College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095, China.
College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China.
Plant Cell Rep. 2015 Oct;34(10):1825-34. doi: 10.1007/s00299-015-1830-9. Epub 2015 Jul 16.
This study identified stable reference genes for normalization of gene expression data in qRT-PCR analysis of leaf and root tissues in creeping bentgrass under four abiotic stresses. Examination of gene expression using quantitative real-time PCR (qRT-PCR) in plant responses to abiotic stresses can provide valuable information for stress-tolerance improvement. Selecting stable reference genes for qRT-PCR analysis is critically important. The objective of this study was to determine the stability of expression for eight candidate reference genes (ACT, EF1a, TUB, UPL7, GAPDH, PP2A, PEPKR1, and CACS) in two tissues (roots and leaves) of a perennial grass species under four abiotic stresses (salt, drought, cold, and heat) using four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). The results showed that (1) the combinations of CACS and UPL7 or PP2A and ACT were stably expressed in salt-treated roots or leaves; (2) the combinations of GAPDH and CACS or PP2A and PEPKR1 were stable in roots and leaves under drought stress; (3) CACS and PP2A exhibited stable expression in cold-treated roots and the combination of EF1a and UPL7 was also stable in cold-treated leaves; and (4) CACS and PP2A were the two most stable reference genes in heat-stressed roots and UPL7 combined with GAPDH and PP2A was stably expressed in heat-stressed leaves. The qRT-PCR analysis of a target gene, AsSAP expression patterns in response to salinity and drought stress, confirmed the reliability of those selected and stable reference genes. Identification of stable reference genes in creeping bentgrass will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.
本研究鉴定了在四种非生物胁迫下匍匐翦股颖叶片和根组织 qRT-PCR 分析中基因表达数据归一化的稳定参考基因。使用定量实时 PCR（qRT-PCR）检测植物对非生物胁迫的反应中的基因表达可以为提高胁迫耐受性提供有价值的信息。选择用于 qRT-PCR 分析的稳定参考基因至关重要。本研究的目的是使用四个程序（GeNorm、NormFinder、BestKeeper 和 RefFinder）确定 8 个候选参考基因（ACT、EF1a、TUB、UPL7、GAPDH、PP2A、PEPKR1 和 CACS）在两种组织（根和叶）中在四种非生物胁迫（盐、干旱、寒冷和热）下的表达稳定性。结果表明：（1）在盐处理的根或叶中，CACS 和 UPL7 或 PP2A 和 ACT 的组合稳定表达；（2）在干旱胁迫下，GAPDH 和 CACS 或 PP2A 和 PEPKR1 的组合在根和叶中稳定；（3）CACS 和 PP2A 在冷处理的根中表现出稳定的表达，EF1a 和 UPL7 的组合在冷处理的叶中也稳定；（4）在热胁迫的根中，CACS 和 PP2A 是两个最稳定的参考基因，UPL7 与 GAPDH 和 PP2A 相结合在热胁迫的叶中稳定表达。目标基因 AsSAP 对盐胁迫和干旱胁迫响应的 qRT-PCR 分析证实了所选和稳定参考基因的可靠性。在匍匐翦股颖中鉴定稳定的参考基因将提高选择胁迫耐受性基因和鉴定赋予该物种胁迫耐受性的分子机制的分析准确性。
