Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses
Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses
复制标题
DOI：
10.1007/s00425-014-2041-2
复制
发表时间：
2014-06-01
期刊：
影响因子：
4.3
通讯作者：
Rombaldi, Cesar Valmor
中科院分区：
文献类型：
--
作者：
Borowski, Joyce Moura;Galli, Vanessa;Rombaldi, Cesar Valmor
内参基因的选择和验证是实时定量聚合酶链反应（RT-qPCR）研究基因表达的第一步。莴苣是世界上最受欢迎的叶菜，它的基因组最近已经被测序；因此，需要合适的内参基因以获得可靠的RT-qPCR分析结果。本研究选取了17个候选内参基因，利用geNorm和NormFinder软件对其在干旱、盐、重金属和UV-C照射条件下以及ABA应用下在生菜叶片中的表达稳定性进行了评价。候选内参基因包括编码蛋白质的传统内参基因和新型内参基因以及microRNAs （miRNAs）。结果表明，表达的稳定性与实验条件有关。新型蛋白编码内参基因在干旱、UV-C照射、重金属等条件下以及ABA的应用下比传统内参基因更适合。只有在盐度条件下，传统的蛋白质编码内参基因比新基因更稳定。此外，mirna主要是MIR169、MIR171/170和MIR172，在评估的非生物胁迫下稳定表达，代表了基因表达数据规范化的合适替代方法。用苯丙氨酸解氨酶（PAL）和4-羟基苯基丙酮酸双加氧酶（HPPD）的表达进一步确认已验证的蛋白编码内参基因，用MIR172和MIR398的表达确认已验证的miRNA基因，表明使用不合适的内参基因会导致错误的结果。本工作是对生菜内参基因稳定性的首次调查，为莴苣研究中获得更准确的RT-qPCR结果提供指导。
The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.