Selección de genes de referencia en expresión génica de Citrus sinensis infectados con CLas o CTV mediante RT-qPCR.

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 RTqPCR 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. [ABSTRACT FROM AUTHOR]