Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes

Abstract

Background Dextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol. Results Comparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis. Conclusions We used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.

Full-Length Transcriptome Sequencing Provides Insights into Flavonoid Biosynthesis in Fritillaria hupehensis

“…The information presented here will be useful for marker-assisted selection to accelerate trait-specific breeding in F. hupehensis. Repeat form analyses of the SSRs demonstrated that the most important components (55.7%) were mono-nucleotide SSRs (22.2%), thus confirming their ubiquity as reported by earlier transcriptome studies in medicinal plants [14,[21][22][23]26,29].…”

“…New groups of LncRNAs, such as promoter-related RNAs and long RNAs, have been discovered as a result of the advancement of modern sequencing technologies [ 22 , 57 ]. Aside from mRNAs, LncRNAs generate a series of transcripts that serve as structural, catalytical, and regulatory RNAs [ 6 , 21 ]. They play a major role in stress response and regulate chromosomal dynamics, RNA editing, splicing, and degradation of mRNAs [ 57 , 58 ].…”

“…yielded 9732 unigenes and novel metabolic pathways [ 19 ]. The multi-component and multi-target mechanisms of secondary metabolites and their regulatory mechanisms have gained prominence in modern Chinese medicinal research [ 19 , 20 , 21 ]. Alkaloids, flavonoids, glycosides, organic acids, saponins, and steroids, and other secondary metabolites are important pharmaceutical and phytochemical enzymes involved in metabolic processes [ 11 , 21 , 22 ].…”

Metabolism and transcriptome profiling provides insight into the genes and transcription factors involved in monoterpene biosynthesis of borneol chemotype of Cinnamomum camphora induced by mechanical damage

“…In an analysis of the full-length transcriptome of the three different chemical types (camphor, linalool, eucalyptol) of C. porrectum, five genes (CpTPS1, CpTPS3, CpTPS4, CpTPS5, and CpTPS9) had specific expression or higher expression in one of the chemotypes and may be involved in monoterpene biosynthesis (Qiu et al, 2019). Our previous transcriptomic study of three different chemotypes of C. burmannii indicated that two monoterpene synthase unigenes, designated CbTPS2 and CbTPS3, were upregulated in the high-borneol group when compared with the low-borneol and borneol-free groups, suggesting they might be vital to the biosynthesis of D-borneol in that species (Yang et al, 2020). The above findings contributed to a better understanding of the mechanism of differential accumulation of specific terpenoids in different chemotypes of Cinnamomum plants.…”

“…To verify the reliability of the obtained RNA sequencing data, we relied on qRT-PCR, as previously described (Yang et al, 2020). The expression levels of 23 DEGs-9 related to the biosynthesis of monoterpenes, 6 TFs, and 8 related to the jasmonic acid (JA) signaling pathway-were evaluated using the 2 − Ct method, with CcActin serving as the reference gene.…”