石蒜属植物实时荧光定量PCR内参基因的选择

Selection of Reference Genes for Quantitative Real-Time PCR in Lycoris

以石蒜属植物换锦花(Lycoris sprengeri)、石蒜(Lycoris radiata)和中国石蒜(Lycoris chinensis Traub)不同组织器官、不同花发育时期以及不同杂交种的幼小鳞茎为研究材料,利用q RT-PCR技术检测Actin、EF-1α、GAPDH、5S r RNA、Ubiquitin和β-Tubulin等6个内参基因的m RNA表达情况,利用ge Norm、Norm Finder、Best Keeper和Reffinder软件综合评价6个内参基因的表达稳定性。结果表明,石蒜属植物种间和种内内参基因表达差异明显,在分析换锦花不同组织基因表达时可选用β-Tubulin、Actin和GAPDH作为内参基因,分析不同花期的基因表达时宜选用5S r RNA、EF-1α、Actin和β-Tubulin作内参基因。中国石蒜不同组织最合适的内参基因是5S r RNA、Ubiquitin、EF-1α和β-Tubulin,而不同花期最合适的内参基因则是Ubiquitin、β-Tubulin。分析石蒜不同组织间基因表达的适宜内参基因是β-Tubulin和5S r RNA,不同花期为Ubiquitin、β-Tubulin以及5S r RNA。不同杂交种鳞茎适宜内参基因为β-Tubulin和Actin。

Different species,different tissues,different development periods of flowers and different hybrids of bulb of Lycoris Herb were taken as materials in this study. The m RNA expression of six housekeeping genes such as Actin,EF-1α,GAPDH,5S r RNA,Ubiquitin and β-Tubulin was detected by q RT-PCR. The stability of expression of the six reference genes were evaluated by ge Norm,Norm Finder,Best Keeper and Reffinder respectively. The results showed that m RNA expression of the reference genes had significant differences during the species and intraspecific in Lycoris. When analyzing the m RNA expression of different organs in Lycoris sprengeri,β-Tubulin,Actin and GAPDH could be reference genes. In the meantime,5S r RNA,EF-1α,Actin and β-Tubulin could be uesed as reference genes in different development periods of flowers. However,5S r RNA,Ubiquitin,EF-1α and β-Tubulin were best choices in different organs of Lycoris chinensis Traub. Meanwhile,Ubiquitin,β-Tubulin were suitable reference genes in different florescence. For Lycoris radiata,the stable m RNA expression in different organs β-Tubulin and 5S r RNA were appropriate,and Ubiquitin,β-Tubulin and 5S r RNA should be chosen in different florescence. While β-Tubulin and Actin should be apply as the reference in different hybrids bulbs. All these results will provide appropriate reference genes for further study,help achieve standardization,decrease experimental errors and increase the reliability of experimental results.