Expression Stabilities of Ten Candidate Reference Genes for RT-qPCR in Zanthoxylum bungeanum Maxim

Abstract

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (<i>Zanthoxylum bungeanum</i> Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (<i>18S</i>, <i>28S</i>, <i>EF</i>, <i>UBA</i>, <i>UBQ</i>, <i>TIF</i>, <i>NTB</i>, <i>TUA</i>, <i>RPS</i>, and <i>TIF5A</i>) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, <i>UBA</i> and <i>UBQ</i> were found to be most stable for the different cultivars and different tissues examined, <i>UBQ</i> and <i>TIF</i> for fruit developmental stage. Meanwhile, <i>EF</i> and <i>TUA</i> were most stable under cold treatment, <i>EF</i> and <i>UBQ</i> under drought treatment and <i>NTB</i> and <i>RPS</i> under salt treatment. <i>UBA</i> and <i>UBQ</i> for all samples evaluated were most stably expressed, but <i>18S</i>, <i>TUA</i> and <i>RPS</i> were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.