Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.
Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy.
复制标题
碱蓬定量 RT-PCR 标准化参考基因的验证，碱蓬是一种一年生盐生植物，具有异形性和 C4 途径，无需 kranz 解剖学
作者：
Cao J;Wang L;Lan H
逆转录实时定量聚合酶链反应（qRT-PCR）是一种用于测量基因表达的强大分析技术，其取决于用于数据标准化的参考基因的稳定性。盐地碱蓬（Suaedaaralocaspica）是一种一年生盐生植物，种子异形，具有C4光合作用途径，不具有Kranz解剖结构，是从转录水平上鉴定和比较植物抗逆性相关基因表达的理想植物。到目前为止，还没有这个物种的分子信息。本研究选择了6个传统上常用的参考基因，研究了它们在两类大豆种子中的表达稳定性。在不同的实验条件下的aralocaspica进行了评价。使用三种分析程序geNorm、Normandy和BestKeeper来评估和排序参考基因表达的稳定性。结果表明，尽管某些参考基因在两种类型种子中的转录谱可能存在差异，但β-TUB和GAPDH在不同发育阶段和不同组织中的转录谱可能是最合适的参考基因。GAPDH是两种种子在不同萌发时间点和盐胁迫条件下的适宜参考基因，ACTIN则适用于两种种子的各种非生物胁迫处理。对于所有样本池，β-TUB作为最稳定的参考基因，而18 S rRNA和28 S rRNA表现不佳，在我们的研究中表现为最不稳定的基因。在不同盐处理下，UBQ似乎不适合作为内对照。此外，还研究了C4途径的光合作用相关基因（PPDK）和S. aralocaspica用于验证最佳性能参考基因。这是第一次系统比较S.这些数据将有助于进一步研究该物种和其他真盐植物的基因表达。
Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds of S. aralocaspica under different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds, β-TUB and GAPDH appeared to be the most suitable references under different developmental stages and tissues. GAPDH was the appropriate reference gene under different germination time points and salt stress conditions, and ACTIN was suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools, β-TUB served as the most stable reference gene, whereas 18S rRNA and 28S rRNA performed poorly and presented as the least stable genes in our study. UBQ seemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK) of C4 pathway and a salt tolerance-related gene (SAT) of S. aralocaspica were used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work in S. aralocaspica and these data will facilitate further studies on gene expression in this species and other euhalophytes.