文献类型：期刊文献

中文题名：扇脉杓兰实时荧光定量PCR内参基因的筛选

英文题名：Reference Gene Selection of Real-time Fluorescence Quantitative PCR in Cypripedium japonicum

作者：段国敏[1,2] 李田园[1] 田敏[1] 王彩霞[1] 张莹[1]

第一作者：段国敏

机构：[1]中国林业科学研究院亚热带林业研究所,浙江杭州311400;[2]南京林业大学风景园林学院,江苏南京210037

年份：2021

卷号：35

期号：3

起止页码：576-585

中文期刊名：核农学报

外文期刊名：Journal of Nuclear Agricultural Sciences

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：浙江省农业(林木)新品种选育重大科技专项(2016C02056-13-2)。

语种：中文

中文关键词：扇脉杓兰;实时荧光定量PCR;内参基因;稳定性评价

外文关键词：Cypripedium japonicum;RT-qPCR;reference genes;expression stability evaluation

分类号：S682.31

摘要：为筛选出扇脉杓兰实时荧光定量PCR(RT-qPCR)中最适内参基因,以扇脉杓兰幼根、茎、叶、唇瓣、种子为研究材料,利用RT-qPCR技术检测12个常见看家基因TIF3I1、CYP22、RPS4、UPL1、UBC2、TUBB3、RPL26B、RAN1、SAMDC、ACT3、PP2A3和EF1的表达情况,结合geNorm、NormFinder和BestKeeper软件综合评价候选内参基因的稳定性;根据分析结果筛选出最佳内参基因和其组合,并通过2个目的基因SERK2和ASK1对内参基因的稳定性进行验证。结果表明,在9个样品材料中,稳定性最好的内参基因为CYP22、UBC2、TUBB3、RPS4;在扇脉杓兰幼根、茎、叶和唇瓣中,PP2A3、TIF3I1表达较为稳定,SAMDC和ACT3最不稳定;TUBB3和UBC2在种子中表达稳定性最佳,RAN1和ACT3稳定性最低。相对稳定的内参基因TUBB3、UBC2、TUBB3+UBC2对目的基因SERK2和ASK1显示出一致的相对表达量;而不稳定的内参基因ACT3并没有对数据进行有效的标椎化,结果存在明显偏差。本研究不仅有助于提高扇脉杓兰基因表达分析的准确性,也为其他杓兰属植物内参基因的筛选提供了理论依据。
In order to select the most stable reference genes in Cypripedium japonicum for real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),the reference genes—IF3I1、CYP22、RPS4、UPL1、UBC2、TUBB3、RPL26B、RAN1、SAMDC、ACT3、PP2A3、EF1 were evaluated by RT-qPCR、geNorm、NormFinder and BestKeeper programs.Furthermore,two target genes SERK2 and ASK1 were selected to verify the reliability of the suitable reference genes.The result showed among 9 samples analyzed,CYP22,UBC2,TUBB3 and RPS4 were the most stable genes.PP2A3,TIF3I1 showed the best expression stability in different tissues,while SAMDC and ACT3 were the least stable genes.The expression stability of TUBB3,UBC2 was the best in seeds and the most unstable genes were RAN1 and ACT3.And then,the target genes were chosen to verify the stability of TUBB3、UBC2、TUBB3+UBC2 and ACT3 in seeds at different developmental stages.The results suggested that the two target genes showed consistent expression profiles when normalized by the two reference genes TUBB3 and UBC2,while it is failed to standardize the expression data with TCT3 This study helps to improve the accuracy of gene expression analysis,and provides a theoretical basis for the selection of other internal reference genes of Cypripedium.