Search

Background Buddleja lindleyana Fort., which belongs to the Loganiaceae with a distribution throughout the tropics, is widely used as an ornamental plant in China. There are several morphologically similar species in the genus Buddleja, but the lack of comprehensive molecular and phylogenetic studies makes it difficult to distinguish related species, which hinders further studies of this genus. Objective Using molecular biology techniques to sequence and analyze the complete chloroplast (cp) genome of B. lindleyana. Methods After sequencing of the genomic DNA using next-generation sequencing, a series of bioinformatics software were used to assemble and analyze the molecular structure of the cp genome of B. lindleyana. Results The complete cp genome of B. lindleyana is a circular 154 487-bp-long molecule with a GC (Guanine and Cytosine) content of 38.1%. It has a quadripartite structure, including a LSC region (85 489 base pair (bp)), a small single-copy region (17 898 bp), and a pair of inverted repeat regions (25 550 bp). A total of 133 genes were identified in this genome, including 86 protein-coding genes, 37 tRNA (transfer Ribonucleic Acid) genes, eight rRNA (ribosomal Ribonucleic Acid) genes, and two pseudogenes. Conclusion These results suggest that the B. lindelyana cp genome could be used as a potential genomic resource to resolve the phylogenetic positions and relationships of Loganiaceae, and will offer valuable information for future research in the identification of Buddleja species and will conduce to genomic investigations into these species. Highlights This paper study the B. lindelyana cp genome and it's structural characteristics, and analyze the phylogeny of Loganiaceae.

Dendrobium officinale caulis is the dried mature stems (Mat) of D. officinale Kimura et Migo. D. officinale protocorms (DOPs) are the conical embryonic tissues formed after seed germination. Previo...

Ardisia Sw. (Primulaceae) is naturally distributed in tropical and subtropical areas. Most of them possess edible and medicinal values and are popular in clinical and daily use in China. However, ambiguous species delineation and genetic information limit the development and utilization of this genus. In this study, the chloroplast genomes of four Ardisia species, namely A. gigantifolia Stapf, A. crenata Sims, A. villosa Roxb. and A. mamillata Hance, were sequenced, annotated, and analyzed comparatively. All the four chloroplast genomes possess a typical quadripartite structure, and each of the genomes is about 156 Kb in size. The structure and gene content of the Ardisia plastomes were conservative and showed low sequence divergence. Furthermore, we identified five mutation hotspots as candidate DNA barcodes for Ardisia, namely, trnT-psbD, ndhF-rpl32, rpl32-ccsA, ccsA-ndhD and ycf1. Phylogenetic analysis based on the whole-chloroplast genomes data showed that Ardisia was sister to Tapeinosperma Hook. f. In addition, the results revealed a great topological profile of Ardisia’s with strong support values, which matches their geographical distribution patterns. Summarily, our results provide useful information for investigations on taxonomic differences, molecular identification, and phylogenetic relationships of Ardisia plants.

Background The borneol chemotype of Cinnamomum camphora (BCC), a monoterpene-rich woody plant species, is the sole source prescribed by the Chinese Pharmacopoeia for the production of natural D-borneol, a major monoterpene in BCC used for millennia as a topical analgesic in China. Nevertheless, the possible gene-regulatory roles of transcription factors (TFs) in BCC’s monoterpenoid biosynthesis remained unknown. Here, a joint analysis of the transcriptome and terpenoid metabolome of BCC induced by mechanical damage (MD) was used to comprehensively explore the interaction between TFs and terpene synthase (TPS) unigenes that might participate in monoterpene biosynthesis in BCC. Results Gas chromatography–mass spectrometry analysis detected 14 monoterpenes and seven sesquiterpenes. All but two monoterpenes underwent a significantly increased accumulation after the MD treatment. RNA sequencing data revealed that 10 TPS, 82 MYB, 70 AP2/ERF, 38 BHLH, 31 WRKY, and 29 bZIP unigenes responded to the MD treatment. A correlation analysis revealed that three monoterpene synthase genes (CcTPS1, CcTPS3, CcTPS4) highly correlated with multiple monoterpenes, namely D-borneol, camphor, and bornyl acetate, which could be responsible for monoterpenoid biosynthesis in BCC. Furthermore, five WRKY, 15 MYB, 10 ERF/AP2, five bZIP, and two BHLH genes had strong, positive correlations with CcTPS1 or CcTPS4, judging by their high coefficient values (R2 > 0.8). The bioinformatics results were verified by quantitative real-time PCR. Conclusion This study provides insight into the genes involved in the biosynthesis and regulation of monoterpene in BCC and thus provides a pool of candidate genes for future mechanistic analyses of how monoterpenes accumulate in BCC.

Dendrobium officinale protocorms (DOPs) are a specific developmental stage of Dendrobium officinale KIMURA et MIGO, which is used in folk medicine to ease skin issues, such as wrinkles and erythema. The purpose of the current study was to evaluate the effect of DOPs on UV irradiation-induced skin damage in bc_nu hairless mice, using matrixyl as a positive control. Hairless mice were randomly separated into 6 groups (8 mice per group). The normal control group received solvent and was not exposed to UV irradiation, while the model control group received solvent and was exposed to UV irradiation. The positive control group was subjected to UV irradiation and then received a 10 mg/mL formulation of matrixyl. The DOPs-treated groups received a transdermal application of a DOPs formulation after 4 weeks of UV irradiation. Relevant indicators, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), thiobarbituric acid reactive substances (TBARS) and matrix metalloproteinases (MMPs), were then used to evaluate the ability of DOPs to repair photodamage. The results indicated that DOPs significantly reduced erythema and protected the skin from dryness and therefore exhibits a significant anti-photoaging effect. In addition, the expression of CAT, SOD, and GSH-Px increased while TBARS and MMPs levels decreased in DOPs-treated mice. This demonstrated that DOPs can inhibit photodamage in the skin of hairless mice. DOPs could be used as a potential therapeutic agent to protect the skin against UV-induced photoaging.

Melastoma dodecandrum, the only creeping species in the Melastoma genus, serves as a medicinal herb in southeast China. It belongs to the huge family Melastomataceae, which contains over 5000 species worldwide. In this study, we used next-generation sequencing to determine the complete chloroplast genome sequences of M. dodecandrum, which is a circular molecule of 156,611 bp in length. After annotation, we identified 131 putative genes in total, comprised of 85 protein-coding genes, 38 transfer RNA genes and 8 ribosomal RNA genes. Genome structure, GC content, repeat sequences and codon usage were investigated to gain a comprehensive understanding of this genome. Furthermore, we conducted comparative genome analyses between the M. dodecandrum genome and that of four other Melastomataceae species. Additionally, a phylogenetic analysis was performed based on available chloroplast genomes of Melastomataceae species and several Myrtaceae species, revealing the taxonomic relationships between M. dodecandrum and related species. In conclusion, our study represents the first look into the complete chloroplast genome of M. dodecandrum, providing abundant information for further studies such as species identification, taxonomy and phylogenetic resolution of Melastomataceae species.

Background The large genus Ficus comprises approximately 800 species, most of which possess high ornamental and ecological values. However, its evolutionary history remains largely unknown. Plastome (chloroplast genome) analysis had become an essential tool for species identification and for unveiling evolutionary relationships between species, genus and other rank groups. In this work we present the plastomes of ten Ficus species. Results The complete chloroplast (CP) genomes of eleven Ficus specimens belonging to ten species were determined and analysed. The full length of the Ficus plastome was nearly 160 kbp with a similar overall GC content, ranging from 35.88 to 36.02%. A total of 114 unique genes, distributed in 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, were annotated in each of the Ficus CP genome. In addition, these CP genomes showed variation in their inverted repeat regions (IR). Tandem repeats and mononucleotide simple sequence repeat (SSR) are widely distributed across the Ficus CP genome. Comparative genome analysis showed low sequence variability. In addition, eight variable regions to be used as potential molecular markers were proposed for future Ficus species identification. According to the phylogenetic analysis, these ten Ficus species were clustered together and further divided into three clades based on different subgenera. Simultaneously, it also showed the relatedness between Ficus and Morus. Conclusion The chloroplast genome structure of 10 Ficus species was similar to that of other angiosperms, with a typical four-part structure. Chloroplast genome sizes vary slightly due to expansion and contraction of the IR region. And the variation of noncoding regions of the chloroplast genome is larger than that of coding regions. Phylogenetic analysis showed that these eleven sampled CP genomes were divided into three clades, clustered with species from subgenus Urostigma, Sycomorus, and Ficus, respectively. These results support the Berg classification system, in which the subgenus Ficus was further decomposed into the subgenus Sycomorus. In general, the sequencing and analysis of Ficus plastomes, especially the ones of species with no or limited sequences available yet, contribute to the study of genetic diversity and species evolution of Ficus, while providing useful information for taxonomic and phylogenetic studies of Ficus.

Background The borneol chemotype of Cinnamomum camphora (BCC), a monoterpene-rich woody plant species, is the sole source prescribed by the Chinese Pharmacopoeia for the production of natural D-borneol, a major monoterpene in BCC used for millennia as a topical analgesic in China. Nevertheless, the possible gene-regulatory roles of transcription factors (TFs) in BCC’s monoterpenoid biosynthesis remained unknown. Here, a joint analysis of the transcriptome and terpenoid metabolome of BCC induced by mechanical damage (MD) was used to comprehensively explore the interaction between TFs and terpene synthase (TPS) unigenes that might participate in monoterpene biosynthesis in BCC. Results Gas chromatography–mass spectrometry analysis detected 14 monoterpenes and seven sesquiterpenes. All but two monoterpenes underwent a significantly increased accumulation after the MD treatment. RNA sequencing data revealed that 10 TPS, 82 MYB, 70 AP2/ERF, 38 BHLH, 31 WRKY, and 29 bZIP unigenes responded to the MD treatment. A correlation analysis revealed that three monoterpene synthase genes (CcTPS1, CcTPS3, CcTPS4) highly correlated with multiple monoterpenes, namely D-borneol, camphor, and bornyl acetate, which could be responsible for monoterpenoid biosynthesis in BCC. Furthermore, five WRKY, 15 MYB, 10 ERF/AP2, five bZIP, and two BHLH genes had strong, positive correlations with CcTPS1 or CcTPS4, judging by their high coefficient values (R2 > 0.8). The bioinformatics results were verified by quantitative real-time PCR. Conclusion This study provides insight into the genes involved in the biosynthesis and regulation of monoterpene in BCC and thus provides a pool of candidate genes for future mechanistic analyses of how monoterpenes accumulate in BCC.

Based on the gradual transformation from wild growth to artificial cultivation, the accurate authentication of licorice seeds contributes to the first committed step of its quality control and is pivotal to ensure the clinical efficacy of licorice. However, it is still challenging to obtain genetically stable licorice germplasm resources due to the multi-source, multi-heterozygous, polyploid, and hybrid characteristics of licorice seeds. Here, a new method for determining the heterozygosity of licorice seed mixture, based on the various sites, and finding the composition characteristics of licorice seed is preliminarily designed and proposed. Namely, high-throughput full-length multiple DNA barcodes(HFMD), based on ITS multi-copy variation exist, the full-length amplicons of ITS2, psbA-trnH and ITS are directly sequenced by rDNA through the next-generation sequence(NGS) and single-molecule real-time (SMRT) technologies. By comparing the three sequencing methods, our results proved that SMRT sequencing successfully identified the complete gradients of complex mixed samples with the best performance. Meanwhile, HFMD is a brilliant and feasible method for evaluating the heterozygosity of licorice seeds. It shows a perfect interpretation of DNA barcoding and can be applied in multi-base multi-heterozygous and polyploid species.

Wurfbainia villosa, which belongs to the huge family Zingiberaceae, is used in the clinic for the treatment of spleen and stomach diseases in southern China. The complete chloroplast genome of W. villosa was sequenced and analyzed using next-generation sequencing technology in the present work. The results showed that the W. villosa chloroplast genome is a circular molecule with 163,608 bp in length. It harbors a pair of inverted repeat regions (IRa and IRb) of 29,820 bp in length, which separate the large single copy (LSC, 88,680 bp) region and the small single copy (SSC, 15,288 bp) region. After annotation, 134 genes were identified in this plastome in total, comprising of 87 protein-coding genes, 38 transfer RNA genes, 8 ribosomal RNA genes and one pseudogene (ycf1). Codon usage, RNA editing sites and single/long sequence repeats were investigated to understand the structural characteristics of the W. villosa chloroplast genome. Furthermore, IR contraction and expansion were analyzed by comparison of complete chloroplast genomes of W. villosa and four other Zingiberaceae species. Finally, a phylogeny study based on the chloroplast genome of W. villosa, along with that of 15 different species, was conducted to further investigate the relationship among these lineages. Overally, our results represented the first insight into the chloroplast genome of W. villosa, and could serve as a significant reference for species identification, genetic diversity analysis and phylogenetic research between W. villosa and other species within Zingiberaceae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-019-00748-3) contains supplementary material, which is available to authorized users.

Dendrobium officinale protocorms (DOPs) are a specific developmental stage of Dendrobium officinale KIMURA et MIGO, which is used in folk medicine to ease skin issues, such as wrinkles and erythema. The purpose of the current study was to evaluate the effect of DOPs on UV irradiation-induced skin damage in bc_nu hairless mice, using matrixyl as a positive control. Hairless mice were randomly separated into 6 groups (8 mice per group). The normal control group received solvent and was not exposed to UV irradiation, while the model control group received solvent and was exposed to UV irradiation. The positive control group was subjected to UV irradiation and then received a 10 mg/mL formulation of matrixyl. The DOPs-treated groups received a transdermal application of a DOPs formulation after 4 weeks of UV irradiation. Relevant indicators, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), thiobarbituric acid reactive substances (TBARS) and matrix metalloproteinases (MMPs), were then used to evaluate the ability of DOPs to repair photodamage. The results indicated that DOPs significantly reduced erythema and protected the skin from dryness and therefore exhibits a significant anti-photoaging effect. In addition, the expression of CAT, SOD, and GSH-Px increased while TBARS and MMPs levels decreased in DOPs-treated mice. This demonstrated that DOPs can inhibit photodamage in the skin of hairless mice. DOPs could be used as a potential therapeutic agent to protect the skin against UV-induced photoaging.

Lycium chinense Mill, an important Chinese herbal medicine, is widely used as a dietary supplement and food. Here the chloroplast (CP) genome of L. chinense was sequenced and analyzed, revealing a size of 155,756 bp and with a 37.8% GC content. The L. chinense CP genome comprises a large single copy region (LSC) of 86,595 bp and a small single copy region (SSC) of 18,209 bp, and two inverted repeat regions (IRa and IRb) of 25,476 bp separated by the single copy regions. The genome encodes 114 genes, 16 of which are duplicated. Most of the 85 protein-coding genes (CDS) had standard ATG start codons, while 3 genes including rps12, psbL and ndhD had abnormal start codons (ACT and ACG). In addition, a strong A/T bias was found in the majority of simple sequence repeats (SSRs) detected in the CP genome. Analysis of the phylogenetic relationships among 16 species revealed that L. chinense is a sister taxon to Lycium barbarum. Overall, the complete sequence and annotation of the L. chinense CP genome provides valuable genetic information to facilitate precise understanding of the taxonomy, species and phylogenetic evolution of the Solanaceae family.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula&mdash, Sanwei-Jili-San (SJS)&mdash, was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

In the last decade, several studies have relied on a small number of plastid genomes to deduce deep phylogenetic relationships in the species-rich Myrtaceae. Nevertheless, the plastome of Rhodomyrtus tomentosa, an important representative plant of the Rhodomyrtus (DC.) genera, has not yet been reported yet. Here, we sequenced and analyzed the complete chloroplast (CP) genome of R. tomentosa, which is a 156,129-bp-long circular molecule with 37.1% GC content. This CP genome displays a typical quadripartite structure with two inverted repeats (IRa and IRb), of 25,824 bp each, that are separated by a small single copy region (SSC, 18,183 bp) and one large single copy region (LSC, 86,298 bp). The CP genome encodes 129 genes, including 84 protein-coding genes, 37 tRNA genes, eight rRNA genes and three pseudogenes (ycf1, rps19, ndhF). A considerable number of protein-coding genes have a universal ATG start codon, except for psbL and ndhD. Premature termination codons (PTCs) were found in one protein-coding gene, namely atpE, which is rarely reported in the CP genome of plants. Phylogenetic analysis revealed that R. tomentosa has a sister relationship with Eugenia uniflora and Psidium guajava. In conclusion, this study identified unique characteristics of the R. tomentosa CP genome providing valuable information for further investigations on species identification and the phylogenetic evolution between R. tomentosa and related species.

In the last decade, several studies have relied on a small number of plastid genomes to deduce deep phylogenetic relationships in the species-rich Myrtaceae. Nevertheless, the plastome of Rhodomyrtus tomentosa, an important representative plant of the Rhodomyrtus (DC.) genera, has not yet been reported yet. Here, we sequenced and analyzed the complete chloroplast (CP) genome of R. tomentosa, which is a 156,129-bp-long circular molecule with 37.1% GC content. This CP genome displays a typical quadripartite structure with two inverted repeats (IRa and IRb), of 25,824 bp each, that are separated by a small single copy region (SSC, 18,183 bp) and one large single copy region (LSC, 86,298 bp). The CP genome encodes 129 genes, including 84 protein-coding genes, 37 tRNA genes, eight rRNA genes and three pseudogenes (ycf1, rps19, ndhF). A considerable number of protein-coding genes have a universal ATG start codon, except for psbL and ndhD. Premature termination codons (PTCs) were found in one protein-coding gene, namely atpE, which is rarely reported in the CP genome of plants. Phylogenetic analysis revealed that R. tomentosa has a sister relationship with Eugenia uniflora and Psidium guajava. In conclusion, this study identified unique characteristics of the R. tomentosa CP genome providing valuable information for further investigations on species identification and the phylogenetic evolution between R. tomentosa and related species.

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.

Lycium chinense Mill, an important Chinese herbal medicine, is emphasized as a healthy food and is widely used as a dietary supplement. Here we sequenced and analyzed the complete chloroplast (CP) genome of the L. chinense, which is 155,756 bp in length and with 37.8% GC content. This CP genome consists of a pair of inverted repeat regions (IRa and IRb) of 25,476 bp, separated by a large single-copy region (LSC) and a small single-copy region (SSC), with length of 86,595 and 18,209 bp, respectively. Annotation results revealed that the L. chinense CP genome contains 114 genes, 16 of which are duplicated genes. Most of the 85 protein-coding genes have a usual ATG start codon, except for 3 genes including rps12, psbL and ndhD. Furthermore, most of the simple sequence repeats (SSRs) are short polyadenine or polythymine repeats that contribute to the high AT content of the chloroplast genome. Revealing of the complete sequences and annotation of the L. chinense chloroplast genome will facilitate phylogenic, population and genetic engineering research investigations involving this particular species.

Melastoma dodecandrum, the only creeping species in the Melastoma genus, serves as a medicinal herb in southeast China. It belongs to the huge family Melastomataceae, which contains over 5000 species worldwide. In this study, we used next-generation sequencing to determine the complete chloroplast genome sequences of M. dodecandrum, which is a circular molecule of 156,611 bp in length. After annotation, we identified 131 putative genes in total, comprised of 85 protein-coding genes, 38 transfer RNA genes and 8 ribosomal RNA genes. Genome structure, GC content, repeat sequences and codon usage were investigated to gain a comprehensive understanding of this genome. Furthermore, we conducted comparative genome analyses between the M. dodecandrum genome and that of four other Melastomataceae species. Additionally, a phylogenetic analysis was performed based on available chloroplast genomes of Melastomataceae species and several Myrtaceae species, revealing the taxonomic relationships between M. dodecandrum and related species. In conclusion, our study represents the first look into the complete chloroplast genome of M. dodecandrum, providing abundant information for further studies such as species identification, taxonomy and phylogenetic resolution of Melastomataceae species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-019-00651-x) contains supplementary material, which is available to authorized users.

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.

Native to Southern China, Ilex pubescens and Ilex asprella are frequently used in traditional Chinese medicine. Both of them produce a large variety of ursane-type triterpenoid saponins, which have been demonstrated to have different pharmacological effects. However, little is known about their biosynthesis. In this study, transcriptomic analysis of I. pubescens and comparison with its closely related specie I. asprella were carried out to identify potential genes involved in triterpenoid saponin biosynthesis. Through RNA sequencing (RNA-seq) and de novo transcriptome assembly of I. pubescens, a total of 68,688 UniGene clusters are obtained, of which 32,184 (46.86%) are successfully annotated by comparison with the sequences in major public databases (NCBI, Swiss-Prot, and KEGG). It includes 128 UniGenes related to triterpenoid backbone biosynthesis, 11 OSCs (oxidosqualene cyclases), 233 CYPs (cytochrome P450), and 269 UGTs (UDP-glycosyltransferases). By homology-based blast and phylogenetic analysis with well-characterized genes involved in triterpenoid saponin biosynthesis, 5 OSCs, 14 CYPs, and 1 UGT are further proposed as the most promising candidate genes. Transcriptomic comparison between two Ilex species using blastp and OrthoMCL method reveals high sequence similarity. All OSCs and UGTs as well as most CYPs are classified as orthologous genes, while only 5 CYPs in I. pubescens and 3 CYPs in I. asprella are species-specific. One of OSC candidates, named as IpAS1, was successfully cloned and expressed in Saccharomyces cerevisiae INVSc1. Analysis of the yeast extract by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) shows IpAS1 is a mixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar to its ortholog IaAS1 from I. asprella. This study is the first exploration to profile the transcriptome of I. pubescens, the generated data and gene models will facilitate further molecular studies on the physiology and metabolism in this plant. By comparative transcriptomic analysis, a series of candidate genes involved in the biosynthetic pathway of triterpenoid saponins are identified, providing new insight into their biosynthesis at transcriptome level.

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.