Ministry of Education, Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Qingdao, 266003, China.
College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
BMC Genomics. 2018 Apr 13;19(1):251. doi: 10.1186/s12864-018-4643-8.
Pyropia yezoensis, a marine red alga, is an ideal research model for studying the mechanisms of abiotic stress tolerance in intertidal seaweed. Real-time quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method to analyze gene expression levels. To accurately quantify gene expression, selection and validation of stable reference genes is required.
We used transcriptome profiling data from different abiotic stress treatments to identify six genes with relatively stable expression levels: MAP, ATPase, CGS1, PPK, DPE2, and FHP. These six genes and three conventional reference genes, UBC, EF1-α, and eif4A, were chosen as candidates for optimal reference gene selection. Five common statistical approaches (geNorm, ΔCt method, NormFinder, BestKeeper, and ReFinder) were used to identify the stability of each reference gene. Our results show that: MAP, UBC, and FHP are stably expressed in all analyzed conditions; CGS1 and UBC are stably expressed under conditions of dehydration stress; and MAP, UBC, and CGS1 are stably expressed under conditions of temperature stress.
We have identified appropriate reference genes for RT-qPCR in P. yezoensis under different abiotic stress conditions which will facilitate studies of gene expression under these conditions.
Pyropia yezoensis 是一种海洋红藻，是研究潮间带海藻非生物胁迫耐受机制的理想研究模式。实时定量聚合酶链反应（RT-qPCR）是分析基因表达水平最常用的方法。为了准确量化基因表达，需要选择和验证稳定的参考基因。
我们使用来自不同非生物胁迫处理的转录组分析数据来鉴定六个具有相对稳定表达水平的基因：MAP、ATPase、CGS1、PPK、DPE2 和 FHP。这六个基因和三个常规参考基因 UBC、EF1-α 和 eif4A 被选为最佳参考基因选择的候选基因。我们使用了五种常见的统计方法（geNorm、ΔCt 方法、NormFinder、BestKeeper 和 ReFinder）来确定每个参考基因的稳定性。结果表明：MAP、UBC 和 FHP 在所有分析条件下均稳定表达；CGS1 和 UBC 在脱水胁迫条件下稳定表达；MAP、UBC 和 CGS1 在温度胁迫条件下稳定表达。
我们已经确定了 P. yezoensis 在不同非生物胁迫条件下进行 RT-qPCR 的合适参考基因，这将有助于研究这些条件下的基因表达。
