Analysis of the expression of β-actin, RuANS and RuMYB10 genes involved in the biosynthesis of anthocyanin using RT-qPCR in Rubus niveus in the Rumiñahui Canton

Abstract

Abstract.BACKGROUND: Berries such as Rubus has caught the attention of scientists and consumers because of their nutritional quality. Furthermore, the presence of phenolic compounds specifically anthocyanins have added potential of antiaging, anticarcinogenic and antioxidant properties to the fruit. Gene expression studies have demonstrated the importance of flavonoids and their biosynthetic pathways, further increasing the research interests alternative and natural sources for these compounds. OBJECTIVE: The aim of this work was to analyze the level of expression of ␤-Actin as a control gene, RuANS gene encoding for anthocyanin synthase and RuMYB10 gene encoding for a transcription factor involved in the biosynthesis of anthocyanins in Rubus niveus. METHODS: To investigate the expression of the genes, the polymerase chain reaction with reverse transcription (RT-qPCR) was performed and the Qubit ® fluorometry equipment for standard curve data. RESULTS: The ANOVA at 95% classified the genes into three homogeneous groups whose means are not statistically different in the gene expression, with an F = 0.46, a p = 0.6348. The results were generated in average: gene ␤-Actin: 174,65 ng/mL, which has greater concentration, followed by the RuMYB10 gene: 167.43 ng/mL and finally the gene RuANS with 163,55. CONCLUSIONS: The Rubus niveus specie presents a level of similar expression among the three analyzed genes.

Isolation and Quantification of three Genes involved in the Expression of Anthocyanins in Rubus glaucus Benth by RT-qPCR

“…The standard curve in acceptable parameters presents an efficiency of 1,922 and an error close to 0, providing certainty in the tests [13].…”

“…To obtain the reference values, logarithmic serial solutions of the complementary DNA obtained previously were prepared, the samples were quantified in the Qubit® 2.0 Fluorometer, with Qubit® dsDNA HS Assay Kit (Life) [13].…”

“…The knowledge of genes as well as their functioning and expression in the various species, allows the opening to a new era of Transcriptomics and biotechnology in Ecuador [13], because it reaches the genomic knowledge not only to understand but also their applications in the field of health, production, quality and improvement of food.…”

Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

“…Due to the accuracy of RT-qPCR results being easily affected by sample difference, tissue specificity, extracted RNA quality, and other factors, the expression analysis of genes requires normalization to reference genes [ 14 ]. Several studies have shown that the β-actin and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) were selected as the RT-qPCR internal reference genes in Rubus niveus and Rubus idaeus , respectively [ 15 , 16 ]. However, studies on the reference genes of Rubus have not been systematically reported.…”

Expression of the ANS, CHS and DFR genes involved in the biosynthesis of anthocyanins in Vaccinium floribundum Kunth from Ecuador, using RT-qPCR.

“…The amplification protocol was programmed in the Light cycler 4.0 software (Roche): initial denaturation at 95 °C, for 1 cycle, then 60 denaturation cycles at 95 °C, hybridization at 60 °C and extension at 72 °C, to finally reach the last step of cooling for 1 cycle at a temperature of 40 °C. Finally, the previously saved external standard curve must be imported to obtain the quantitative data 16 .…”