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Abstract:

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.

Registro:

Documento: Artículo
Título:Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method
Autor:da Cruz Cabral, L.; Delgado, J.; Andrade, M.J.; Rodríguez, M.; Rodríguez, A.
Filiación:Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Avda. de las Ciencias, s/n, Cáceres, 10003, Spain
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica, Laboratorio de Microbiología de Alimentos, Buenos Aires, Argentina
Palabras clave: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; Rho1 gene; sensitivity and specificity; species; sporogenesis; wall stress; analysis; cell wall; food contamination; food control; gene expression; gene expression regulation; genetics; metabolism; procedures; real time polymerase chain reaction; reverse transcription; Antifungal Agents; Aspergillus flavus; Base Sequence; Cell Wall; DNA Primers; Food Contamination; Food Microbiology; Fungal Proteins; Gene Expression; Gene Expression Regulation, Fungal; Penicillium; Real-Time Polymerase Chain Reaction; Reverse Transcription; rho GTP-Binding Proteins
Año:2018
Volumen:275
Página de inicio:17
Página de fin:23
DOI: http://dx.doi.org/10.1016/j.ijfoodmicro.2018.03.020
Título revista:International Journal of Food Microbiology
Título revista abreviado:Int. J. Food Microbiol.
ISSN:01681605
CODEN:IJFMD
CAS:protein, 67254-75-5; Antifungal Agents; DNA Primers; Fungal Proteins; rho GTP-Binding Proteins
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01681605_v275_n_p17_daCruzCabral

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Citas:

---------- APA ----------
da Cruz Cabral, L., Delgado, J., Andrade, M.J., Rodríguez, M. & Rodríguez, A. (2018) . Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method. International Journal of Food Microbiology, 275, 17-23.
http://dx.doi.org/10.1016/j.ijfoodmicro.2018.03.020
---------- CHICAGO ----------
da Cruz Cabral, L., Delgado, J., Andrade, M.J., Rodríguez, M., Rodríguez, A. "Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method" . International Journal of Food Microbiology 275 (2018) : 17-23.
http://dx.doi.org/10.1016/j.ijfoodmicro.2018.03.020
---------- MLA ----------
da Cruz Cabral, L., Delgado, J., Andrade, M.J., Rodríguez, M., Rodríguez, A. "Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method" . International Journal of Food Microbiology, vol. 275, 2018, pp. 17-23.
http://dx.doi.org/10.1016/j.ijfoodmicro.2018.03.020
---------- VANCOUVER ----------
da Cruz Cabral, L., Delgado, J., Andrade, M.J., Rodríguez, M., Rodríguez, A. Detection of changes in mould cell wall stress-related gene expression by a novel reverse transcription real-time PCR method. Int. J. Food Microbiol. 2018;275:17-23.
http://dx.doi.org/10.1016/j.ijfoodmicro.2018.03.020