滇牡丹种子实时荧光定量PCR分析中内参基因的筛选与验证

doi:10.13560/j.cnki.biotech.bull.1985.2019-1208

摘要/Abstract

摘要： 通过实时荧光定量PCR（Real-time quantitative PCR,RT-qPCR）分析滇牡丹（Paeonia delavayi）不同发育时期的种子中内参基因的表达稳定性,筛选出滇牡丹种子成熟过程中最稳定的内参基因。利用实时荧光定量PCR技术,分析8个传统内参基因GAPC、PEPC、CYC、ACT、EF2α、TUB、ACP1和RPL1在滇牡丹种子不同发育时期的表达差异情况,利用3个分析软件GeNorm、Norm Finder 和BestKeeper分别评价上述内参基因在滇牡丹不同发育阶段的种子中的表达稳定性。荧光定量及软件分析结果显示,8个内参基因的稳定性各异,表达最为稳定的内参基因是CYC和EF2α,表达最不稳定的内参基因是ACT。对滇牡丹油脂代谢合成途径中部分相关基因进行RT-qPCR分析,得到了相同的表达趋势,验证了CYC和EF2α适合作为内参基因。得到了两个最适用于滇牡丹种子的内参基因CYC和EF2α。

关键词: 滇牡丹, 种子, 荧光定量PCR, 内参基因

Abstract: The objective of this work is to analyze the expression stability of internal reference genes in the seeds at different developmental stages of Paeonia delavayi by real-time quantitative PCR（RT-qPCR）,and to screen the most stable reference genes in the maturation of seeds. RT-qPCR was used to analyze their expression differences of 8 housekeeping internal reference genes such as GAPC,PEPC,CYC,ACT,EF2α,TUB,ACP1 and RPL1 in P. delavayi seeds at different developmental stages. Three software（GeNorm,Norm Finder and BestKeeper）were applied to evaluate the expression stabilities of above genes in the seeds at different developmental stages of P. delavayi. The results from RT-qPCR and software demonstrated that the expression stabilities of 8 reference genes varied,the most stable ones were CYC and EF2,while the least sable one was ACT. RT-qPCR analysis of some related genes in the lipid metabolism and synthesis pathway of P. delavayi presented the same expression trend,which verified that CYC and EF2α were suitable as reference genes. In conclusion,2 internal reference genes,CYC and EF2α,are obtained to be the suitable internal reference genes for the seeds of P. delavayi.

Key words: Paeonia delavayi, seed, RT-q PCR, reference gene

潘沫晗, 陆添权, 田波. 滇牡丹种子实时荧光定量PCR分析中内参基因的筛选与验证[J]. 生物技术通报, 2020, 36(9): 218-226.

PAN Mo-han, LU Tian-quan, TIAN Bo. Selection and Validation of Reference Genes in the Seeds of Paeonia delavayi in Quantitative Real-time PCR Analysis[J]. Biotechnology Bulletin, 2020, 36(9): 218-226.

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