College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang 471023, China.
Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China.
Int J Mol Sci. 2023 Oct 11;24(20):15087. doi: 10.3390/ijms242015087.
Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in under abiotic stresses (including drought, salt, and ZnSO treatments), seven different tissues of , as well as the roots, stems, and leaves of under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. and were the best reference genes under drought stress. Under salt stress, , , , and were the most stable reference genes. Under heavy metal stress, and were the most suitable reference genes. and were the most suitable internal reference genes in the different tissues of . In addition, , , , and were the most suitable internal reference genes for the abiotic stresses and the different tissues of . The expression patterns of and were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in .
实时荧光定量 PCR（RT-qPCR）具有高灵敏度和强特异性，广泛应用于基因表达分析。选择合适的内参基因是通过 RT-qPCR 准确分析靶基因表达变化的关键。为了找出最适合研究非生物胁迫（包括干旱、盐和 ZnSO4 处理）下 基因表达的内参基因，以 7 种不同组织和非生物胁迫下的 根、茎和叶为试验材料，基于转录组数据通过 RT-qPCR 筛选了 15 个候选内参基因。然后，通过 geNorm（v3.5）、NormFinder（v0.953）、BestKeeper（v1.0）和 RefFinder 软件综合评估候选基因的表达稳定性。根据分析结果筛选出最佳内参基因及其组合。在干旱胁迫下，和 是最佳的参考基因。在盐胁迫下，、、、和 是最稳定的参考基因。在重金属胁迫下，和 是最合适的参考基因。在不同组织中，和 是最适合的内参基因。此外，、、、和 是最适合非生物胁迫和不同组织的内参基因。通过使用筛选出的稳定和不稳定的内参基因分析 和 的表达模式。这进一步验证了筛选出的内参基因的可靠性。本研究为 基因的功能分析和调控机制研究奠定了基础。
