楸树逆境胁迫下的实时荧光定量PCR内参基因筛选

Selection of Optimal Reference Genes for Quantitative Real-time PCR in Catalpa bungei under Different Stress Conditions

楸树是中国特有的珍贵树种,具有较高的材用、药用及观赏价值,但在其功能基因方面的研究报道较少。为使后续楸树相关功能基因在表达分析的定量实验结果有更加准确的参照,需要筛选表达稳定性高的基因作为内参基因。本研究数据采用楸树二代转录组测序,CYP40-1、CYP40-2、EF1A、EF1B、MDH、PP2A、TUBA、TUBB、UBC2和H3-1共10个内参基因初步筛选为候选内参基因。对楸树组培苗进行低温、高温、干旱及高盐胁迫处理后,利用实时荧光定量PCR(RT-qPCR)技术检测上述候选内参基因的表达变化情况,并利用geNorm、Normfinder和BestKeeper 3个分析软件综合评估了这些内参基因的稳定性。结果表明,4℃、37℃、300 mmol/L甘露醇和100 mmol/L NaCl处理下表达量最稳定的内参基因分别为UBC2、CYP40-2、PP2A和CYP40-1。本研究所筛选到的表达稳定的内参基因及设计的特异引物为后续楸树抗逆相关基因表达分析提供了理论基础。

Catalpa bungei C.A.Meyer.is a precious and unique tree species in China,with high material,medicinal and ornamental values,but there are few reports on its functional genes.In order to provide a more accurate reference for the quantitative experimental results of subsequent expression analysis of related functional genes in Catalpa bungei,it is necessary to screen genes with high expression stability as internal reference genes.In this study,based on the transcriptome data from Catalpa bungei,ten housekeeping genes,including CYP40-1,CYP40-2,EF1A,EF1B,MDH,PP2A,TUBA,TUBB,UBC2 and H3-1,were selected as candidate internal reference genes.Then,the cultured Catalpa bungei seedlings were treated by low temperature,high temperature,drought and high salinity,respectively,and then the expression levels of ten candidate genes were monitored by quantitative real-time PCR.Finally,analysis of the gene expression stability by three methods(geNorm,Normfinder and BestKeeper)suggested that UBC2,CYP40-2,PP2A and CYP40-1 were the best reference genes under 4℃,37℃,300 mmol/L mannitol,and 100 mmol/L NaCl,respectively.The most stable internal reference genes and the corresponding primer sequences provided in this study will facilitate further gene expression analysis in Catalpa bungei under different stress conditions.