阳春砂实时荧光定量PCR内参基因的筛选

Screening of Reference Genes for Real-time Fluorescence Quantitative PCR in Amomum villosum Lour.

【目的】筛选稳定表达的内参基因,用于阳春砂不同发育时期的果皮和种子团中基因表达量实时荧光定量PCR(qRTPCR)检测的校正。【方法】以阳春砂3个不同发育时期的果实(分为果皮和种子团)为材料,根据高通量测序得到的转录组和表达谱数据,选择5个表达稳定的常用内参基因β-actin、EF-1α、GAPDH、PGK和TUA作为候选基因,并利用qPCR技术检测它们在不同样品中表达水平的变化,使用GeNorm和NormFinder软件对基因的稳定性进行分析。【结果】5个内参基因在不同发育时期的果皮和种子团中的表达稳定性有明显的差异,其中GeNorm分析得到的内参基因的稳定顺序为:EF-1α=TUA>PGK>GAPDH>β-actin,NormFinder的分析结果中稳定性最好的是EF-1α,其次为TUA,其他3个内参基因稳定性的排列顺序与GeNorm软件的结果一致。【结论】可选用EF-1α和TUA作为阳春砂果实发育过程中基因表达量差异分析的双内参基因。

Objective To identify the reliable reference genes for gene expression analysis of the pericarp and seed of A momum 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-lc~ = TUA 〉PGK〉GAPDH〉^-actin. The results of NormFinder revealed that EF-la 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-1α and 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.