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BMC Mol Biol. 2010 Jan 20;11:8. doi: 10.1186/1471-2199-11-8.
Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use.
Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field.
This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.
黑麦草（Lolium perenne L.）是一种重要的牧草和草坪草。生物技术，如基因表达研究，正被用于改良这种温带牧草的特性。定量逆转录聚合酶链反应（qRT-PCR）是目前用于确定基因表达变化的最佳方法之一。在分析靶基因表达之前，选择合适的标准化策略来控制样品之间的非特异性变化是至关重要的。具有在不同生物学和生理状态下稳定表达的参考基因可以有效地用于标准化；然而，在使用之前必须验证其表达稳定性。
查询了现有的基因表达序列分析数据，以鉴定出六个在一年中表达相对稳定的中度表达基因。这六个候选参考基因（真核延伸因子 1α，eEF1A；TAT 结合蛋白同源物 1，TBP-1；真核翻译起始因子 4α，eIF4A；YT521-B 样蛋白家族蛋白，YT521-B；组蛋白 3，H3；泛素结合酶，E2）在 442 个不同的黑麦草（Lolium perenne L.）样本中进行了 qRT-PCR 标准化验证，这些样本来自田间和实验室种植的植物，在广泛的实验条件下。真核 EF1A 是由多个基因家族成员编码的，这些成员表现出不同的表达，需要对不同的 eEF1A 编码基因进行表达分析；一个高度表达的 eEF1A（h），一个中度但稳定表达的 eEF1A（s），以及多基因 eEF1A 的组合表达（m）。NormFinder 确定 eEF1A（s）和 YT521-B 是田间不同刈割管理下黑麦草基因表达数据标准化的最佳两基因组合。
本研究在测试样本的数量上是独特的，包括了许多田间生长的样本，为在田间条件下进行多年生生物量作物的基因表达研究铺平了道路。从我们的研究中，已经验证了几个稳定表达的参考基因。这为在除本研究测试条件以外的条件下选择黑麦草的参考基因提供了有用的候选基因。
