苹果实时荧光定量PCR分析中内参基因的筛选

【目的】为筛选苹果实时荧光定量PCR实验中最适内参基因,【方法】应用实时荧光定量PCR技术,分析5个传统内参基因18SrRNA、ACTB、GAPDH、UBQ、TUB在苹果不同基因型、不同组织、果实不同发育时期的mRNA表达差异情况。供试的6个不同基因型苹果分别为:新疆野生苹果、八棱海棠、丽江山荆子、‘津轻’、‘国庆’、‘金冠’;6种不同组织为果皮、果肉、叶片、愈伤组织、花瓣、种子;5个果实发育不同时期为花后28、50、74、95、115 d。【结果】经geNorm程序分析发现5种内参基因的表达稳定性各异,UBQ在果实不同基因型和不同发育时期的基因表达分析中最稳定;ACTB和UBQ在6种不同组织中表达均稳定。【结论】UBQ在参试样品中表达均比较稳定,是研究苹果基因表达分析中理想的内参基因。

Screening of reference genes for Real-time Fluorescence Quantitative PCR in apple(Malus×domestica)

【Objective】The Objective of the study is to select the most stable reference genes of apple for RT-qPCR.【Method】To analyze the expression of five traditional reference genes(18SrRNA,ACTB,GAPDH,UBQ,TUB) in different genotypes,different tissues and apples in different developmental stages,respectively,at the mRNA level by using the real-time quantitative PCR(real-time qPCR).Six different genotypes: Malus sieversii,Malus robusta(Carr.)Rehd.,Malus rockii Schneid.,'Tsugaru','Guoqing','Golden Delicious';Six different tissues: peel,pulp,leaf,callus,petals,seed;Five different development stages of fruits: 28,50,74,95,115 days after anthesis(DAA).【Result】 According to the analysis by geNorm program,the stabilities of the five candidate reference genes are different,UBQ was stably expressed in different genotypes and fruit developmental stages.ACTB and UBQ were both stably expressed in six different tissues.【Conclusion】This results showed that UBQ was the most suitable reference gene among all the tested samples.