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.

Risk assessment of combined exposure to lead, cadmium, and total mercury among the elderly in Shanghai, China.

Lead (Pb), cadmium (Cd) and total mercury (THg) are toxic heavy metals (THMs) that are widely present in the environment and can cause substantial health problems. However, previous risk assessment studies have rarely focused on the elderly population and have usually targeted a single heavy metal, which might underestimate the long-term accumulative and synergistic effects of THMs in humans. Based on the food frequency questionnaire and inductively coupled plasma mass spectrometry, this study assessed external and internal exposures to Pb, Cd and THg in 1747 elderly people in Shanghai. Probabilistic risk assessment with the relative potential factor (RPF) model was used to assess the neurotoxicity and nephrotoxicity risks of combined THMs exposures. The mean external exposures of Pb, Cd and THg in Shanghai elderly were 46.8, 27.2 and 4.9 µg/day, respectively. Plant-based foods are the main source of Pb and THg exposure, while Cd is mainly from animal-based foods. The mean concentrations of Pb, Cd and THg were 23.3, 1.1 and 2.3 µg/L in the whole blood, and 6.2, 1.0 and 2.0 µg/L in the morning urine, respectively. Combined exposure to THMs leading to 10.0 % and 7.1 % of Shanghai elderly at risk of neurotoxicity and nephrotoxicity. The results of this study have important implications for understanding the profiles of Pb, Cd and THg exposure in the elderly living in Shanghai and provide data support for risk assessment and control of nephrotoxicity and neurotoxicity from combined THMs exposure in the elderly.

Isolation and identification of novel phenolic and lignan glycosides from Swertia davidii Franch.

Phytochemical analyses of Swertia davidii Franch. extracts using column chromatography and semi-preparative HPLC were performed. Two novel phenolic glycosides named swertiosides A and B (compounds 1 and 2, respectively) were isolated and characterized. Four known phenolic glycosides were also extracted (compounds 3-6). The structural characteristics of these novel compounds were analyzed using 1D, 2D NMR, and HRMS. All six compounds have never been isolated from this particular plant species before this study. Subsequent assessment of bioactive properties suggested that compounds 1 and 2 exhibited moderate levels of cytotoxicity.

Structures and Anti-Inflammatory Evaluation of Phenylpropanoid Derivatives from the Aerial Parts of Dioscorea polystachya.

Seven undescribed phenylpropanoid constituents, including three new bibenzyl derivatives (1-3) along with four new benzofuran stilbene derivatives (4-7), were isolated from the aerial parts of Dioscorea polystachya. The structures of these compounds were elucidated using a combination of spectroscopic analyses, including UV, IR, HRESIMS, 1D, and 2D NMR. Further, all the compounds were evaluated on the anti-inflammatory activity for their inhibition of nitric oxide (NO) production by RAW 264.7 macrophages cells, and some of them (1-3 and 6) displayed inhibitory activity with IC50 values in the range of 9.3-32.3 µM. Moreover, compound 3 decreased the expression of iNOS in Western blot analysis, suggesting compound 3 is mediated via the suppression of an LPS-induced NF-κB inflammasome pathway.

Phenylpropanoid Derivatives from the Tuber of Asparagus cochinchinensis with Anti-Inflammatory Activities.

Three undescribed phenylpropanoid derivatives, including two new bibenzyl constituents (1-2), one new stilbene constituent (3), together with five known compounds stilbostemin F (4), dihydropinosylvin (5), 2-(4-hydroxyphenyl)ethyl benzoate (6), 1-(4-hydroxybenzoyl)ethanone (7), and 4-hydroxy-3-prenylbenzoic acid (8), were isolated from the tuber of Asparagus cochinchinensis. The structures of 1-8 were elucidated according to UV, IR, HRMS, 1D and 2D-NMR methods together with the published literature. All of the isolated compounds were assessed for anti-inflammatory activity by acting on lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells in vitro. The results showed that compounds 2 and 5 were found to inhibit the production of nitric oxide (NO) with the IC50 value of 21.7 and 35.8 µM, respectively. In addition, further studies found that compound 2 demonstrated concentration-dependent suppression of the protein expression of iNOS and exerted anti-inflammatory activity via the NF-κB signalling pathway. The present data suggest that phenylpropanoid derivatives from the tuber of A. cochinchinensis might be used as a potential source of natural anti-inflammatory agents.

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.

[Effect of total flavonoids in Scutellaria barbata in mediating autophagy in tumor cells via PI3K/AKT/mTOR pathway].

To investigate the effect of the total flavonoids in Scutellaria barbata(TF-SB) against autophagy in tumor cells in vivo, and further determine whether the mechanism is correlated with the PI3K/AKT/mTOR pathway, which lead to autophagy-induced tumor cell death. Melanoma-bearing mice were prepared and divided into control group, rapamycin group (Rap 1.5 mg•kg⁻¹), and high, middle and low-dose TF-SB (200, 100, 50 mg•kg⁻¹) groups. The groups were given drugs once a day for successively 11 days. The inhibitory effect of TF-SB on the growth of melanoma was determined by measuring tumor volume and tumor inhibition rate. TUNEL method was used to detect the apoptosis of tumor cells to further verify the antitumor activity of TF-SB. The protein expressions of LC3-Ⅰ and LC3-Ⅱ were detected by Western blot, and the relative expression of LC3-Ⅱ was calculated based on LC3-Ⅱ/LC3-Ⅰ. In addition, the effect of TF-SB on autophagy of tumor cells, the underlying molecular mechanism of TF-SB in inducing autophagy and PI3K/AKT/mTOR pathway marker protein phosphorylation were also studied. According to the results, TF-SB effectively inhibited melanoma growth in mice, reduced tumor volume, increased the tumor inhibition rate, and significantly increased tumor cell apoptosis index and the ratio of LC3-Ⅱ/LC3-I (P<0.05, P<0.01 or P<0.001). The protein expressions of p-PI3K, p-AKT and p-mTOR were also suppressed dramatically compared with those in control group (P<0.05, P<0.01 or P<0.001). In conclusion, the total flavonoids in S. barbata could inhibit the growth of melanoma in vivo by inducing autophagy and apoptosis of tumor cells, which may be correlated with suppression of PI3K/AKT/mTOR pathway.

[Total flavonoids in Scutellaria barbata prevents NLRP3 inflammasome expression in tumor cells by affecting autologous pathway].

This paper was aimed to investigate the relationship between autophagy and NLRP3 inflammasome activation by studying the effect oftotal flavonoids in Scutellaria barbata (TF-SB) on autophagy in tumor cells and NLRP3 inflammasome, and to provide experimental evidence for further study of the anti-tumor mechanism of TF-SB. Mielanoma models were established by inoculating B16-F1 cell line to mice, and then were randomly divided into 5 groups (n=10 in each group): model control, positive control control(Rap, 1.5 mg•kg⁻¹), and TF-SB low, middle and high groups (50, 100 and 200 mg•kg⁻¹). Meanwhile, healthy C57BL/6J mice were used as normal control group (n=10). The drugs were given once daily for 2 weeks consecutively. Thirty minutes after last treatment, the determinations at endpoint were performed; pathological changes of tumor tissue were evaluated by using HE staining; protein expressions of LC3-II/LC3-I or NLRP3inflammasome/caspase-1/IL-1ß and IL-18 in tumor tissues were detected by using Western-blot; and serum levels of IL-1ß and IL-18 were detected by using Elisa kit. The results showed that the tumor cells in model group showed obvious atypia and malignant proliferation; the invasion of tumor tissue was significantly reduced, the tumor necrosis area was significantly increased, and the inflammatory reaction was significantly alleviated in positive control group and various TF-SB groups. As compared with model control group, LC3-II/LC3-I was significantly increased, while NLRP3/caspase-1/IL-1ßand IL-18 protein expressions were significantly decreased in positive control group and TF-SB groups. Serum IL-1ß and IL-18 levels in model control group were found higher than those in control group (P<0.001), but they were significantly lowered in positive control group and TF-SB groups (P<0.05, P<0.01 or P<0.001). Taken together, total flavonoids in S. barbata could effectively alter the tumor growth micro-environment by inhibiting the expression of NLRP3 inflammasome, and its anti-tumor effect may be associated with the induction of tumor cell autophagy.

Effective recognition design in 8-ball billiards vision systems for training purposes based on Xception network modified by improved Chaos African Vulture Optimizer.

This research paper presents a comprehensive investigation into the utilization of color image processing technologies and deep learning algorithms in the development of a robot vision system specifically designed for 8-ball billiards. The sport of billiards, with its various games and ball arrangements, presents unique challenges for robotic vision systems. The proposed methodology addresses these challenges through two main components: object detection and ball pattern recognition. Initially, a robust algorithm is employed to detect the billiard balls using color space transformation and thresholding techniques. This is followed by determining the position of the billiard table through strategic cropping and isolation of the primary table area. The crucial phase involves the intricate task of recognizing ball patterns to differentiate between solid and striped balls. To achieve this, a modified convolutional neural network is utilized, leveraging the Xception network optimized by an innovative algorithm known as the Improved Chaos African Vulture Optimization (ICAVO) algorithm. The ICAVO algorithm enhances the Xception network's performance by efficiently exploring the solution space and avoiding local optima. The results of this study demonstrate a significant enhancement in recognition accuracy, with the Xception/ICAVO model achieving remarkable recognition rates for both solid and striped balls. This paves the way for the development of more sophisticated and efficient billiards robots. The implications of this research extend beyond 8-ball billiards, highlighting the potential for advanced robotic vision systems in various applications. The successful integration of color image processing, deep learning, and optimization algorithms shows the effectiveness of the proposed methodology. This research has far-reaching implications that go beyond just billiards. The cutting-edge robotic vision technology can be utilized for detecting and tracking objects in different sectors, transforming industrial automation and surveillance setups. By combining color image processing, deep learning, and optimization algorithms, the system proves its effectiveness and flexibility. The innovative approach sets the stage for creating advanced and productive robotic vision systems in various industries.

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.

Screening of Platycodonis Radix Fractions for Antiobesity Activities and Elucidation of Its Molecular Mechanisms in High-Fat Diet-Fed C57BL/6 Mice.

Obesity is a threat to public health and effective new medications are required. Platycodonis Radix (PR) is a traditional medicinal/dietary plant with activities against obesity. Using mice given a diet rich in fat, the antiobesity components of PR were identified and their molecular mechanisms were clarified further in this investigation. Initially, the impacts of PR fractions on liver histology and biochemical markers were assessed. Subsequently, the degrees of lipogenic and lipolytic gene and protein expressions were determined. Oral administration of PR polysaccharides (PG) (0.80 g/kg body weight) improved liver function (alanine aminotransferase and aspartate aminotransferase) and its antioxidant activities (total superoxide dismutase, glutathione peroxidase, and malondialdehyde), as well as alleviated blood lipid (total cholesterol, total triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol) values, inflammatory systemic (TNF-α and IL-1ß), and histological abnormalities within the liver. Furthermore, PG administration downregulated the expression for lipogenic genes (ACC and FAS) and upregulated the expression for the lipolytic gene (PPARα, LPL, CPT1, and HSL). Importantly, PG raised AMPK phosphorylation and decreased SREBP-1c protein synthesis. Thus, it is possible that PG stimulates the AMPK-LPL/HSL path (lipolytic route) plus the AMPK-ACC/PPARα-CPT1 path (associated to ß-oxidation of fatty acids), while inhibiting the AMPK/(SREBP-1c)-ACC/FAS path (lipogenic route). In summary, PG has the ability to regulate lipid metabolism, and it may be useful to pharmacologically activate AMPK with PG to prevent and cure obesity.

Genome-wide analysis of UDP-glycosyltransferases family and identification of UGT genes involved in drought stress of Platycodon grandiflorus.

Introduction: The uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs). Methods: The genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis. Results: A total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements. Discussion: PgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.

Two new N-containing heterocyclic compounds from the roots of Platycodon grandiflorus.

Two unusual N-containing heterocyclic compounds, Plagranlines B-C, were isolated from the roots of Platycodon grandiflorus. Plagranline B (1) was consisted of neolignane and monomeric quinoline constituent units and plagranline C (2) possessed pyridinone ring that was not commonly discovered in natural product. Their planar structures were elucidated based on analysis of NMR and HRESIMS spectroscopy data, and their absolute configurations were determined by quantum chemical calculations, including GIAO 13C NMR (DP4+) calculation and ECD calculation. In addition, extensive activity screening including glycosidases, oestrogen-like, and NO inhibitory assays were performed, compounds 1 and 2 possessed the weak activities.

Analysis of the influence of surgical robot drilling parameters on the temperature of skull drilling based on Box-Behnken design.

It is easy to cause thermal damage to the bone tissue when the surgical robot performs skull drilling to remove bone flaps, due to the large diameter of the drill bit, the large heat-generating area, and the long drilling time. Therefore, in order to reduce the thermal damage during the robot-assisted skull drilling process, the relationship between the drilling parameters and the drilling temperature during the skull drilling was studied in this paper. Firstly, a dynamic numerical simulation model of the skull drilling process was established by ABAQUS, and a temperature simulation plan for skull drilling was designed based on the Box-Behnken method. Then according to the simulation results, a quadratic regression model of drill diameter, feed rate, drill speed, and drilling temperature was established by using the multiple regression method. By analyzing the regression model, the influence of drilling parameters on the drilling temperature was clarified. Finally, the bone drilling experiment was carried out, and the error percentage was lower than 10.5% through the experiment to verify the reliability of the conclusion, and a safety strategy was proposed to ensure the safety of the surgical drilling process based on this experiment.

New stilbene metabolites isolated from the tuber of Bletilla striata and their biological assessment.

A phytochemical investigation of the tuber of Bletilla striata afforded eight stilbene derivatives, including a new bibenzyl (1) and two new phenanthrene (2 and 3). The structures of these compounds were elucidated using a combination of spectroscopic analyses. Further, the anti-inflammatory activity of all the compounds was evaluated to assess their capability to inhibit nitric oxide (NO) production by RAW 264.7 macrophages. The bioactive screening revealed that compounds 4 and 7 exhibited moderate inhibitory effects against NO production with the IC50 values 38.7 and 16.7 µM, respectively.

Effect of muscle activation on dynamic responses of neck of pilot during emergency ejection: a finite element study.

To determine the effect of muscle activation on the dynamic responses of the neck of a pilot during simulated emergency ejections. A complete finite element model of the pilot's head and neck was developed and dynamically validated. Three muscle activation curves were designed to simulate different activation times and levels of muscles during pilot ejection: A is the unconscious activation curve of the neck muscles, B is the pre-activation curve, and C is the continuous activation curve. The acceleration-time curves obtained during ejection were applied to the model, and the influence of the muscles on the dynamic responses of the neck was investigated by analyzing both angles of rotation of the neck segments and disc stresses. Muscle pre-activation reduced fluctuations in the angle of rotation in each phase of the neck. Continuous muscle activation caused a 20% increase in the angle of rotation compared to pre-activation. Moreover, it resulted in a 35% increase in the load on the intervertebral disc. The maximum stress on the disc occurred in the C4-C5 phase. Continuous muscle activation increased both the axial load on the neck and the posterior extension angle of rotation of the neck. Muscle pre-activation during emergency ejection has a protective effect on the neck. However, continuous muscle activation increases the axial load and rotation angle of the neck. A complete finite element model of the pilot's head and neck was established and three neck muscle activation curves were designed to investigate the effects of muscle activation time and level on the dynamic response of the pilot's neck during ejection. This increased insights into the protection mechanism of neck muscles on the axial impact injury of the pilot's head and neck.

A Thermoelectric MEMS Microwave Power Sensor with Inline Self-Detection Function.

In this paper, the design, fabrication and measurement of a thermoelectric MEMS microwave power sensor with the terminal load inline self-detection function is proposed. The structure of the sensor mainly includes a coplanar waveguide, a thermopile, two terminal load resistors and two calibration resistors. In order to realize the inline self-detection function, the load and calibration resistors are designed to form a voltage divider circuit. The fabrication of this sensor is compatible with the GaAs MMIC technology. The on-chip performance is tested by using a microwave experimental platform. The measured reflection loss is less than -10 dB at 0.1-10 GHz. When the bias voltage is not applied, the sensitivity of the sensor is 47.39 µV/mW@5 GHz and 32.58 µV/mW@10 GHz, respectively, and when the bias voltage is applied, the sensitivity is 47.50 µV/mW@5 GHz and 32.73 µV/mW@10 GHz, respectively. The difference between the two cases is less than 0.5% at the same frequency, which indicates that whether or not to apply the bias voltage has little effect on the sensitivity. In addition, when the calibration resistance is increased from 50 to 100 Ω, the current flowing through the load resistance is decreased under the same bias voltage. Therefore, the DC power consumed on the load resistance will be significantly reduced. This makes the measured and theoretical results show better agreement, thus verifying the validity of the design.

Novel phenolic metabolites isolated from plant endophytic fungus Fusarium guttiforme.

Two new phenolic compounds Fusagunolics A (1) and B (2) were isolated from the plant endophytic fungus Fusarium guttiforme, in addition to the previously known metabolites (3-6). The structures of these compounds was elucidated using a combination of spectroscopic analyses, including UV, HRESIMS, 1 D, and 2 D NMR, as well as electronic circular dichroism (ECD) and the optical rotatory dispersion (ORD). Further, the anti-inflammatory activity of all the compounds was evaluated to assess their capability to inhibit nitric oxide (NO) production by RAW 264.7 macrophages. The bioactive screening revealed that compounds 2 and 6 exhibited moderate inhibitory effects against NO production with the IC50 values 28.6 and 37.6 µM, respectively.

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.

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.