Identification of reference genes for quantitative real-time PCR in different developmental stages and under refrigeration conditions in soursop fruits (Annona muricata L.)

摘要

The quantitative real-time polymerase chain reaction (qRT-PCR) is a technique to quantify the gene expression. However, data normalization needs suitable references genes that show a constant expression under different conditions. Up to date, no reference genes have been evaluated in soursop fruits (Annona muricata L). The objective of this work was to analyze the transcript stability of some genes in different developmental stages and under refrigeration conditions in soursop fruits. We created a database from a public transcriptome data of soursop leaf, identify homologous gene in soursop and designed primers to amplify the reference genes: Ubiquitin carrier-like protein (UBCc), Ubiquitin-conjugating enzyme E2 2 (UBCg), Elongation factor 1 alpha (EF1 alpha), beta-tubulin (TUB) and 18 S ribosomal RNA (18S). Total RNA was extracted from soursop mesocarp at 0, 3 and 6 days from fruits stored at 25 +/- 1 degrees C and 0, 3, 6 and 9 days from fruits stored at 15 +/- 1 degrees C. cDNA was synthesized through SuperScript III kit following the manufacturer instructions. The gene expression was analyzed by qRT-PCR using the Rotor-Gene Q equip, the geNorm and RefFinder web-tool were used to classify the most stable transcript for each condition. The results indicated that the UBCc was the most stable transcript during the fruit development at 25 +/- 1 degrees C. On the other hand, EF1 alpha showed the highest stability in soursop fruits stored at 15 +/- 1 degrees C. Global analysis of both conditions demonstrated that UBCc and EF1 alpha were the most stable transcripts and can be used as reference genes for the normalization of the data in soursop during fruit development and under refrigeration.