# Biosynthesis of Camphane Volatile terpenes in _Annomum_ _villosum_ Lour: Involved Genes and Enzymes
## 3 Discussion  
Volatile terpenes (monoterpene and sesquiterpene), not separate monomers, are the medicinal ingredient in Chinese medicine. Fructus Amomi, which contains volatile terpenes such as bornyl acetate, borneol, and camphor, belongs to the homogeneous medicine and food that is prevalent in Southeast Asia. Because of the length of the harvest (July to September) and lack of light and temperature, some fruits cannot mature completely. Provided that the fruits at different stages of ripening are mixed with unripe fruits, they cannot avoid the influence of the efficacy of Fructus Amomi. In this study, morphological changes and fruit size were measured, and quantitative analyses of bornyl acetate at different growth stages were established by GC-MS. The results demonstrate that the increase in weight and volume of _A. villusum_ fruits occurs on the 5 th-24 th day (Figure 1A,B), and tends to stabilize after 24 days. The content of bornyl acetate was altered in the different growth stages. The significant change occurred from 7 to 30 DAP (\(p<0.05\)), in particular. The content of bornyl acetate at 30 DAP increased sharply, then remained stable up to the 80 th day (Figure 1C). From the results, we could clearly discriminate the timing difference of bornyl acetate in fruits at different development stages, and we could provide scientific evidence for timing the fruit harvest. Furthermore, it was significant that we could further research the molecular regulatory mechanism to improve volatile terpen yield in the crucial stages of fruit development. De novo transcriptome assembly of RNA-Seq involves the full transcriptome, and screening of genes with different expression levels could be conducted to explore obscure biological process [39, 40]. Transcriptome profile analysis is an effective approach to obtain functional genes and regulation pathways [41], such as some genes and pathways involved in the response to water loss in _Taxillusi chinensis_ (DC.) Dansers were identified [42]. To fully regulate the gene expression of _A. villusum_ fruits in different growth stages, several transcriptomes were carried out in materials with different treatments [43, 44]. However, significant progress has not yet been made in molecular research on this plant species. Only a few key genes involved in the biosynthesis of camphane volatile terpenes have been characterized and functionally analyzed, and knowledge of the whole process of volatile terpenes is incomplete. Therefore, it is desirous to acquire further information on various modifications in the biosynthesis pathway downstream. In the study, we obtained three libraries with two biological repeats using the Illumina 4000 platform, producing 214,320 unigenes, which is larger than the previous transcriptome databases. As shown in this study, a large number (135 and 62 respectively) of screening genes were produced based on sequence alignment with _BDH_ and _BPPS_ in _S. officinalis_ and _L. angustifolia_ subsp. All of them might play roles in the catalytic reactions of camphane volatile terpene backbone formation at different times, but usually several of them are crucial. The labor required to characterize the enzymes actually engaged in this pathway increases due to the increased screening findings. Three _BDHs_ and nine _BPPSs_ were selected as the candidates most likely associated with volatile terpene biosynthesis through analysis of the sequence alignment and different expressions of unigenes at three different development stages. RNA-Seq combined with different expressions is an effective tool for the functional gene excavation of the response under adversity stress [45, 46], the secondary metabolism pathway [47, 48], the metamorphosis process [49], etc. The transcriptome on the different development stages in the fruit of _A. villusum_ upgraded the transcriptome resources and is regarded as a significant cornerstone in the further investigation of the secondary metabolite biosynthesis pathway.