Quantitative Proteomics, Institute of Molecular Biology (IMB), Mainz, Germany.
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.
PLoS One. 2019 Jul 30;14(7):e0220518. doi: 10.1371/journal.pone.0220518. eCollection 2019.
Perennial ryegrass (Lolium perenne) is a forage and amenity grass species widely cultivated in temperate regions worldwide. As such, perennial ryegrass populations are exposed to a range of environmental conditions and stresses on a seasonal basis and from year to year. One source of potential stress is limitation on water availability. The ability of these perennial grasses to be able to withstand and recover after periods of water limitation or drought can be a key component of grassland performance. Thus, we were interested in looking at changes in patterns of gene expression associated with increasing water stress. Clones of a single genotype of perennial ryegrass were grown under non-flowering growth room conditions in vermiculite supplemented with nutrient solution. Leaf and root tissue was sampled at 4 times in quadruplicate relating to estimated water contents of 35%, 15%, 5% and 1%. RNA was extracted and RNAseq used to generate transcriptome profiles at each sampling point. Transcriptomes were assembled using the published reference genome sequence and differential gene expression analysed using 3 different programmes, DESeq2, edgeR and limma (with the voom transformation), individually and in combination, deriving Early, Middle and Late stage comparisons. Identified differentially expressed genes were then associated with enriched GO terms using BLAST2GO. For the leaf, up-regulated differentially expressed genes were strongly associated with GO terms only during the Early stage and the majority of GO terms were associated with only down-regulated genes at the Middle or Late stages. For the roots, few differentially expressed genes were identified at either Early or Middle stages. Only one replicate at 1% estimated water content produced high quality data for the root, however, this indicated a high level of differential expression. Again the majority of enriched GO terms were associated with down-regulated genes. The performance of the different analysis programmes and the annotations associated with identified differentially expressed genes is discussed.
多年生黑麦草（Lolium perenne）是一种饲用和观赏草种，广泛种植于世界各地的温带地区。因此，多年生黑麦草种群会受到季节性和年度性的一系列环境条件和压力的影响。潜在压力源之一是水分供应的限制。这些多年生草种在经历水分限制或干旱期后能够承受并恢复的能力，是草地表现的一个关键组成部分。因此，我们有兴趣研究与水分胁迫增加相关的基因表达模式的变化。从单个多年生黑麦草基因型的克隆开始，在添加营养溶液的蛭石中，在非开花生长室条件下进行培养。在 4 个时间点，以 4 倍体重复的方式从叶片和根部组织取样，与估计的含水量 35%、15%、5%和 1%相对应。提取 RNA 并使用 RNAseq 在每个采样点生成转录组图谱。使用已发表的参考基因组序列组装转录组，并使用 3 种不同的程序（DESeq2、edgeR 和 limma（带有 voom 转换））单独和组合分析差异基因表达，得出早期、中期和晚期比较。然后使用 BLAST2GO 将鉴定出的差异表达基因与富集的 GO 术语相关联。对于叶片，上调的差异表达基因主要与早期阶段的 GO 术语强烈相关，而大多数 GO 术语仅与中期或晚期的下调基因相关联。对于根部，在早期或中期阶段很少鉴定出差异表达基因。只有在估计含水量为 1%的一个重复中，根部产生了高质量的数据，但这表明差异表达水平很高。同样，大多数富集的 GO 术语与下调基因相关联。讨论了不同分析程序的性能以及与鉴定出的差异表达基因相关联的注释。
