高温胁迫下茄子qRT-PCR内参基因筛选及稳定性分析

Selection and Stability Analysis of Reference Gene for qRT-PCR in Eggplant Under High Temperature Stress

为筛选茄子(Solanum melongena L.)高温胁迫下稳定表达的内参基因,以不同茄子品系(种)为研究对象,利用实时荧光定量PCR技术对来自茄子高温胁迫转录组数据库8个候选内参基因(SmEF1a、SmEF2、Sm40s RPS29、Sm60s RPL24、SmTRX、SmCK I、SmDAHPS I、SmUCP)和SmActin在不同试验情况下进行表达检测,并结合GeNorm、Norm Finder、Best Keeper和ReFinder软件综合评价9个内参基因的表达稳定性。结果表明,在茄子热敏品系05-1和耐热品系05-4经高温处理不同时间的样品和不同组织样品以及10个耐热性不同的茄子品系(种)中,9个内参基因的表达丰度及稳定性存在差异;茄子热敏品系05-1和耐热品系05-4高温胁迫处理不同时间样品中表达稳定性最好的是SmEF1a和SmUCP;其不同组织中表达水平最稳定的是SmEF1a和SmTRX;高温胁迫下不同茄子品系(种)中以SmTRX和SmEF2的表达稳定性最好。综合来看,SmEF1a和SmTRX在所有茄子试验样品中的表达稳定性最好,而SmActin和SmCK I的表达稳定性较差。

The present study aimed at selecting the stable reference genes to ensure the reliability and accuracy in gene expression analysis of eggplant （Solanum melongena L.） under high temperature stress. By real-time fluorescence quantitative RT-PCR technology, we investigated the expression stability of eight candidatereferencegenes （SmEFla, SmEF2, Sm40sRPS29, Sm60sRPL24, SmTRX, StuCK I,SmDAHPS I, SmUCP） from RNA-Seq database under high temperature stress and SmActin, and the stability of expression of nine reference genes were evaluated by GeNorrn, NormFinder, B estKeeper and ReFinder, respectively. The results showed that, at different periods, different tissues and different lines of eggplant under high temperature, the expression abundance and stability of these nine reference genes are different. When analyzing the gene expression of thermo-sensitive line 05-1 and thermo-resistant line 05-4 under high temperature stress, SmEFla and SmUCP were the best reference genes for different periods , and SmEFla and SmTRX could be used as best reference genes for different tissues. Meanwhile, SmTRX and SmEF2 could be used as best reference genes in lines or varieties with different thermo-tolerance. On the whole, SmEFla and SmTRX exhibited the most stable expression among all of the tested samples, while SmActin and StuCK/exhibited the least stable expression under most of the experimental conditions. It was found to be feasible and efficient to identify stably expressed genes from transcriptome database under high temperature stress as candidate reference genes, which will be helpful for the study of expression of genes response to high temperature and the mechanisms of thermo-tolerance in eggplant.