## 3 Discussion
The FPKM values in transcriptome sequencing can reflect the expression levels of genes, and qRT PCR is used to perform secondary validations of the expression trends in pathway genes. A combination of these two methods can obtain more reliable trends in the expression levels of pathway genes. Changes in gene expression levels can cause changes in the expression levels of corresponding enzymes, which directly affect changes in secondary metabolites. During the development process of plants, there are significant changes in secondary metabolites, and therefore, gene expression patterns can be associated with plant development, and relevant pathway genes can be identified through changes in secondary metabolites. In this study, 18 genes related to the limonin synthesis pathway were identified using this method. The expected results were that the quantitative expression levels of the pathway genes obtained through the qRT-PCR analysis would be consistent with the trends in the FPKM values in the transcriptome and that the expression levels of _HMGCR_, _SQE_, _CYP450_ would be continuously decreasing, consistent with the trends in the changes in the limonin contents. The expression patterns of some genes in the MEP and MVA pathways do not completely align with the changing trends in limonin. This discrepancy may arise because these genes are simultaneously involved in the biosynthesis of evodiamine, rutacarpin, and other compounds. The accumulation patterns of these other compounds in _E. rutacarpa_ var. _officialis_ could differ from that of limonin, or it could be due to their distant relationship with limonin synthesis and their weaker correlations. The expression levels of _HMGCR_, _SQE_, and _CYP450_ were at their highest on 17 July, coinciding with the peak contents of limonin. This observation suggested that these genes were likely participants in the limonin synthesis pathway, contributing to the accelerated synthesis of limonin in plants. As the fruits ripened, the limonin contents notably decreased, accompanied by significant reductions in the expression levels of these genes. As the limonin contents reached appropriate levels, limonin biosynthesis became regulated by the reduced gene expression, leading to a gradual decline in limonin accumulation. In the quantitative results using qRT-PCR, there was an additional DXS gene with the same trend as the change in limonin content, and most of the genes had the same trend as the transcriptome, which met the expected results. The expression trends of most genes were to first increase and then decrease, which may be consistent with the metabolic law of plants. Before fruit ripening, the metabolism in fruit needs to be continuously enhanced to achieve the goal of ripening. After a fruit ripens, it begins to age, with the reduced expression levels of pathway genes and the weakened metabolism of the MVA and MEP pathways. After screening candidate genes, it becomes essential to conduct a functional verification of these genes. From the perspectives of forward genetics and reverse genetics, these genes can be overexpressed or knocked out. Subsequently, changes in the secondary metabolite contents can be detected. Alternatively, an expression vector can be constructed and transformed into _Saccharomyces cerevisiae_ or _Escherichia coli_. Inducing protein expression followed by substrate feeding can help determine whether product generation occurs. Most researchers opt for _Citrus_ of the Rutaceae family as the starting point when investigating the biosynthesis mechanism of limonin. _E. rutacarpa_ var. _officialis_ belongs to the Rutaceae family as well, yet there has been limited research on the molecular mechanism of secondary metabolite biosynthesis, such as that of limonin. Investigating the molecular mechanism of limonin biosynthesis in _E. rutacarpa_ var. _officialis_ is beneficial for revealing the comprehensive limonin biosynthesis pathway.