Selection of Reference Genes in Siraitia siamensis and Expression Patterns of Genes Involved in Mogrosides Biosynthesis.

Siraitia siamensis is a traditional Chinese medicinal herb. In this study, using S. siamensis cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of cucurbitadienol synthase, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that CDC6 and NCBP2 expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in S. siamensis in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways.

Genome-Wide Identification of Seven in Absentia E3 Ubiquitin Ligase Gene Family and Expression Profiles in Response to Different Hormones in Uncaria rhynchophylla.

SINA (Seven in absentia) E3 ubiquitin ligases are a family of RING (really interesting new gene) E3 ubiquitin ligases, and they play a crucial role in regulating plant growth and development, hormone response, and abiotic and biotic stress. However, there is little research on the SINA gene family in U. rhynchophylla. In this study, a total of 10 UrSINA genes were identified from the U. rhynchophylla genome. The results of multiple sequence alignments and chromosomal locations show that 10 UrSINA genes were unevenly located on 22 chromosomes, and each UrSINA protein contained a SINA domain at the N-terminal and RING domains at the C-terminal. Synteny analysis showed that there are no tandem duplication gene pairs and there are four segmental gene pairs in U. rhynchophylla, contributing to the expansion of the gene family. Furthermore, almost all UrSINA genes contained the same gene structure, with three exons and two introns, and there were many cis-acting elements relating to plant hormones, light responses, and biotic and abiotic stress. The results of qRT-PCR show that most UrSINA genes were expressed in stems, with the least expression in roots; meanwhile, most UrSINA genes and key enzyme genes were responsive to ABA and MeJA hormones with overlapping but different expression patterns. Co-expression analysis showed that UrSINA1 might participate in the TIA pathway under ABA treatment, and UrSINA5 and UrSINA6 might participate in the TIA pathway under MeJA treatment. The mining of UrSINA genes in the U. rhynchophylla provided novel information for understanding the SINA gene and its function in plant secondary metabolites, growth, and development.

Genome-Wide Mining of CULLIN E3 Ubiquitin Ligase Genes from Uncaria rhynchophylla.

CULLIN (CUL) protein is a subtype of E3 ubiquitin ligase that is involved in a variety of biological processes and responses to stress in plants. In Uncaria rhynchophylla, the CUL gene family has not been identified and its role in plant development, stress response and secondary metabolite synthesis has not been studied. In this study, 12 UrCUL gene members all contained the typical N-terminal domain and C-terminal domain identified from the U. rhynchophylla genome and were classified into four subfamilies based on the phylogenetic relationship with CULs in Arabidopsis thaliana. They were unevenly distributed on eight chromosomes but had a similar structural composition in the same subfamily, indicating that they were relatively conserved and potentially had similar gene functions. An interspecific and intraspecific collinearity analysis showed that fragment duplication played an important role in the evolution of the CUL gene family. The analysis of the cis-acting elements suggests that the UrCULs may play an important role in various biological processes, including the abscisic acid (ABA) response. To investigate this hypothesis, we treated the roots of U. rhynchophylla tissue-cultured seedlings with ABA. The expression pattern analysis showed that all the UrCUL genes were widely expressed in roots with various expression patterns. The co-expression association analysis of the UrCULs and key enzyme genes in the terpenoid indole alkaloid (TIA) synthesis pathway revealed the complex expression patterns of 12 UrCUL genes and some key TIA enzyme genes, especially UrCUL1, UrCUL1-likeA, UrCUL2-likeA and UrCUL2-likeB, which might be involved in the biosynthesis of TIAs. The results showed that the UrCULs were involved in the response to ABA hormones, providing important information for elucidating the function of UrCULs in U. rhynchophylla. The mining of UrCULs in the whole genome of U. rhynchophylla provided new information for understanding the CUL gene and its function in plant secondary metabolites, growth and development.

Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla.

Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the charactertics of GATA TFs in U. rhynchophylla and how different light qualities affect the expression of GATA and key enzyme genes, thereby affecting the changes in U. rhynchophylla alkaloids have not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIAs content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content. In this research, we comprehensively analyzed the UrGATA TFs, and offered insight into the involvement of UrGATA TFs from U. rhynchophylla in TIAs biosynthesis.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis.

E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the "full-length transcriptome + expression profile + metabolome" data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis.

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

[Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis].

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factors in Siraitia siamensis.

WRKY transcription factors, as the largest gene family in higher plants, play an important role in various biological processes including growth and development, regulation of secondary metabolites, and stress response. In this study, we performed genome-wide identification and analysis of WRKY transcription factors in S. siamensis. A total of 59 SsWRKY genes were identified that were distributed on all 14 chromosomes, and these were classified into three major groups based on phylogenetic relationships. Each of these groups had similar conserved motifs and gene structures. We compared all the S. siamensis SsWRKY genes with WRKY genes identified from three diverse plant species, and the results implied that segmental duplication and tandem duplication play an important roles in the evolution processes of the WRKY gene family. Promoter region analysis revealed that SsWRKY genes included many cis-acting elements related to plant growth and development, phytohormone response, and both abiotic and biotic stress. Expression profiles originating from the transcriptome database showed expression patterns of these SsWRKY genes in four different tissues and revealed that most genes are expressed in plant roots. Fifteen SsWRKY genes with low-temperature response motifs were surveyed for their gene expression under cold stress, showing that most genes displayed continuous up-regulation during cold treatment. Our study provides a foundation for further study on the function and regulatory mechanism of the SsWRKY gene family.

The levels of bioactive ingredients in Citrus aurantium L. at different harvest periods and antioxidant effects on H2 O2 -induced RIN-m5F cells.

BACKGROUND: Citrus aurantium L. (Aurantii fructus) is a multi-purpose citrus fruit with high medicinal and nutritional value, but currently there are no data that can be used to investigate the appropriate harvest time to obtain high-quality citrus bioactive ingredients from it. RESULTS: Phytochemicals and the levels of the main bioactive ingredients were investigated by ultra high performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF/MS). The flavanone, polymethoxyflavone, coumarin, synephrine, and limonin content in the citrus fruit was analyzed at different harvest periods, and significant differences, ranging from 0.03 ± 0.01 to 116.26 ± 40.20 g kg-1 (DW), were shown. These compounds were present in higher amounts in June and then decreased gradually, while the biomass accumulation of most of them showed an increasing tendency around harvest time. The H2 O2 -induced RIN-m5F cells model was employed to evaluate their antioxidant capacity. Citrus fruit harvested from June 11 to July 7 possessed an excellent antioxidant capacity by inhibiting the intensity of intracellular reactive oxygen species (ROS) (P < 0.01) and improving superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) activity (P < 0.01). The chemical composition and antioxidant capacity of citrus leaves, stems, and roots were also evaluated, and these showed great variation compared with other citrus fruits. Multivariate statistical analysis indicated that harvesting time was related closely to the phytochemical contents and antioxidant capacity. CONCLUSION: Citrus fruit can be appropriately harvested from June to early July when the levels of bioactive ingredients and antioxidant activity reach higher values. This research provides practical information for producing high-quality citrus products. © 2020 Society of Chemical Industry.