Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method

The cell wall integrity (CWI) pathway is activated in response to cell wall stresses due to different food-related environments. Rho1 is one of the main regulators within such pathway. The objective of this work was to design an easy-to-use RT-qPCR technique for the evaluation of the Rho1 gene expre...

Descripción completa

Guardado en:
Detalles Bibliográficos
Publicado: 2018
Materias:
Cell wall integrity
Filamentous fungi
Rho1 gene
Stress
protein
protein PgAFP
unclassified drug
antifungal agent
fungal protein
primer DNA
Rho guanine nucleotide binding protein
Article
Aspergillus flavus
computer model
controlled study
fungal cell wall
fungal gene
fungus growth
gene expression
gene overexpression
genetic conservation
mould
nonhuman
nucleotide sequence
Penicillium
Penicillium chrysogenum
Penicillium polonucum
quantitative analysis
real time polymerase chain reaction
reverse transcription polymerase chain reaction
sensitivity and specificity
species
sporogenesis
wall stress
analysis
cell wall
food contamination
food control
gene expression regulation
genetics
metabolism
procedures
reverse transcription
Antifungal Agents
Base Sequence
Cell Wall
DNA Primers
Food Contamination
Food Microbiology
Fungal Proteins
Gene Expression
Gene Expression Regulation, Fungal
Real-Time Polymerase Chain Reaction
Reverse Transcription
rho GTP-Binding Proteins
Acceso en línea:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01681605_v275_n_p17_daCruzCabral
http://hdl.handle.net/20.500.12110/paper_01681605_v275_n_p17_daCruzCabral
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
Descripción
Sumario:The cell wall integrity (CWI) pathway is activated in response to cell wall stresses due to different food-related environments. Rho1 is one of the main regulators within such pathway. The objective of this work was to design an easy-to-use RT-qPCR technique for the evaluation of the Rho1 gene expression useful to measure responses to the presence of cell wall stressors such as the antifungal protein PgAFP. Two primer pairs were designed from published conserved regions. Their specificity initially was determined by in silico analysis for several fungal species. After optimising the qPCR, the primer pair Rho1-F1/R2 was selected due to the lowest Cq values obtained and its specificity. The qPCR method showed efficiencies between 97.5% and 100.5%. Applicability of the designed qPCR method was evaluated in the presence of the stressor PgAFP. The PgAFP-resistant Penicillium polonicum and the PgAFP-sensitive Aspergillus flavus showed Rho1 gene over- and under- expression, respectively, indicating that the CWI pathway is activated in the former species but not activated in the latter one in response to the stress caused by PgAFP. This novel qPCR methodology able to detect changes in CWI-related gene expression in filamentous fungi will be useful in future studies to evaluate physiological mould responses to different food environmental challenges. © 2018 Elsevier B.V.

Ejemplares similares