# Biosynthesis of Camphane Volatile terpenes in _Annomum_ _villosum_ Lour: Involved Genes and Enzymes
## 2 Results
### Unigenes Involved in Backbone Biosynthesis of Camphane Volatile Terpenes  
The isoprenoid route is used to create camphane volatile terpenes (mainly involving monoterpenes and sesquiterpenes) by cyclizing geranyl diphosphate, primarily to generate the backbone. After that, the backbone goes through a number of alterations (oxidation, reduction, and substitution), which are aided by a number of enzymes, e.g., geranyl diphosphate, dehydrogenase and acyltransferases, to create the volatile terpene [32] (Figure 6). Evidence has shown that both the cytosolic MVA pathway and the plasidial MEP pathway can produce isopentenyl diphosphate (IPP) in the early stage of active isoprene unit formation, which is then converted to its allylic isomer [33]. The upstream genes in the backbone biosynthesis of camphane volatile terpenes were mapped in the KOG classification. Bornyl diphosphate synthase (BPPS), bornyl pyrophosphate hydrolase (BPPH), bornel dehydrogenase (BDH), and BAHD acyltransferases in the downstream need further research for screening. A wide family of enzymes known as BAHD acyltransferases is responsible for the acyl-CoA dependent acylation of secondary metabolites, which commonly produces esters and amides [34]. The family name of BAHD acyltransferases is composed of the first four characterized members (BEAT or benzylalcohol O-acetyltransferase from Clarkia breweri; AHCTs or anthocyanin O-hydroxycinnamoyltransferases from Petunia, Senecio, Gentiana, Perilla, and Lavandula; HCBT or anthranilate N-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus; and DAT or deacetylylundoline 4-O-acetyltransferase from Catharanthus roseus) [35; 36]. It has been reported that monoterpene acetates were produced by acetyltransferase in _Lavandula x intermedia_[37]. Fortunately, three candidate _BDHs_ (unigene c96075_g1, unigene c105839_g1, and unigene c85128_g2) were found to be close to genes encoding a short-chain dehydrogenase in _Artemisia amua_ and _L x intermedia_. The sequence of unigene c105839_g1 was blasted to the National Center for Biotechnology Information (NCBI) and corresponded with the _Zinggiber zerumbet zsd1_ mRNA for short-chain dehydrogenase/reductase1 (accession: AB480831.1) [38]. Nine candidate _BPPSs_ (unigene c103210_g1, unigene c95418_g1, unigene c102189_g1, unigene c46547_g1, unigene c105905_g3, unigene c82785_g1, unigene c98804_g1, unigene c91238_g1, and unigene c106371_g2) were matched with genes encoding the bornyl diphosphate synthase in _Salvia officinalis_ and _Lavandula angustifolia_ subsp [22]. Moreover, seven unigenes (unigene c160888_g1, unigene c76165_g1, unigene c1599426_g1, unigene c150006_g1, unigene c92359_g2, unigene c23572_g1, and unigene c33340_g2) were annotated with BAHD acyltransferases, but only unigene c92359_g2 belongs to the DEGs. However, unigenes involved in the BPPH were not found in the sequence annotation against seven databases, and little research was available for reference. The process transforming bornyl diphosphate to bornyl acetate might be intricate. In the KOG classification of DEGs, 106 DEGs were assigned to eight biosynthesis pathways for terpenoids and polykeptides. The largest subcategory was 'Terpenoid backbone biosynthesis' (35), followed by 'Carotenoid biosynthesis' (21), 'Limonene and pinene degradation' (12), and 'Zeatin biosynthesis' (12) (Figure 7A). Excitingly, 32 DEGs in the 'Terpenoid backbone biosynthesis' mapped into the backbone biosynthesis of camphane volatile terpenes (Table S3). The expression of DEGs in the backbone biosynthesis pathway of camphane volatile terpenes might account for the regulation of transcription factors (TFs). Furthermore, we analyzed the DEGs annotated in the transcription factor. In total, 143 DEGs of TFs were divided into 18 subfamilies, including MYB, bHLH, AP2/ERF, WRKY, bZIPs, and so on (Figure 7B). According to the findings, several TFs engaged in the biosynthesis of terpenoids that were downregulated in G3 may be in conflict with the expression of genes involved in the production pathway of terpenoids.