Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

实时定量逆转录聚合酶链式反应（Quantitative RT-PCR）是一种灵敏度极高、功能强大的基因差异表达检测技术。非生物胁迫诱导的基因表达变化复杂且多维度，这使得筛选用于数据标准化的稳定表达基因颇具难度。为筛选适用于大豆非生物胁迫研究的最优内参基因，本研究从已有文献中获取13个候选基因，采用ΔCT法与geNorm算法，评估其在脱水、高盐、低温及脱落酸（abscisic acid，ABA）处理下的表达稳定性。内参基因验证结果表明，最优内参基因具有组织特异性与胁迫特异性。针对脱水胁迫处理，大豆幼苗根组织与地上组织中表达稳定性最高的基因对分别为Fbox/ABC与Fbox/60s；而在脱水处理的根与地上组织混合样本中，Fbox与60s基因是最适内参基因。在盐胁迫下，大豆幼苗根组织与地上组织中稳定性最优的基因对分别为ELF1b/IDE与Fbox/ELF1b；两类组织的混合样本中，60s/Fbox为最佳基因对。针对低温胁迫研究，根组织与地上组织中分别适用的优良内参基因对为IDE/60s与Fbox/Act27。而就脱落酸处理下的基因表达分析而言，当仅分析根组织、仅分析地上组织，或分析两类组织的混合样本时，最适内参基因对分别为60s/ELF1b、ELF1b/Fbox与60s/ELF1b。在本研究涉及的各类胁迫处理下，根组织、地上组织及两类混合组织中稳定性最优的基因对分别为ELF1b/60s、60s/Fbox与60s/Fbox。在所有受试基因中，60s是适用于不同组织与各类胁迫条件下的最优内参基因。本研究筛选得到的高稳定性内参基因，可有效检测到表达速率的细微差异；若使用稳定性较差的内参基因，这类差异将被遗漏。