Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress
Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress
复制标题
DOI：
10.1016/j.plantsci.2008.12.005
复制
发表时间：
2009-03-01
期刊：
影响因子：
5.2
通讯作者：
Martin, Ruth C.
中科院分区：
文献类型：
--
作者：
Dombrowski, James E.;Martin, Ruth C.
多花黑麦草Lolium temulentum L(Darnel Ryegras)是研究牧草和草坪草逆境胁迫的有价值的模式草种。定量实时RT-PCR的基因表达分析依赖于使用适当的内部标准。本研究的目的是寻找和评价用于L非生物胁迫实时定量RT-PCR研究的参考基因。根据近缘种L看家基因设计简并引物，通过RT-PCR法获得9个看家基因的部分序列。根据这些部分序列设计了定量RT-PCR的引物。对这些看家基因在遭受不同形式非生物胁迫的植物样品中的表达稳定性进行了评估。分析发现，在测试的基因中，EEF-1α和UBQ5最稳定，Act11最不稳定。GeNorm分析表明，两个最稳定表达的看家基因(EEF-1α和UBQ5)应该被利用，并足以在L胁迫相关研究中实现基因表达的正常化。爱思唯尔爱尔兰有限公司出版。
Lolium temulentum L (Darnel ryegrass) is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real-time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR for abiotic stress studies in L temulentum. Partial sequences of nine L temulentum housekeeping genes were obtained by RT-PCR using degenerate primers designed from the corresponding genes in closely related species. Primers for quantitative RT-PCR were designed based on these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to different forms of abiotic stress. The analysis found that eEF-1 alpha and UBQ5 were the most stable and ACT11 was the least stable of the genes tested. Analysis by geNorm indicated that the two most stably expressed housekeeping genes (eEF-1 alpha and UBQ5) should be utilized and are sufficient for normalization of gene expression during stress-related studies in L temulentum. Published by Elsevier Ireland Ltd.