Abstract

TRIANA-GUTIERREZ, Rosa Isela et al. Housekeeping genes selection for gene expression on Citrus sinensis infected with CLas or CTV using RT-qPCR. Rev. mex. fitopatol [online]. 2021, vol.39, n.2, pp.354-370.  Epub Nov 03, 2021. ISSN 2007-8080.  https://doi.org/10.18781/r.mex.fit.2102-2.

A possible alternative for the control of CTV and CLas in Citrus sinensis is the use of systemic acquired resistance. The study of this mechanism requires normalizing with reference genes. Therefore, a gene expression quantification protocol using RT-qPCR was developed to evaluate the potential use of GAPDH, ACTIN, F-BOX, COX, and 18S rRNA as reference genes in citrus. A total of nine plants infected with CTV (3), CLas (3), and healthy (3) were used to select a composed sample of eight leaves/plant. An in-house protocol was developed and optimized in all its stages, varying the concentration of reagents, primers, and reaction template. Extraction of total RNA with 2% CTAB was in the range of 200-1000 ng µL-1. The reverse transcription produced on average 1069 ng µL-1 cDNA. The reference gene products GAPDH, ACTINA, and F-BOX exhibited melting curves without dimer products, Ct ≤ 28, and reaction efficiencies in the range of 90-110%. In all gene expressions, CTV infected samples had the higher Ct’s (25-27) followed by healthy samples (24-25). However, GAPDH and ACTIN had the most stable gene expression (ln 1/M = 2.83), therefore these genes are proposed for normalization. The RT-qPCR protocol was also specific and efficient for the CDR13 gene, putatively associated with systemic acquired resistance to CLas, which suggest its viability in resistance studies of C. sinensis/C. aurantium in response to CTV and CLas infection.

Keywords : Transcriptome; Orange; CDR13; ACTIN; GAPDH.

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