Identification and Functional Verification of the Glycosyltransferase Gene Family Involved in Flavonoid Synthesis in Rubus chingii Hu.

Glycosylation is catalyzed by UDP-glycosyltransferase (UGT) and plays an important role in enriching the diversity of flavonoids. Rubus plants contain a lot of natural flavonoid glycosides, which are important plants with a homology of medicine and food. However, information about the Rubus UGT gene family is very limited. In this study, we carried out genome-wide analysis and identified the 172, 121, 130, 121 UGT genes in R. chingii, R. corchorifolius, R. idaeus, and R. occidentalis, respectively, and divided them into 18 groups. The analysis of the protein motif and gene structure showed that there were structural and functional conservations in the same group, but there were differences among different groups. Gene replication analysis showed that raspberry and dicotyledons had a higher homology. The expansion of the UGTs gene family was mainly driven by tandem replication events, and experienced purified selection during the long evolution of the raspberry. Cis-acting element analysis showed that they were related to plant growth and development, hormone regulation, and stress response. In addition, according to a comprehensive analysis of the co-expression network constructed by transcriptome data and phylogenetic homology, RchUGT169 was identified as a flavonoid glucosyltransferase. Through the transient expression in tobacco, it was verified that RchUGT169 could catalyze the conversion of kaempferol and quercetin to the corresponding flavonoid glycosides. In conclusion, this research enriched the understanding of the diversity of UGTs in Rubus and determined that RcUGT169 can catalyze flavonoids.

[Comparative analysis of plastomes of Rubus chingii and R. chingii var. suavissimus].

Rubus chingii and R. chingii var. suavissimus are unique dual-purpose plant resources, with significant nutraceutical, pharmaceutical, and economic value, as well as promising prospects for further development. To investigate the genetic structure and evolutionary characteristics of these two varieties, this study conducted plastome sequencing using the Illumina HiSeq XTen sequencing platform. Subsequently, the study performed assembly, annotation, and characterization of the genomes, followed by a comparative plastome and phylogenetic analysis using bioinformatics techniques. The results revealed that the plastomes of R. chingii and R. chingii var. suavissimus exhibited a tetrad structure, comprising a large single-copy region(LSC), a small single-copy region(SSC), and two inverted repeat regions(IRs). The study identified a total of 56 simple sequence repeats(SSRs) after comparative analysis, predominantly consisting of A and T. Furthermore, the structure of the IR boundary genes in both varieties was found to be highly conserved, with only minor nucleotide variations. Additionally, the study identified three highly variable regions: rps16-trnQ-psbK, trnR-atpA, and trnT-trnL, which held promise as potential identification marks for further development and utilization. Phylogenetic analysis results obtained by the maximum likelihood and Bayesian inference methods demonstrated a close clustering of R. chingii and R. chingii var. suavissimus(100% support), with their closest relatives being R. trianthus. This study, focusing on plastome-level genetic distinctions between these two varieties, lays a foundation for future species protection, development, and utilization.

Genetic Diversity Analysis and Core Germplasm Construction of Rubus chingii Hu.

Rubus chingii Hu is the only species that is used for both edible and medicinal purposes among the 194 species of the genus Rubus in China. It is well known for its sweet and sour fresh fruits that are rich in vitamins and for its dried immature fruits that are used to treat kidney-related ailments. This study aims to evaluate genetic diversity and population structure and build a core germplasm repository of 132 R. chingii accessions from the provinces of Jiangxi and Fujian, using Hyper-seq-derived single-nucleotide polymorphism (SNP) markers. This is the first genetic study of R. chingii based on SNP molecular markers, and a total of 1,303,850 SNPs and 433,159 insertions/deletions (InDels) were identified. Low values for observed heterozygosity, nucleotide diversity (Pi) and fixation indexes (Fis) indicated low genetic diversity within populations, and an analysis of molecular variance (AMOVA) showed that 37.4% and 62.6% of the variations were found between populations and within samples, respectively. Four main clusters were identified by means of neighbor-joining (NJ) trees, the ADMIXTURE program and principal component analysis (PCA). Based on the genetic diversity, we finally constructed 38 representative core collections, representing 50% of the total core germplasm samples and 95.3% of the genotypes. In summary, the results of our study can provide valuable information on the genetic structure of R. chingii germplasm resources, which is helpful for further explorations of potential high-quality genes and for formulating future breeding and conservation strategies.

Genome-Wide Identification of Expansins in Rubus chingii and Profiling Analysis during Fruit Ripening and Softening.

Improving fruit size or weight, firmness, and shelf life is a major target for horticultural crop breeding. It is associated with the depolymerization and rearrangement of cell components, including pectin, hemicellulose, cellulose, and other structural (glyco)proteins. Expansins are structural proteins to loosen plant cell wall polysaccharides in a pH-dependent manner and play pivotal roles in the process of fruit development, ripening, and softening. Rubus chingii Hu, a unique Chinese red raspberry, is a prestigious pharmaceutical and nutraceutical dual-function food with great economic value. Thirty-three RchEXPs were predicted by genome-wide identification in this study, containing twenty-seven α-expansins (EXPAs), three ß-expansins (EXPBs), one expansin-like A (EXPLA), and two expansin-like B (EXPLBs). Subsequently, molecular characteristics, gene structure and motif compositions, phylogenetic relationships, chromosomal location, collinearity, and regulatory elements were further profiled. Furthermore, transcriptome sequencing (RNA-seq) and real-time quantitative PCR assays of fruits from different developmental stages and lineages showed that the group of RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expanding and ripening, while another group of RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening. They were regulated by both abscisic acid and ethylene and were collinear with phylogenetic relationships in the same group. Our new findings laid the molecular foundation for improving the fruit texture and shelf life of R. chingii medicinal and edible fruit.

Effects of different drying methods on Rubus chingii Hu fruit during processing.

In this study, the dried fruits of Rubus chingii Hu (Chinese name: Fu-Pen-Zi; FPZ) were processed and dried by three methods-in the shade, the sun, and the oven. The composition regarding the standard ingredient, color, and antioxidant capacities were investigated pro- and post-processing. The technique of headspace-solid-phase-microextraction-gas-chromatography-mass spectrometry (HS-SPME-GC-MS) and flavoromics were used to analyze the flavor-conferring metabolites of FPZ. The results obtained revealed that the highest use value and antioxidant capacities were detected in the FPZ fruits processed and dried in the shade. A total of 358 metabolites were detected from them mainly consisting of terpenoids, heterocyclic compounds, and esters. In differential analysis, the down-regulation of the metabolites was much greater than their up-regulation after all three drying methods. In an evaluation of the characteristic compounds and flavors produced after the three methods, there were variations mainly regarding the green and fruity odors. Therefore, considerable insights may be obtained for the development of novel agricultural methods and applications in the pharmaceutical and cosmetic industries by analyzing and comparing the variations in the chemical composition detected pre- and post-processing of the FPZ fruits. This paper provides a scientific basis for quality control in fruits and their clinical applications.

Ent-labdane-type diterpene glycosides obtained from Rubus chingii Hu and their inhibitory effects on PDE5A activity.

In this study, 16 new ent-labdane-type diterpene glycosides, designated as goshonosides J1-J16 (1-16), along with nine previously known diterpene glycosides (17-25) were extracted from the fruits of Rubus chingii Hu. The structures of goshonosides J1-J16 were elucidated using various analytical techniques, such as nuclear magnetic resonance, electron capture detector ECD, high-resolution electrospray ionization mass spectrometry HREIMS, single-crystal X-ray diffraction, and hydrolysis. Furthermore, the isolates' efficacy in inhibiting the activity of phosphodiesterase type 5 A was evaluated. Goshonosides J1, J2, and G effectively inhibited the activity of the aforementioned enzyme (IC50 values: 6.15 ± 1.76, 3.27 ± 0.65, and 9.61 ± 2.36 µM, respectively). Our findings highlight the remarkable structural diversity of bioactive compounds in R. chingii Hu and offer insights into the use of this shrub.

Constituents of the fruits of Rubus chingii Hu and their neuroprotective effects on human neuroblastoma SH-SY5Y cells.

Rubi fructus (Rubus chingii Hu) is a fruit of Rubus genus and is used in medicine and food applications. In this study, eight new phenylpropanoids (1-8) and seven known compounds (9-15) were isolated from the dried fruits of Rubus chingii Hu, and their structures were characterized through high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Electronic circular dichroism (ECD) experiments were performed, and the results were compared with ECD spectra. Compound 3 was characterized through extensive single crystal X-ray diffraction. Evaluation of the neuroprotective pharmacological activities revealed that compounds 6, 7, 9, and 14 exerted protective effects against H2O2-induced neurotoxicity by reducing the reactive oxygen species levels at concentrations of 50 and 100 µM. Moreover, the three compounds 6, 9, and 14 significantly inhibited the expression of the Casp3 gene at a concentration of 50 µM. Compounds 7 and 9 effectively repressed the expression of the MYC gene. Compounds 6 and 9 obviously upregulated the ratio of Bcl2/Bax in SH-SY5Y cells and inhibited cell apoptosis. The study results can be used as a reference for the development of R. chingii products to realize their neuroprotective functions in the future.

Integrated analysis of multiple metabolome and transcriptome revealed the accumulation of flavonoids and associated molecular regulation mechanisms in Rubus chingii Hu at different developmental stages.

The traditional Chinese herb Rubus chingii Hu (R. chingii) is widely used in clinical practice due to its beneficial effects. Flavonoids are the important class of pharmacological substances in R. chingii, however, the molecular mechanism underlying the differences in active flavonoid contents in R. chingii at different developmental stages remain poorly understood. In this experiment, we selected four developmental stages (GG, GY, YR, RR) of R. chingii as the research material. We studied the untargeted and targeted metabolic profiles of flavonoids in different periods of R. chingii, combining full-length and comparative transcriptome analyses. Functional analyses were conducted on genes implicated in flavonoid differences. GG and RR displayed relatively higher and lower contents of flavonols, flavones, flavanols, flavanones, and isoflavonoid, respectively. RNA-seq analyses showed structural genes such as RcPAL, RcC4H, Rc4CL, RcCHS, RcCHI, RcF3H, RcF3'H, and RcFLS in flavonoid biosynthesis pathway were upregulated in GG, which were essential for the accumulation flavanones, flavones, and flavonols (effective components). qRT-PCR analyses investigated that six structural genes RcCHI, RcF3H, 2 RcCHS, and 2 Rc4CL, two TFs RcMYB308 and RcMYB123 had a consistent expression pattern with which in transcriptome. Also, an interaction network showed that the RcMYB308 could positively regulate Ka3R, Qu, Qu3G, AS, Hy, Ti through RcF3H. Furthermore, Subcellular localization analysis revealed that RcMYB308 was localization to the nucleus. In tobacco, RcMYB308 was overexpressed, resulting in higher flavonoids, RcF3H, RcF3'H, RcCHI, and RcFLS. RcMYB308 upregulated RcF3H in dual-luciferase assays. These results provide new insights for further understanding the molecular mechanism regulating flavonol biosynthesis in R. chingii fruit, and also provide a potential MYB regulator for molecular breeding of R. chingii.

Rubus chingii Hu relieved the polycystic ovary syndrome with enhanced insulin sensitivity through inhibiting TXNIP/NLRP3 inflammasome signaling.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders in gynecology with severe metabolic abnormalities. Therefore, identifying effective treatments and drugs for PCOS is important. We aimed to investigate effect of the traditional Chinese medicine (TCM) Rubus chingii Hu (R. chingii) on ovarian function and insulin resistance (IR) of PCOS rat models, and to explore the underlying mechanisms. METHODS: A PCOS rat model was established by subcutaneous injection of dehydroepiandrosterone (DHEA) solution for 20 days. PCOS rats were randomly divided into a control group (CON), model group (MOD), metformin group (MET), TCM R. chingii group (RCG), and RCG + Ad-TXNIP groups. After 28 days of treatment, the samples were collected for subsequent experiments. RESULTS: R. chingii treatment alleviated hormone imbalance and IR while improving ovarian pathology in the PCOS model. R. chingi inhibited the activation of the thioredoxin-interacting protein (TXNIP)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in the ovarian tissue of PCOS rats. Furthermore, TXNIP overexpression hindered the protective effect of R. chingii intervention in PCOS rats, as evidenced by the increase of homeostasis model assessment of insulin resistance (HOMA-IR), luteinizing hormone (LH), testosterone (T), C-reactive protein (CRP) levels, and atretic follicles. CONCLUSION: R. chingii intervention improved ovarian polycystic development by suppressing the TXNIP/NLRP3 inflammasome, which may be an effective treatment for PCOS.

Insights into the metabolite profiles of Rubus chingii Hu at different developmental stages of fruit.

The fruits of Rubus chingii Hu have high medicinal and nutritional values. However, the metabolite profiles of R. chingii, especially the alterations during different development stages of fruit, have not been comprehensively analyzed, hindering the effective utilization of the unique species. In this study, we comprehensively analyzed the metabolites of R. chingii fruit at four developmental stages using systematic untargeted and targeted liquid chromatography-mass spectrometry metabolomics analysis and identified 682 metabolites. Significant changes were observed in metabolite accumulation and composition in fruits during the different developmental stages. The contents of the index components, kaempferol-3-O-rutinoside and ellagic acid, were the highest in immature fruit. The analysis identified 64 differentially expressed flavonoids and 39 differentially expressed phenolic acids; the accumulation of most of these differentially expressed metabolites decreased with the developmental stages of fruit from immaturity to maturity. These results confirmed that the developmental stages of fruit are a critical factor in determining its secondary metabolite compositions. This study elucidated the metabolic profile of R. chingii fruit at different stages of development to understand the dynamic changes in metabolites.

Rubochingosides A - J, labdane-type diterpene glycosides from leaves of Rubus chingii.

Ten previously undescribed labdane-type triterpenoids, rubochingosides A - J (1-10), were isolated from the leaves of Rubus chingii Hu. Their structures were elucidated by spectroscopic and chemical methods. The cytotoxicity of all isolated compounds was tested against five human tumor cell lines (Bel-7402, Caski, BGC-823, A2780, and HCT-116). Among them, compounds 4 and 8 showed cytotoxic activities against Bel-7402 and A2780 cells with IC50 values of 10.43 ± 0.51 and 29.03 ± 2.94 µM, respectively; Compound 8 exhibited cytotoxic activities against Bel-7402 and HCT-116 cells with IC50 values of 17.78 ± 1.54 and 26.23 ± 6.14 µM, respectively.

Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu.

Rubus chingii Hu is a berry plant of the genus Rubus of the Rosaceae family, which has high nutritional and medicinal value and is rich in flavonoids. Flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) compete for the common substrate dihydroflavonols to regulate the metabolic flux of flavonoids. However, the competition between FLS and DFR based on enzyme is rarely reported. Here, we isolated and identified two FLS genes (RcFLS1 and RcFLS2) and one DFR gene (RcDFR) from Rubus chingii Hu. RcFLSs and RcDFR were highly expressed in stems, leaves, and flowers, although the flavonol accumulation in these organs was significantly higher than that of proanthocyanidins (PAs). The recombinant RcFLSs demonstrated bifunctional activities via hydroxylation and desaturation at the C-3α position having a lower Michaelis constant (Km) for dihydroflavonols than RcDFR. We also found that a low concentration of flavonols could significantly inhibit RcDFR activity. To investigate the competitive relationship between RcFLSs and RcDFR, we used a prokaryotic expression system (E. coli) to co-express these proteins. The transgenic cells expressing recombinant proteins were incubated with substrates, and the reaction products were analyzed. Furthermore, two transient expression systems (tobacco leaves and strawberry fruits) and a stable genetic system (Arabidopsis thaliana) were used to co-express these proteins in vivo. The results showed that RcFLS1 was dominant in the competition with RcDFR. Our results demonstrated that the competition between FLS and DFR regulated the metabolic flux distribution of flavonols and PAs, which will be of great significance for the molecular breeding of Rubus plants.

Diterpenoid glucosides with anti-inflammatory activity from Rubi Fructus.

For centuries, dried unripe fruits of Rubus chingii Hu (Chinese name "Fu-pen-zi") have been widely used in traditional Chinese medicine for the treatment of various diseases, commonly associated with kidney deficiency. Rubi Fructus is an edible berry suitable for consumption either directly or in the form of juice and jam. The phytochemical investigation focused on the bioactive non-nutrient ingredient from the fruit of R. chingii, especially diterpenoid compounds. Seven diterpenoid glucosides, including three new (1-3) and four known (4-7) compounds, were obtained from the fruits of R. chingii. The structures along with the absolute configurations of the new compounds were determined by extensive NMR spectroscopic analysis. Compounds 1, 2, 4, 5, and 7 showed anti-inflammatory activity against LPS-induced NO production in RAW 264.7 macrophages. Further, the preliminary structure-activity relationships of these active compounds have been scientifically evaluated and discussed in this study.

Characteristics and phylogenetic analysis of the complete chloroplast genome of Rubus chingii Hu 1925 from the family Rosaceae.

Rubus chingii Hu 1925 is an important medicinal vine shrub in the Rosaceae family, widely distributed in China and Japan. In this study, the complete chloroplast genome of R. chingii was sequenced and identified. The chloroplast genome was 155,563 bp in size with a total GC content of 37.06%. Two 25,749-bp inverted repeat (IRA and IRB) regions divided the genome as four sections, with the remainder forming a large single-copy (LSC, 85,322 bp) and a small single-copy (SSC, 18,743 bp) regions. This genome contained a total of 131 genes, of which 86 were protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic analysis showed that R. chingii, along with several other R. longisepalus, R. tsangii, R. hirsutus, R. taiwanicola, R. rubroangustifolius, and R. glandulosopunctatus, formed the monophylic group. Interestingly, the chloroplast genome structure we reported was different from the previously reported structure and provided richer phylogenetic analysis information in the Rubus genus compared to previous studies. The genome information reported in this paper will provide some useful information for further investigation on the evolution of the family Rosaceae.

Anti-inflammatory Effect of a Novel Pectin Polysaccharide From Rubus chingii Hu on Colitis Mice.

Rubus chingii Hu has been used as a functional food for a long time. A novel pectin polysaccharide named RCHP-S from R. chingii Hu was structurally identified and explored its anti-inflammatory effect on colitis mice. RCHP-S was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. NMR spectroscopy and methylation analysis showed that RCHP-S was mainly composed of HG-type pectin domains but also contains a small amount of RG-I. The anti-inflammatory tests indicated that the mouse macrophage RAW 264.7 cells pretreated with RCHP-S could show a significant inhibitory effect on the mRNA level of iNOS, IL-1ß, IL-6, and TNF-α in vitro. Polysaccharide RCHP-S reduced the enteritis symptoms in dextran sulfate sodium (DSS)-induced colitis mice by inhibiting released inflammatory factors. These results indicated that the R. chingii Hu polysaccharide can be used as food additives for the treatment of intestinal inflammation.

Rubus chingii Hu. unripe fruits extract ameliorates carbon tetrachloride-induced liver fibrosis and improves the associated gut microbiota imbalance.

BACKGROUND: The unripe fruits of Rubus chingii Hu. ("Fu-peng-zi" in Chinese) is a well-known herbal tonic in traditional Chinese medicine (TCM) for tonifying liver and kidney. However, little is known regarding its therapeutic efficacy against liver fibrosis and the underlying mechanism. METHODS: The current research aims to explore the potential of Rubus chingii Hu. unripe fruits extract (RF) in the treatment of liver fibrosis and explore the underlying mechanism. RF was administered (450 and 900 mg·kg- 1 of body weight per day) orally to male C57BL/6 mice with CCl4-induced liver fibrosis for 3 weeks. The histopathological changes and fibrosis stage in liver tissue were assessed using hematoxylin and eosin (H&E) and Sirius red staining. The distribution of α-SMA and Col1A1 in the liver was analyzed to determine the hepatic stellate cells (HSCs) activation using immunohistochemistry and immunofluorescent analysis. Various biochemical markers in serum (ALT, AST) and liver (Hyp, IL1-ß, IL6, TNF-α and MCP-1) were observed to assess the liver's injury, fibrosis, and inflammation. In liver tissue, fibrosis-associated proteins including α-SMA, TGF-ß1, Smad2/3, p-Smad2/3, and Smad4 were detected through a Western blot assay. Pyrosequencing-based analysis of bacterial 16 S ribosomal RNA from variable regions V3-V4 of fecal samples characterized the gut microbiota. Spearman's rank correlation analysis was performed for the association between altered bacterial genera by RF and pharmacodynamics parameters. RESULTS: Three weeks of RF treatment can significantly lower liver inflammatory levels, pathological abnormalities, and collagen fibrous deposition in mice with CCl4-induced liver fibrosis. The expressions of α-SMA and Col1A1 were lowered by RF, while the expression levels of TGF-ß/Smads signaling pathway-related proteins, including TGF-ß1, p-Smad2/3, and Smad4, were dramatically decreased by RF. The RF treatment significantly increased or reduced 18 different bacterial species, restoring the CCl4-induced gut microbiota imbalance to the normal group's levels. According to correlation analysis, the bacterial genera Bifidobacterium and Turicibacter were the most significant in restoring CCl4-induced liver fibrosis. CONCLUSIONS: RF can reduce liver damage and delay the onset of liver fibrosis through modulating TGF-ß/Smads signaling pathway. Furthermore, RF's anti-liver fibrosis effect was related to balancing the gut microbial community, partly attained by increasing Bifidobacterium and Turicibacter in liver fibrosis.

Antibacterial effect of Rubus chingii flower extract against multidrug-resistant bacteria.

Rubus chingii is a well reputed member of Chinese traditional medicine system and is used for managing different ailments since historic times. The present report elucidates the growth impeding effect of R. chingii flower extract against multidrug resistant Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The extracts were prepared using standard Soxhlet extraction method using ethanol, methanol and acetone as a solvent. The extracts were further subjected to agar streak method and the stains that showed their sensitivity were further evaluated for minimum inhibitor concentration (MIC) assessment through TCC method. Subsequently the MIC was further used for well diffusion assay. All the strains used in the study showed their sensitivity towards R. chingii flowers extract in respective solvents. Highest antibacterial activity was seen against E. coli. and S. aureus whereas the lowest activity was recorded against K. pneumoniae. Thus the study reported herewith provided an insight into the antibacterial efficacy of R. chingii flower extract against MDR E. coli., S. aureus and K. pneumoniae.

Bioactive components, pharmacological effects, and drug development of traditional herbal medicine Rubus chingii Hu (Fu-Pen-Zi).

Rubus chingii Hu (Chinese Raspberry), known as Fu-Pen-Zi in Chinese, a woody perennial plant of the genus Rubus in the Rosaceae family, has specific nutritional and medicinal values, which is considered food-medicine herb in China for thousands of years to treat impotence, premature ejaculation, enuresis, frequent urination, and other diseases. This review aims to summarize recent advances in the bioactive components, pharmacological effects, and drug development and utilization of Rubus chingii Hu, hoping to provide useful support for its further research and clinical application. The bioactive components in Rubus chingii Hu contain mainly terpenoids, flavonoids, alkaloids, phenolic acids, polysaccharides, and steroids. The main pharmacological effects are their anti-oxidant, anti-inflammatory, and anti-tumor capacity on human health. Rubus chingii Hu is a very valuable food-medicine herb. The development of Rubus chingii Hu-related drugs is relatively single, which is limited to traditional Chinese medicine and prescriptions. Therefore, it is vital to pay interest to Rubus chingii Hu and its bioactive components in the future and extend its scientific application.

Combined transcriptomic and metabolic analyses reveal potential mechanism for fruit development and quality control of Chinese raspberry (Rubus chingii Hu).

KEY MESSAGE: Combined transcriptomic and metabolic analyses reveal that fruit of Rubus chingii Hu launches biosynthesis of phenolic acids and flavonols at beginning of fruit set and then coordinately accumulated or converted to their derivatives. Rubus chingii Hu (Chinese raspberry) is an important dual functional food with nutraceutical and pharmaceutical values. Comprehensively understanding the mechanisms of fruit development and bioactive components synthesis and regulation could accelerate genetic analysis and molecular breeding for the unique species. Combined transcriptomic and metabolic analyses of R. chingii fruits from different developmental stages, including big green, green-to-yellow, yellow-to-orange, and red stages, were conducted. A total of 89,188 unigenes were generated and 57,545 unigenes (64.52%) were annotated. Differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly involved in the biosynthesis of secondary metabolites. The fruit launched the biosynthesis of phenolic acids and flavonols at the very beginning of fruit set and then coordinately accumulated or converted to their derivatives. This was tightly regulated by expressions of the related genes and MYB and bHLH transcription factors. The core genes products participated in the biosynthesis of ellagic acid (EA) and kaempferol-3-O-rutinoside (K-3-R), such as DAHPS, DQD/SDH, PAL, 4CL, CHS, CHI, F3H, F3'H, FLS, and UGT78D2, and their corresponding metabolites were elaborately characterized. Our research reveals the molecular and chemical mechanisms of the fruit development of R. chingii. The results provide a solid foundation for the genetic analysis, functional genes isolation, fruit quality improvement and modifiable breeding of R. chingii.