Wang Xia, Ma Xiao, Huang Linkai, Zhang Xinquan
Grassland Science Department, Sichuan Agriculture University, Chengdu 611130, China.
Molecules. 2015 Mar 16;20(3):4833-47. doi: 10.3390/molecules20034833.
Annual ryegrass (Lolium multiflorum) is a cool-season annual grass cultivated worldwide for its high yield and quality. With the areas of saline soil increasing, investigation of the molecular mechanisms of annual ryegrass tolerance under salt stress has become a significant topic. qRT-PCR has been a predominant assay for determination of the gene expression, in which selecting a valid internal reference gene is a crucial step. The objective of present study was to evaluate and identify suitable reference genes for qRT-PCR in annual ryegrass under salt stress. The results calculated by RefFinder indicated that eEF1A(s) was the most stable reference gene in leaves, whereas EF1-a was the least stable; meanwhile, TBP-1 was the most optimal in roots and in all samples, and the eIF-5A shouldn't be utilized for normalization of the gene expression. eEF1A(s) is more suitable than TBP-1 as reference gene in leaves when verified with P5CS1 and Cyt-Cu/Zn SOD genes. We should choose optimal reference genes in specific tissues instead of the most stable one selected from different conditions and tissues.
一年生黑麦草（多花黑麦草）是一种冷季型一年生禾本科植物，因其高产和优质而在全球范围内广泛种植。随着盐渍土面积的增加，研究一年生黑麦草在盐胁迫下的耐受分子机制已成为一个重要课题。实时荧光定量PCR（qRT-PCR）一直是测定基因表达的主要方法，其中选择一个有效的内参基因是关键步骤。本研究的目的是评估和鉴定盐胁迫下一年生黑麦草中适合用于qRT-PCR的内参基因。RefFinder计算结果表明，eEF1A(s)是叶片中最稳定的内参基因，而EF1-a最不稳定；同时，TBP-1在根和所有样品中是最适宜的，而eIF-5A不应用于基因表达的标准化。在用P5CS1和细胞色素铜/锌超氧化物歧化酶（Cyt-Cu/Zn SOD）基因验证时，eEF1A(s)比TBP-1更适合作为叶片中的内参基因。我们应该在特定组织中选择最佳内参基因，而不是从不同条件和组织中选出的最稳定的内参基因。
