杂交兰实时荧光定量PCR内参基因的筛选

Reference Gene Selection of Real-time Quantitative PCR in Cymbidium Hybrid

为了给杂交兰的基因功能表达和调控研究提供内参基因,本研究采取同源克隆方法和RT-PCR技术,以杂交兰‘黄金小神童’叶片为材料,分离杂交兰Ch18S r RNA、Ch28S r RNA、Ch ACT、Ch TUA、Ch TUB、Ch UBQ、Ch EF-1α和Chrpo B基因的片段,以10个不同品种杂交兰叶片及‘黄金小神童’花器官不同部位为材料,利用实时荧光定量PCR(Real-time quantitative PCR,q PCR)检测分析各引物的扩增效率和相对表达量,采用ge Norm、Norm Finder和Best Keeper软件对各个候选内参基因的表达稳定性进行分析,并通过研究杂交兰花器官不同部位Ch PDS基因的表达模式来验证筛选得到的内参基因的可靠性。结果显示,各引物扩增的片段长度分别为171 bp、148 bp、183 bp、146 bp、130 bp、147 bp、203 bp、139 bp,扩增效率分别为1.94、2.19、1.88、1.99、2.12、2.20、2.11、1.98。综合3个软件的评价结果发现,杂交兰不同品种叶片中最佳内参基因为Ch ACT,不同花器官部位中最稳定内参基因为Ch ACT、Ch TUB、Ch UBQ和Ch EF-1α;而对于实验中所有样品来说,内参基因稳定性最高的为Ch TUB;说明不同的实验条件下,所需的内参基因不同。Ch PDS基因相对表达水平分析结果证实了所筛选内参基因的可靠性,以Ch UBQ、Ch EF-1α、Ch TUB、Ch ACT及Ch UBQ和Ch EF-1α基因组合进行校正的Ch PDS基因的相对表达量均为花瓣>唇瓣>蕊柱。

To provide reference genes for the studies of gene function expression and regulation, sequence of Ch18S rRNA, Ch28S rRNA, ChACT, ChTUA, ChTUB, ChUBQ, ChEF-la and ChrpoB genes were cloned by RT- PCR using the leaf of Cymbidium Golden Elf＇ Sun-dust＇ as materials, Real-time quantitative PCR （qPCR） analyses were employed to determine the primer amplification efficiency and their relative quantities for each gene in leaves of ten different Cymbidium hybrid cultivars, geNorrn, NormFinder and BestKeeper software programs were used to analyze the expression stability of each candidate gene and the expression pattern of ChPDS in different parts of the flower organs was analyzed to verify the reference genes selected in this study. The RT-PCR and qPCR results showed that the length of each candidate gene fragments were 171 bp, 148 bp, 183 bp, 146 bp, 130 bp, 147 bp, 203 bp and 139 bp, while the primer efficiencies were 1.94, 2.19, 1.88, 1.99, 2.12, 2.20, 2.11 and 1.98, respectively. The evaluation results of three software programs showed that ChA CT was the optimal reference gene from the different eultivars, ChACT, ChTUB, ChUBQ and ChEF-1α were the optimal reference genes from the different parts of the flower organs, but, for all the samples in the experiment, the most stable internal gene was ChTUB. The results indicated that different reference gene was required in different test conditions. Analysis results of the relative expression level for ChPDS confirmed the reliability of the reference genes selected, the relative expression of ChPDS using ChUBQ, ChEF-1α, ChTUB, ChACT, the combination of ChUBQ and CHEF-1α for normalization was petal〉labellum〉gynandrium.