Screening of Reference Genes for Real-time Fluorescence Quantitative PCR in Amomum villosum Lour / 广州中医药大学学报

ABSTRACT

Objective To identify the reliable reference genes for gene expression analysis of the pericarp and seed of Amomum villosum Lour. by using real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR). Methods Using the fruits ( separated into peels and seeds) of A. villosum at three different developmental periods as the experimental material, 5 candidate reference genes (β-actin, EF-1α, GAPDH, PGK, TUA) with steady expression were screened out by the high throughout sequencing of transcriptome and expression profile data. The qRT-PCR technique was applied to study the expression levels of 5 candidate reference genes in different samples. The stability of the candidate reference genes were evaluated by GeNorm and NormFinder software. Results The 5 reference genes had different stabilities in the pericarp and seed of A. villosum Lour. at different development periods . The order of the steadiness of reference genes showed by GeNorm was EF-1α = TUA>PGK>GAPDH>β-actin. The results of NormFinder revealed that EF-1α was the most stable, followed by TUA, and the order of the other three genes was as same as the results of GeNorm. Conclusion EF-1αand TUA could be used as double reference genes for the normalization of gene expression in A. villosum fruits at different developmental periods by using qRT-PCR.