## 1 Introduction  
_Wuzhuyu_, as one of the traditional Chinese medicines, refers to the dry and nearly mature fruit of _Evodia nutacearpa_ ([PERSON].) Benth, _Evodia nutacearpa_ ([PERSON].) Benth. var. _officinalis_ ([PERSON]) [PERSON], or _Evodia nutacearpa_ ([PERSON].) Benth. var. _bodinieri_ ([PERSON]) [PERSON]. It mainly contains alkaloids, volatile oil, flavonoids, and limionoids and was originally used to treat headaches, amenorneka, postpartum bleeding [1], and gastrointestinal diseases [2; 3]. The latest research shows that it also has therapeutic effects on dysmerorrhea [4] and Alzheimer's disease [5]. According to the Chinese Pharmacopoeia, the contents of evodiamime and rutacearpin in the dry products of _E. nutacearpa_ var. _officinalis_ must not be less than 0.15% and those of limonin must not be less than 0.20% [6]. _E. nutacearpa_ var. _officinalis_ is mainly distributed in the south of China. Most of the research on _E. nutacearpa_ var. _officinalis_ has focused on the extraction, separation [7], and identification of its chemical components [8] and the pharmacology [9] and toxicology of its effective components [10]. Limonin, found in _E. nutacearpa_ var. _officinalis_, exhibits anti-inflammatory, antibacterial, and antioxidant properties [11] and has a wide range of effective anti-cancer effects [12]. The method for improving the limonin content in _E. nutacearpa_ var. _officinalis_ through traditional cultivation and breeding is inefficient and costly. The genes involved in the biosynthesis of limonin in _E. nutacearpa_ var. _officinalis_ have not yet been investigated. Analyzing its molecular mechanism represents an effective means to improve the content of limonin in _E. nutacearpa_ var. _officinalis_. There have not yet been studies on the molecular mechanism of the biosynthesis of the active ingredients in _E. nutacearpa_ var. _officinalis_. The biosynthesis pathway of limonin has a long history of research, but no one has taken _E. nutacearpa_ var. _officinalis_ as the research object. This study is the first to study limonin in _E. nutacearpa_ var. _officinalis_. It can provide a new objective and approach for many researchers to study limonin. Limonin is a tetracyclite triterpene compound. Terpenoids are compounds, and their derivatives are based on isoprene as the basic unit. Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are two precursors of isoprenoids. These precursors can be synthesized via two different pathways: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythnitol 4-phosphate (MEP) pathway. The biosynthesis of limonin begins with the generation of IPP and DMAPP through both the MVA and MEP pathways. In the MVA pathway, the substrate acetyl CoA is successively catalyzed by ACAT, HMGCS, HMGCR, MVK, MVAK, and MVD to form IPP. In the MEP pathway, the substrate D-Glyceraldehyde-3-phosphate undergoes catalysis by DXS, DXR, ISPD, ISPE, ISPF, ISPG, and ISPH to form DMAPP. Both IPP and DMAPP are then catalyzed by geranylpyrophosphate synthetase (GPS) to produce geranylpyrophosphate (GPP). Subsequently, IPP and GPP react under the catalysis of farnesyl pyrophosphate synthetase (FPS) to form farnesyl pyrophosphate (FPP). Two molecules of FPP can combine to generate presqualene diphosphate under the catalysis of squalene synthase (SQS). Presqualene pyrophosphate is further catalyzed by squalene epoxidase (SQE) to form 2,3-oxidosqualene [13]. The pathways of limonoids have been elucidated, but limonin's have not yet been elucidated. In _E. nutacearpa_ var. _officinalis_, limonin is obtained from intermediates such as melanol, nomilin, and obacunone. It is possible that CYP450 enzymes are involved in the intermediate steps of limonin synthesis from melanol [14]. The potential biosynthesis pathway of limonin in _E. nutacearpa_ var. _officinalis_ is illustrated in Figure 1.  
In order to study the enzyme genes related to the biosynthesis pathway of limonin in _E. nutacearpa_ var. _officinalis_, it is necessary to validate the functions of the candidate genes.