Real-time reverse quantitative transcription PCR (RT-qPCR) is a commonly used method for gene expression analysis. However, the accuracy of RT-qPCR analysis greatly depends on the stability of the reference genes selected for data normalization. Unfortunately, there have been no systematic evaluation studies on reference genes in the unicellular green alga Auxenochlorella protothecoides. Ten genes (ACT, COX6B, CYB5R4, EF1α, GAPDH, rbcL, RPL19, RPL32, RPS10 and TUA) were chosen as candidate reference genes. The expression stability of each reference gene was determined by applying geNorm, NormFinder, BestKeeper and comprehensive ranking based on geomean ranking values. In order to validate the suitability and reliability of the candidate reference genes selected for data normalization, two key functional genes related to lipid metabolism were also selected as target genes. The results revealed that RPL32 was the most stably expressed and suitable reference genes under all experimental conditions of the analyses; CYB5R4 was also a suitable reference gene under continuous heterotrophic culture condition, and RPL19 was top-ranked and stably expressed under both low and high temperature stress. These results provided a list of suitable reference genes for accurate normalization of heterotrophic A. protothecoides time-course samples under different temperature stress in qPCR experiments. This study will be of very great reference value to assess the stability of candidate reference genes in other heterotrophically grown algae.

实时逆定量转录PCR（RT-qPCR）是基因表达分析的常用方法。但是，RT-qPCR分析的准确性很大程度上取决于选择用于数据标准化的参考基因的稳定性。不幸的是，在单细胞绿藻原小球藻中没有关于参考基因的系统评价研究。十个基因（ACT，COX6B，CYB5R4，EF1α，GAPDH，rbcL，RPL19，RPL32，RPS10和TUA选择）作为候选参考基因。通过应用geNorm，NormFinder，BestKeeper和基于geomean排名值的综合排名来确定每个参考基因的表达稳定性。为了验证用于数据标准化的候选参考基因的适用性和可靠性，还选择了与脂质代谢有关的两个关键功能基因作为靶基因。结果表明，在所有实验条件下，RPL32是表达最稳定，最合适的参考基因。CYB5R4也是连续异养培养条件下的合适的参考基因，和RPL19在低温和高温胁迫下均排名第一并稳定表达。这些结果提供了合适的参考基因的列表，用于在qPCR实验中在不同温度胁迫下准确地对原养曲霉A. thetothecoides时程样品进行标准化。这项研究对于评估其他异养藻类中候选参考基因的稳定性具有非常重要的参考价值。