioluminescence of Fungi, Millipede and Marine Organisms: Chemical and Biological Aspects
Grantee:Douglas Moraes Mendel Soares
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 19/21782-3 - Caffeic Acid Cycle (CAC) in fungal bioluminescence: luciferin antioxidant effect, oxyluciferin recycling and oxidative stress
Grantee:Caio Klocke Zamuner
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 17/22501-2 - Electronic chemiexcitation in biological systems: bioluminescence and photochemistry in the dark
Grantee:Etelvino José Henriques Bechara
Support Opportunities: Research Projects - Thematic Grants