## 1 Introduction
The research on gene expression is a crucial approach for understanding gene functions. Conducting gene expression studies requires reliable reference genes. The optimal reference genes can vary and exhibit fluctuations across different species, among diverse tissues, across various anatomical regions, across different developmental stages, and under distinct treatments within the same species. It is necessary to screen for reference genes under different conditions. The stability of these reference genes must be analyzed. Real-time quantitative PCR (qRT-PCR) is commonly used for gene expression analysis due to its high accuracy, strong specificity,and simple operation [15; 16; 17]. With the application of qRT-PCR technology, the reference genes were identified in various plant species. For instance, in _Fragaria chiloensis_, _chloroplastic BYASS regulatory factor (FcCHP) 2_ and _FcACTIN1_ were identified as the most stable reference genes in vegetative samples, while _FcDBP (DNA binding protein)_ and _FcCHP1_ were found to be stable in ripening fruit samples [18]. Similarly, in _Kengyilia melanthera_, _translationally controlled tumor protein (TCTP)_ and _TIP41-like protein (TIPRL)_ were the most stable reference genes under abscisic acid (ABA) treatment, _TIP41-like protein (CACS)_ and _TCTP_ were the most stable reference genes under cold treatment, _CACS_ and _F-box only protein 6-like (FBXO6L)_ were the most stable reference genes under both heat and drought treatment, and _TIPRL_ and _F-box only protein 6-like (CYPA3)_ were the most stable reference genes under salt treatment [19]. _F1-ATPase/ATP synthase/ACCase (Acetyl CoA Carboxylase)_ were identified as the best reference genes in _S. rosmarinus_[20]. In _ginseng_, different vegetative organs have shown variations in the most stable reference genes. For the roots, _GAPDH_ and _30S ribosomal protein S20 (30 SRPS20)_ were found to be the best reference genes; in the stems, _cyclophilin (CYP)_ and _(60S ribosomal protein L13 (60 SRPL13)_ were top-ranked; and in the leaves, _CYP_ and _ubiquol-cytochrome C reductase (QCR)_ exhibited the highest levels of stability [21]. Figure 1: The putative biosynthesis pathway of limonin. (DXS: 1-deoxy-D-xylulose-5-phosphate synthase. DXR: 1-deoxy-D-xylulose-5-phosphate reductase. ISPD: 2-C-methyl-D-erythhitol-phosphate cytidylyltransferase. ISPE: 4-diphosphocytidyl-2-C-methyl-D-erythhitol kinase. ISPF: 2-C-methyl-D-erythhitol 2,4-cyclodextrin diphosphate synthase. ISPG: (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase. ISPH: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase. FPS/GPS: Geranyl pyrophosphate synthetase/Famesyl pyrophosphate synthetase. ACAT: acetyl-CoA C-acetyltransferase. HMGCS: hydroxymethylglutaryl-CoA synthetase. HMGCR: hydroxymethylglutaryl-CoA reductase. MVK: mexulonate kinase. MVAK: phosphomevalonate kinase. MVD: diphosphomevalonate decarboxylase. IDI: isopentenyl-diphosphate Delta-isomerase. SQS: squalene synthase. SQE: squalene epoxidase. CYP450: cytochromeP450). The full sequences of all the genes are available in the Supplementary Materials. In this study, the stability of ten candidate reference genes was evaluated, and the two most stable reference genes were used to relatively quantify the genes related to the limonin biosynthesis pathway that were screened according to the \"full length transcriptome + expression profile + metabolome\", and the expression profiles of the genes related to the limonin biosynthesis pathway were obtained. Based on the changes in gene expression, it was inferred that several genes may be involved in the biosynthesis of limonin in _E. rutuacarpa_ var. _officialis_.