## 3 Discussion  
Research into the selection and validation of reference genes in plants is expanding. The proper selection of reference genes is a prerequisite for gene expression research. This study represents the first attempt at screening the reference genes in _E. rutaecarpa_ var. _officialis_. Among the ten commonly considered candidate reference genes in plants (_Act1_, _UBQ_, _His3_, _g-TUB6_, _GAPDH_, _UBQ10_, _Act100_, _a-TUB_, _g-TUB2_, and _CHS1_), the stability of these genes in the various tissues and fruits at the different developmental stages of _E. rutacarpa_ var. _officialis_ was comprehensively evaluated using GeNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder. _UBQ_ ranked second in terms of stability among the fruits at the different developmental stages, rendering it suitable as a reference gene. Its stability diminished in the different tissues, thereby making it unsuitable for reference gene usage in that context. _Act100_ exhibited low stability rankings in both the fruits at the different periods and the different tissues, thereby making it unsuitable as a reference gene. Regardless of whether we were considering the fruits at the various developmental stages or the different tissues, _Act1_ consistently secured the first rank in terms of stability, establishing it as the most suitable reference gene. Nevertheless, GeNorm stipulated the determination of the requisite number of reference genes when calculating the relative expression levels. Consequently, when assessing the relative expression levels in the fruits and tissues across the different development periods, the utilization of two reference genes was recommended. Many researchers have conducted relatively quantitative comparisons between the most stable and unstable reference genes, and they have often obtained significant differences. The use of unstable reference genes leads to inaccurate quantitative results [30; 31; 32]. Specifically, in the case of fruits at different developmental stages, the two most stable reference genes, _Act1_ and _UBQ_, should be employed, while _Act1_ and _His3_ are advisable choices for different tissues. The expression levels of reference genes vary among different plants and under different treatments. There is no reference gene that can be stably expressed under all conditions. For instance, in the context of the strawberry defense response, _Act1_ was recommended as a suitable reference gene [33]. _Act_ also performs well in cucumber for treatments involving cold and heat stress [34]. In kiwifruit, _Act1_ is considered the most suitable reference gene for fruit development. However, _Act_ is found to be unstable in tea plants across various experimental conditions [35], and it was ranked as the least stable reference gene in staminate and pistellate flower samples of jatropha [36]. In quinoa treated with ABA, the expression of _Act1_ is also found to be unstable [37]. The identification of stable reference genes in _E. rutaecarpa_ var. _officialis_ holds significant importance as it lays the foundation for the subsequent gene expression analysis. Using the \"Full length transcriptome + expression profile + metabolome\" correlation analysis method for screening genes related to enzymes in biosynthesis pathways can narrow the scope of gene research. This approach is widely employed to study the synthesis mechanisms of secondary metabolites in plants. [PERSON] et al. identified the key genes involved in flavonoid biosynthesis in the _Dendrobium_ species [38]. [PERSON] et al. identified the key genes contributing to amino acid biosynthesis in _Torreya grandis_[39]. From a pool of 80 CYP450 and 90 UDPG genes, [PERSON] et al. screened 7 _CYP450_ and 5 _UDP-glucosyltransferase (UDPG)_ candidate genes that may participate in the biosynthesis of mgroside V in _Siraitia grosvenorii_. One or two of these candidates were verified through prokaryotic expression or yeast expression [40; 41; 42].