Cloning, Expression Characteristics of Farnesyl Pyrophosphate Synthase Gene from Platycodon grandiflorus and Functional Identification in Triterpenoid Synthesis.

Platycodon grandiflorus is a medicinal plant whose main component is platycodins, which have a variety of pharmacological effects and nutritional values. The farnesyl pyrophosphate synthase (FPS) is a key enzyme in the isoprenoid biosynthesis pathway, which catalyzes the synthesis of farnesyl diphosphate (FPP). In this study, we cloned the FPS gene from P. grandiflorus (PgFPS) with an ORF of 1260 bp, encoding 419 amino acids with a deduced molecular weight and theoretical pI of 46,200.98 Da and 6.52, respectively. The squalene content of overexpressed PgFPS in tobacco leaves and yeast cells extract was 1.88-fold and 1.21-fold higher than that of the control group, respectively, and the total saponin content was also increased by 1.15 times in yeast cells extract, which verified the biological function of PgFPS in terpenoid synthesis. After 48 h of MeJA treatment and 6 h of ethephon treatment, the expression of the PgFPS gene in roots and stems reached its peak, showing a 3.125-fold and 3.236-fold increase compared to the untreated group, respectively. Interestingly, the expression of the PgFPS gene in leaves showed a decreasing trend after exogenous elicitors treatment. The discovery of this enzyme will provide a novel perspective for enhancing the efficient synthesis of platycodins.

Genome-wide analysis of bZIP transcription factors and their expression patterns in response to methyl jasmonate and low-temperature stresses in Platycodon grandiflorus.

Background: Platycodon grandiflorus belongs to the genus Platycodon and has many pharmacological effects, such as expectorant, antitussive, and anti-tumor properties. Among transcription factor families peculiar to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important, which exists widely in plants and participates in many biological processes, such as plant growth, development, and stress responses. However, genomic analysis of the bZIP gene family and related stress response genes has not yet been reported in P. grandiflorus. Methods: P. grandiflorus bZIP (PgbZIP) genes were first identified here, and the phylogenetic relationships and conserved motifs in the PgbZIPs were also performed. Meanwhile, gene structures, conserved domains, and the possible protein subcellular localizations of these PgbZIPs were characterized. Most importantly, the cis-regulatory elements and expression patterns of selected genes exposed to two different stresses were analyzed to provide further information on PgbZIPs potential biological roles in P. grandiflorus upon exposure to environmental stresses. Conclusions: Forty-six PgbZIPs were identified in P. grandiflorus and divided into nine groups, as displayed in the phylogenetic tree. The results of the chromosomal location and the collinearity analysis showed that forty-six PgbZIP genes were distributed on eight chromosomes, with one tandem duplication event and eleven segmental duplication events identified. Most PgbZIPs in the same phylogenetic group have similar conserved motifs, domains, and gene structures. There are cis-regulatory elements related to the methyl jasmonate (MeJA) response, low-temperature response, abscisic acid response, auxin response, and gibberellin response. Ten PgbZIP genes were selected to study their expression patterns upon exposure to low-temperature and MeJA treatments, and all ten genes responded to these stresses. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that the expression levels of most PgbZIPs decreased significantly within 6 h and then gradually increased to normal or above normal levels over the 90 h following MeJA treatment. The expression levels of all PgbZIPs were significantly reduced after 3 h of the low-temperature treatment. These results reveal the characteristics of the PgbZIP family genes and provide valuable information for improving P. grandiflorus's ability to cope with environmental stresses during growth and development.

Genome-wide identification and expression profiling of the WRKY gene family reveals abiotic stress response mechanisms in Platycodon grandiflorus.

The WRKY family of transcription factors (TFs) is an important gene family involved in abiotic stress responses. Although the roles of WRKY TFs in plant abiotic stress responses are well studied, little is known about the stress-induced changes in WRKY family in Platycodon grandiflorus. 42 PgWRKY genes in seven subgroups were identified in the P. grandiflorus genome. The content of eight platycodins in P. grandiflorus was investigated under cold, heat, and drought stresses. Platycodin D levels significantly increased under three abiotic stresses, while the content changes of other platycodins varied. Transcriptome analysis showed that different WRKY family members exhibited varied expression patterns under different abiotic stresses. PgWRKY20, PgWRKY26, and PgWRKY39 were identified as three key candidates for temperature and drought stress responses, and were cloned and analysed for sequence characteristics, gene structure, subcellular localisation, and expression patterns. The RT-qPCR results showed that PgWRKY26 expression significantly increased after heat stress for 48 h, cold stress for 6 h, and drought stress for 2 d (DS_2 d). The PgWRKY39 expression level significantly increased at DS_2 d. This study provides a theoretical basis for clarifying the molecular mechanism of the abiotic stress responses of the WRKY gene family in P. grandiflorus.

Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Chromosome-Scale Genome Assembly and Triterpenoid Saponin Biosynthesis in Korean Bellflower (Platycodon grandiflorum).

Platycodon grandiflorum belongs to the Campanulaceae family and is an important medicinal and food plant in East Asia. However, on the whole, the genome evolution of P. grandiflorum and the molecular basis of its major biochemical pathways are poorly understood. We reported a chromosome-scale genome assembly of P. grandiflorum based on a hybrid method using Oxford Nanopore Technologies, Illumina sequences, and high-throughput chromosome conformation capture (Hi-C) analysis. The assembled genome was finalized as 574 Mb, containing 41,355 protein-coding genes, and the genome completeness was assessed as 97.6% using a Benchmarking Universal Single-Copy Orthologs analysis. The P. grandiflorum genome comprises nine pseudo-chromosomes with 56.9% repeat sequences, and the transcriptome analysis revealed an expansion of the 14 beta-amylin genes related to triterpenoid saponin biosynthesis. Our findings provide an understanding of P. grandiflorum genome evolution and enable genomic-assisted breeding for the mass production of important components such as triterpenoid saponins.

Genome-wide identification and expression profiling analysis of the trihelix gene family and response of PgGT1 under abiotic stresses in Platycodon grandiflorus.

The trihelix gene family plays an important role in plant growth and abiotic stress responses. Through the analysis of genomic and transcriptome data, 35 trihelix family members were identified for the first time in Platycodon grandiflorus; they were classified into five subfamilies: GT-1, GT-2, SH4, GTγ, and SIP1. The gene structure, conserved motifs and evolutionary relationships were analyzed. Prediction of physicochemical properties of the 35 trihelix proteins founded, the number of amino acid molecules is between 93 and 960, theoretical isoelectric point is between 4.24 and 9.94, molecular weight is between 9829.77 and 107435.38, 4 proteins among them were stable, and all GRAVY is negative. The full-length cDNA sequence of the PgGT1 gene of the GT-1 subfamily was cloned by PCR. It is a 1165 bp ORF encoding a 387 amino acid protein, with a molecular weight of 43.54 kDa. The predicted subcellular localization of the protein in the nucleus was experimentally verified. After being treated with NaCl, PEG6000, MeJA, ABA, IAA, SA, and ethephon, the expression of PgGT1 gene showed an up-regulated trend except for the roots treated with NaCl and ABA. This study laid a bioinformatics foundation for the research of trihelix gene family and the cultivation of excellent germplasm of P. grandiflorus.

Identification and expression analysis of YABBY family genes in Platycodon grandiflorus.

Platycodon grandiflorus set ornamental, edible, and medicinal plant with broad prospects for further application development. However, there are no reports on the YABBY transcription factor in P. grandiflorus. Identification and analysis of the YABBY gene family of P. grandiflorus using bioinformatics means. Six YABBY genes were identified and divided into five subgroups. Transcriptome data and qRT-PCR were used to analyze the expression patterns of YABBY. YABBY genes exhibited organ-specific patterns in expression in P grandiflorus. Upon salt stress and drought induction, P. grandiflorus presented different morphological and physiological changes with some dynamic changes. Under salt treatment, the YABBY gene family was down-regulated; PgYABBY5 was up-regulated in leaves at 24 h. In drought treatment, PgYABBY1, PgYABBY2, and PgYABBY3 were down-regulated to varying degrees, but PgYABBY3 was significantly up-regulated in the roots. PgYABBY5 was up-regulated gradually after being down-regulated. PgYABBY5 was significantly up-regulated in stem and leaf at 48 h. PgYABBY6 was down-regulated at first and then significantly up-regulated. The dynamic changes of salt stress and drought stress can be regarded as the responses of plants to resist damage. During the whole process of salt and drought stress treatment, the protein content of each tissue part of P grandiflorus changed continuously. At the same time, we found that the promoter region of the PgYABBY gene contains stress-resistant elements, and the regulatory role of YABBY transcription factor in the anti-stress mechanism of P grandiflorus remains to be studied. PgYABBY1, PgYABBY2, and PgYABBY5 may be involved in the regulation of saponins in P. grandiflorus. PgYABBY5 may be involved in the drought resistance mechanism in P. grandiflorus stems and leaves. This study may provide a theoretical basis for studying the regulation of terpenoids by the YABBY transcription factor and its resistance to abiotic stress.

Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Effect of Platycodon grandiflorus Polysaccharide on M1 Polarization Induced by Autophagy Degradation of SOCS1/2 Proteins in 3D4/21 Cells.

M1-polarized macrophages can improve the body's immune function. This study aimed to explore the mechanism of Platycodon grandiflorus polysaccharide (PGPSt) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPSt significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1ß, iNOS, CD80, and CD86, indicating that PGPSt promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPSt autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPSt significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPSt degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPSt upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPSt treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPSt in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus.

Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis.

Integrated metabolomic and transcriptomic analysis reveals variation in the metabolites and genes of Platycodon grandiflorus roots from different regions.

INTRODUCTION: Platycodon grandiflorum root (PG), a popular traditional Chinese medicine, contains considerable chemical components with broad pharmacological activities. The complexity and diversity of the chemical components of PG from different origins contribute to its broad biological activities. The quality of southern PG is superior to that of northern PG, but the mechanisms underlying these differences remain unclear. OBJECTIVES: In order to study variation in the differentially accumulated metabolites (DAMs), differentially expressed genes (DEGs), as well as their interactions and signalling pathways among PG from Anhui and Liaoning. METHODS: The metabolomes based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PGn and PGb. RESULTS: A total of 6515 DEGs and 83 DAMs from the comparison of PG from Anhui and Liaoning were detected. Integrated analysis of metabolomic and transcriptomic data revealed that 215 DEGs and 57 DAMs were significantly enriched in 48 pathways according to KEGG pathway enrichment analysis, and 15 DEGs and 10 DAMs significantly enriched in the main pathway sesquiterpenoid and triterpenoid and phenylpropanoid biosynthesis might play a key role in complex response or regulatory processes. CONCLUSION: Differences in PG from southern and northern China might thus stem from differences in environmental factors, such as precipitation, light duration, and humidity. The results of our study provide new insight into geographic variation in gene expression and metabolite accumulation and will enhance the utilisation of PG resources.

Transcriptome analysis identifies putative genes involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of ß-amyrin synthase, the key synthase of ß-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

A candidate gene identified in converting platycoside E to platycodin D from Platycodon grandiflorus by transcriptome and main metabolites analysis.

Platycodin D and platycoside E are two triterpenoid saponins in Platycodon grandiflorus, differing only by two glycosyl groups structurally. Studies have shown ß-Glucosidase from bacteria can convert platycoside E to platycodin D, indicating the potential existence of similar enzymes in P. grandiflorus. An L9(34) orthogonal experiment was performed to establish a protocol for calli induction as follows: the optimal explant is stems with nodes and the optimum medium formula is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L to obtain callus for experimental use. The platycodin D, platycoside E and total polysaccharides content between callus and plant organs varied wildly. Platycodin D and total polysaccharide content of calli was found higher than that of leaves. While, platycoside E and total polysaccharide content of calli was found lower than that of leaves. Associating platycodin D and platycoside E content with the expression level of genes involved in triterpenoid saponin biosynthesis between calli and leaves, three contigs were screened as putative sequences of ß-Glucosidase gene converting platycoside E to platycodin D. Besides, we inferred that some transcription factors can regulate the expression of key enzymes involved in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus. Totally, a candidate gene encoding enzyme involved in converting platycoside E to platycodin D, and putative genes involved in polysaccharide synthesis in P. grandiflorus had been identified. This study will help uncover the molecular mechanism of triterpenoid saponins biosynthesis in P. grandiflorus.

Polyacylated Anthocyanins in Bluish-Purple Petals of Chinese Bellflower, Platycodon grandiflorum.

The bluish-purple petals of Chinese bellflower, Platycodon grandiflorum (kikyo in Japanese), contain platyconin (1) as the major anthocyanin. Platyconin (1) is a polyacylated anthocyanin with two caffeoyl residues at the 7-position, and its color is stable in a diluted, weakly acidic aqueous solutions. HPLC analysis of the fresh petal extract showed the presence of several minor pigments. Photo-diode array detection of minor pigments suggested that some of these were polyacylated anthocyanins. To establish the relationship between structure and stability of the acylated anthocyanins and to obtain information on their biosynthetic pathways, minor pigments were isolated from the petals, and their structures were determined by MS and NMR analyses. Four known (2-5) and three new anthocyanins (6-8) were identified, which contained a delphinidin chromophore, and four of these (5-8) were diacylated anthocyanins, in which the acyl-glucosyl-acyl-glucosyl chain was attached at the 7-O-position of the delphinidin chromophore. These diacylated anthocyanins exhibited a bluish-purple color at pH 6, which was stable for more than a week.

[Transcriptome analysis of Platycodon grandiflorum at different growth years and discovery of key genes for platycodin biosynthesis].

Platycodon grandiflorum is a medicinal and edible medicinal material. Our study is aimed to explore the differences in the gene expression of P. grandiflorum in different growth years, and the expression rules of key genes in the biosynthesis of the main active substances of P. grandiflorum. Illumina Hiseq 4000 sequencing platform was used to sequence the transcriptome of P. grandiflorum in different years. Then, 59 654 unigenes were obtained through filtering, assembly, splicing and bioinformatics analysis of the sequencing data, of which 1 671 unigenes were differentially expressed between at least two samples. The results of cluster analysis showed that there was a great difference in the gene expression of P. grandiflorum from one-year-old to two/three-year-old. There were 1 128 different genes between one-and three-year old P. grandiflorum, and only 57 different genes between two-and three-year-old P. grandiflorum. KEGG enrichment results showed that the differential genes of P. grandiflorum in different years were mainly concentra-ted in the biosynthesis of sesquiterpenes and triterpenes, and the biosynthesis of terpenoid skeletons. In the triterpenoid biosynthesis-related pathways, a total of 15 unigenes were identified, involving 5 enzymes. The expression levels of ACAT, HMGR, FDFT1, SQLE decreased with the increase of the growth year of P. grandiflorum. The expression of HMGS was the highest in the one-year-old P. grandiflorum, followed by the three-year-old sample. This study provides useful data for the development of P. grandiflorum, and also provides a basis for the study of related genes in the biosynthetic pathway of platycodin.

Transcriptome analysis of Platycodon grandiflorum at different growth years and discovery of key genes for platycodin biosynthesis / 中国中药杂志

Platycodon grandiflorum is a medicinal and edible medicinal material. Our study is aimed to explore the differences in the gene expression of P. grandiflorum in different growth years, and the expression rules of key genes in the biosynthesis of the main active substances of P. grandiflorum. Illumina Hiseq 4000 sequencing platform was used to sequence the transcriptome of P. grandiflorum in different years. Then, 59 654 unigenes were obtained through filtering, assembly, splicing and bioinformatics analysis of the sequencing data, of which 1 671 unigenes were differentially expressed between at least two samples. The results of cluster analysis showed that there was a great difference in the gene expression of P. grandiflorum from one-year-old to two/three-year-old. There were 1 128 different genes between one-and three-year old P. grandiflorum, and only 57 different genes between two-and three-year-old P. grandiflorum. KEGG enrichment results showed that the differential genes of P. grandiflorum in different years were mainly concentra-ted in the biosynthesis of sesquiterpenes and triterpenes, and the biosynthesis of terpenoid skeletons. In the triterpenoid biosynthesis-related pathways, a total of 15 unigenes were identified, involving 5 enzymes. The expression levels of ACAT, HMGR, FDFT1, SQLE decreased with the increase of the growth year of P. grandiflorum. The expression of HMGS was the highest in the one-year-old P. grandiflorum, followed by the three-year-old sample. This study provides useful data for the development of P. grandiflorum, and also provides a basis for the study of related genes in the biosynthetic pathway of platycodin.

accD nuclear transfer of Platycodon grandiflorum and the plastid of early Campanulaceae.

BACKGROUND: Campanulaceae species are known to have highly rearranged plastid genomes lacking the acetyl-CoA carboxylase (ACC) subunit D gene (accD), and instead have a nuclear (nr)-accD. Plastid genome information has been thought to depend on studies concerning Trachelium caeruleum and genome announcements for Adenophora remotiflora, Campanula takesimana, and Hanabusaya asiatica. RNA editing information for plastid genes is currently unavailable for Campanulaceae. To understand plastid genome evolution in Campanulaceae, we have sequenced and characterized the chloroplast (cp) genome and nr-accD of Platycodon grandiflorum, a basal member of Campanulaceae. RESULTS: We sequenced the 171,818 bp cp genome containing a 79,061 bp large single-copy (LSC) region, a 42,433 bp inverted repeat (IR) and a 7840 bp small single-copy (SSC) region, which represents the cp genome with the largest IR among species of Campanulaceae. The genome contains 110 genes and 18 introns, comprising 77 protein-coding genes, four RNA genes, 29 tRNA genes, 17 group II introns, and one group I intron. RNA editing of genes was detected in 18 sites of 14 protein-coding genes. Platycodon has an IR containing a 3' rps12 operon, which occurs in the middle of the LSC region in four other species of Campanulaceae (T. caeruleum, A. remotiflora, C. takesimana, and H. asiatica), but lacks accD, clpP, infA, and rpl23, as has been found in these four species. Platycodon nr-accD contains about 3.2 kb intron between nr-accD.e1 and nr-accD.e2 at the same insertion point as in other Campanulaceae. The phylogenies of the plastid genomes and accD show that Platycodon is basal in the Campanulaceae clade, indicating that IR disruption in Campanulaceae occurred after the loss of accD, clpP, infA, and rpl23 in the cp genome, which occurred during plastid evolution in Campanulaceae. CONCLUSIONS: The plastid genome of P. grandiflorum lacks the rearrangement of the IR found in T. caeruleum, A. remotiflora, C. takesimana, and H. asiatica. The absence of accD, clpP, infA, and rpl23 in the plastid genome is a synapomorphic characteristic of Campanulaceae. The chloroplast genome phylogeny supports the hypothesis that chloroplast genomic arrangement occurred after accD nuclear transfer and loss of the four genes in the plastid of early Campanulaceae as a lineage of asterids.

Cytochrome P450 Monooxygenase CYP716A141 is a Unique ß-Amyrin C-16ß Oxidase Involved in Triterpenoid Saponin Biosynthesis in Platycodon grandiflorus.

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces ß-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the ß-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on ß-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of ß-amyrin at C-16ß. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.

Identification and Characterization of Diploid and Tetraploid in Platycodon grandiflorum.

Platycodon grandiflorum (PG), a species of herbaceous flowering perennial plant of the family Campanulaceae, has been used as a traditional oriental medicine for bronchitis, asthma, pulmonary tuberculosis, diabetes, hepatic fibrosis, bone disorders and many others similar diseases and as a food supplement. For the primary profiling of PG gas chromatography coupled with high resolution - time of flight mass spectrometry (GC/HR-TOF MS) was used as an analytical tool. A comparison of optimal extraction of metabolites was carried out with a number of solvents [hexane, methylene chloride, methanol, ethanol, methanol: ethanol (70:30, v:v)]. In extracts with methanol: ethanol (70:30 v:v) were detected higher amounts of metabolites than with other solvents. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) plots showed significant differences between the diploid and tetraploid metabolite profiles. Extracts of tetraploid showed higher amounts of amino acids, while extracts of diploid contained more organic acids and sugars. Graphical Abstract ᅟ.