Integrated component identification, network pharmacology, and experimental verification revealed mechanism of Dendrobium officinale Kimura et Migo against lung cancer.

BACKGROUND: Dendrobium officinale Kimura et Migo (DO), a valuable Chinese herbal medicine, has been reported to exhibit potential effects in the prevention and treatment of lung cancer. However, its material basis and mechanism of action have not been comprehensively analyzed. PURPOSE: The objective of this study was to preliminarily elucidate the active components and pharmacological mechanisms of DO in treating lung cancer, according to UPLC-Q/TOF-MS, HPAEC-PAD, network pharmacology, molecular docking, and experimental verification. METHODS: The chemical components of DO were identified via UPLC-Q/TOF-MS, while the monosaccharide composition of Dendrobium officinale polysaccharide (DOP) was determined by HPAEC-PAD. The prospective active constituents of DO as well as their respective targets were predicted in the combined database of Swiss ADME and Swiss Target Prediction. Relevant disease targets for lung cancer were searched in OMIM, TTD, and Genecards databases. Further, the active compounds and potential core targets of DO against lung cancer were found by the C-T-D network and the PPI network, respectively. The core targets were then subjected to enrichment analysis in the Metascape database. The main active compounds were molecularly docked to the core targets and visualized. Finally, the viability of A549 cells and the relative quantity of associated proteins within the major signaling pathway were detected. RESULTS: 249 ingredients were identified from DO, including 39 flavonoids, 39 bibenzyls, 50 organic acids, 8 phenanthrenes, 27 phenylpropanoids, 17 alkaloids, 17 amino acids and their derivatives, 7 monosaccharides, and 45 others. Here, 50 main active compounds with high degree values were attained through the C-T-D network, mainly consisting of bibenzyls and monosaccharides. Based on the PPI network analysis, 10 core targets were further predicted, including HSP90AA1, SRC, ESR1, CREBBP, MAPK3, AKT1, PIK3R1, PIK3CA, HIF1A, and HDAC1. The results of the enrichment analysis and molecular docking indicated a close association between the therapeutic mechanism of DO and the PI3K-Akt signaling pathway. It was confirmed that the bibenzyl extract and erianin could inhibit the multiplication of A549 cells in vitro. Furthermore, erianin was found to down-regulate the relative expressions of p-AKT and p-PI3K proteins within the PI3K-Akt signaling pathway. CONCLUSIONS: This study predicted that DO could treat lung cancer through various components, multiple targets, and diverse pathways. Bibenzyls from DO might exert anti-lung cancer activity by inhibiting cancer cell proliferation and modulating the PI3K-Akt signaling pathway. A fundamental reference for further studies and clinical therapy was given by the above data.

In vitro assessment of the pesticide bioaccessibility in Dendrobium officinale Kimura et Migo.

Bioaccessibility (BA) is a crucial factor for evaluating the absorption of pollutants in the human digestion system, which is of vital importance for risk assessment of pollutants via food intake. Multi-pesticides were detected in Dendrobium officinale Kimura et Migo (D. officinale), a popular dual-use plant with both botanical medicine and food applications. Nevertheless, the BA of pesticides in D. officinale remains unknown, restricting its market size. Herein, the BA of 19 pesticides with varying properties was evaluated by using an in vitro digestion model, showing BA values between 27.4 and 96.8%. The BA was controlled by the hydrophobicity and water solubility of pesticides, since the significant correlation between these two factors and BA values was observed. Moreover, co-ingested food ingredients could influence the BA, wherein the effect was significant for pesticides of logKow values no less than 3. Lipids enhanced the BA by 9-66%, whereas proteins or carbohydrates decreased BA values by 6-28%. In particular, considering the BA, the risk quotient values were reduced by 3-73%. Clearly, this work suggested that traditional risk assessment without considering the BA would seriously overestimate the actual risk of pesticides in food.

Metabolomics-Based Analysis of the Effects of Different Cultivation Strategies on Metabolites of Dendrobium officinale Kimura et Migo.

Dendrobium officinale Kimura et Migo is a famous plant with a high medicinal value which has been recorded in the Chinese Pharmacopoeia (2020 Edition). The medicinal properties of D. officinale are based on its chemical composition. However, there are no reports on how different cultivation methods affect its chemical composition. In order to reveal this issue, samples of the D. officinale were collected in this study through tree epiphytic cultivation, stone epiphytic cultivation, and greenhouse cultivation. Polysaccharides were determined by phenol sulfuric acid method and secondary metabolites were detected by the UPLC-MS technique. In addition, with regards to metabolomics, we used multivariate analyses including principal component analysis (PCA) and orthogonal partial least squares analysis (OPLS-DA) to screen for differential metabolites which met the conditions of variable importance projection values >1, fold change >4, and p < 0.05. The differential metabolites were taken further for metabolic pathway enrichment analysis, which was based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and validated by antioxidant activity. Comparing the three groups of samples according to the standards of the ChP (2020 edition), the results showed that the polysaccharide content of the samples from stony epiphytic cultivation and greenhouse cultivation was significantly higher than that of the samples from live tree epiphytic cultivation. Metabolomic analysis revealed that there were 185 differential metabolites among the 3 cultivation methods, with 99 of the differential metabolites being highest in the stone epiphytic cultivation. The results of the metabolic pathway enrichment analysis showed that the different cultivation strategies mainly effected four carbohydrate metabolic pathways, five secondary metabolite synthesis pathways, six amino acid metabolic pathways, one nucleotide metabolism pathway, three cofactor and vitamin metabolism pathways, and one translation pathway in genetic information processing. Furthermore, D. officinale from stone epiphytic cultivation which had the best antioxidant activity was implicated in differential metabolite production. This study revealed the effects of different cultivation methods on the chemical composition of D. officinale and also provided a reference for establishing the quality control standards to aid its development and utilization.

RNA sequencing-based exploration of the effects of far-red light on microRNAs involved in the shade-avoidance response of D. officinale.

Dendrobium officinale (D. officinale) has remarkable medicinal functions and high economic value. The shade-avoidance response to far-red light importantly affects the D. officinale productivity. However, the regulatory mechanism of miRNAs involved in the far-red light-avoidance response is unknown. Previous studies have found that, in D. officinale, 730 nm (far-red) light can promote the accumulation of plant metabolites, increase leaf area, and accelerate stem elongation. Here, the effects of far-red light on D. officinale were analysed via RNA-seq. KEGG analysis of miRNA target genes revealed various far-red light response pathways, among which the following played central roles: the one-carbon pool by folate; ascorbate and aldarate; cutin, suberine and wax biosynthesis; and sulfur metabolism. Cytoscape analysis of DE miRNA targets showed that novel_miR_484 and novel_miR_36 were most likely involved in the effects of far-red light on the D. officinale shade avoidance. Content verification revealed that far-red light promotes the accumulation of one-carbon compounds and ascorbic acid. Combined with qPCR validation results, the results showed that miR395b, novel_miR_36, novel_miR_159, novel_miR_178, novel_miR_405, and novel_miR_435 may participate in the far-red light signalling network through target genes, regulating the D. officinale shade avoidance. These findings provide new ideas for the efficient production of D. officinale.

RNA sequencing-based exploration of the effects of blue laser irradiation on mRNAs involved in functional metabolites of D. officinales.

Dendrobium officinale Kimura et Migo (D. officinale) has promising lung moisturizing, detoxifying, and immune boosting properties. Light is an important factor influencing functional metabolite synthesis in D. officinale. The mechanisms by which lasers affect plants are different from those of ordinary light sources; lasers can effectively address the shortcomings of ordinary light sources and have significant interactions with plants. Different light treatments (white, blue, blue laser) were applied, and the number of red leaves under blue laser was greater than that under blue and white light. RNA-seq technology was used to analyze differences in D. officinale under different light treatments. The results showed 465, 2,107 and 1,453 differentially expressed genes (DEGs) in LB-B, LB-W and W-B, respectively. GO, KEGG and other analyses of DEGs indicated that D. officinale has multiple blue laser response modes. Among them, the plasma membrane, cutin, suberine and wax biosynthesis, flavone and flavonol biosynthesis, heat shock proteins, etc. play central roles. Physiological and biochemical results verified that blue laser irradiation significantly increases POD, SOD, and PAL activities in D. officinale. The functional metabolite results showed that blue laser had the greatest promoting effect on total flavonoids, polysaccharides, and alkaloids. qPCR verification combined with other results suggested that CRY DASH, SPA1, HY5, and PIF4 in the blue laser signal transduction pathway affect functional metabolite accumulation in D. officinale through positively regulated expression patterns, while CO16 and MYC2 exhibit negatively regulated expression patterns. These findings provide new ideas for the efficient production of metabolites in D. officinale.

Medical Plant Extract Purification from Cadmium(II) Using Modified Thermoplastic Starch and Ion Exchangers.

Pure compounds extracted and purified from medical plants are crucial for preparation of the herbal products applied in many countries as drugs for the treatment of diseases all over the world. Such products should be free from toxic heavy metals; therefore, their elimination or removal in all steps of production is very important. Hence, the purpose of this paper was purification of an extract obtained from Dendrobium officinale Kimura et Migo and cadmium removal using thermoplastic starch (S1), modified TPS with poly (butylene succinate); 25% of TPS + 75% PBS (S2); 50% of TPS + 50% PLA (S3); and 50% of TPS + 50% PLA with 5% of hemp fibers (S4), as well as ion exchangers of different types, e.g., Lewatit SP112, Purolite S940, Amberlite IRC747, Amberlite IRC748, Amberlite IRC718, Lewatit TP207, Lewatit TP208, and Purolite S930. This extract is used in cancer treatment in traditional Chinese medicine (TCM). Attenuated total reflectance-Fourier transform infrared spectroscopy, thermogravimetric analysis with differential scanning calorimetry, X-ray powder diffraction, gel permeation chromatography, surface analysis, scanning electron microscopy with energy dispersive X-ray spectroscopy, and point of zero charge analysis were used for sorbent and adsorption process characterization, as well as for explanation of the Cd(II) sorption mechanism.

Dissipation, occurrence, and risk assessment of 12 pesticides in Dendrobium officinale Kimura et Migo.

The residual behaviors and dietary risk probability of 12 pesticides in Dendrobium officinale Kimura et Migo cultivated at two representative locations under green house conditions were investigated using liquid chromatography-tandem mass spectrometry. Field trials showed that the half-lives of 12 pesticides ranged from 0.9 to 14.4 days in fresh D. officinale stems. Based on maximum residue levels (MRLs), the ultimate residues of imidacloprid, dimethomorph, metalaxyl, tebuconazole, and cyazofamid at a pre-harvest interval (PHI) of 28 days were within acceptable limits. For abamectin, indoxacarb, and difenoconazole, 35-day PHIs were needed. The PHIs of trifloxystrobin and fluopyram were 42 days, the time required for their residues to be reduced to an MRL of 4 mg/kg. The chronic and acute risk quotients of target pesticides at PHIs of 28-42 days were below 5.929% and 0.532%, respectively, showing that the evaluated D. officinale exhibited an acceptably low dietary risk to the general population.

RNA sequencing-based exploration of the effects of far-red light on lncRNAs involved in the shade-avoidance response of D. officinale.

Dendrobium officinale (D. officinale) is a valuable medicinal plant with a low natural survival rate, and its shade-avoidance response to far-red light is as an important strategy used by the plant to improve its production efficiency. However, the lncRNAs that play roles in the shade-avoidance response of D. officinale have not yet been investigated. This study found that an appropriate proportion of far-red light can have several effects, including increasing the leaf area and accelerating stem elongation, in D. officinale. The effects of different far-red light treatments on D. officinale were analysed by RNA sequencing technology, and a total of 69 and 78 lncRNAs were differentially expressed in experimental group 1 (FR1) versus the control group (CK) (FR1-CK) and in experimental group 4 (FR4) versus the CK (FR4-CK), respectively. According to GO and KEGG analyses, most of the differentially expressed lncRNA targets are involved in the membrane, some metabolic pathways, hormone signal transduction, and O-methyltransferase activity, among other functions. Physiological and biochemical analyses showed that far-red light promoted the accumulation of flavonoids, alkaloids, carotenoids and polysaccharides in D. officinale. The effect of far-red light on D. officinalemight be closely related to the cell membrane and Ca2+ transduction. Based on a Cytoscape analysis and previous research, this study also found that MSTRG.38867.1, MSTRG.69319.1, and MSTRG.66273.1, among other components, might participate in the far-red light signalling network through their targets and thus regulate the shade-avoidance response of D. officinale. These findings will provide new insights into the shade-avoidance response of D. officinale.

Differences in the chemical composition of Dendrobium officinale Kimura et Migo and Dendrobium crepidatum Lindl based on UPLC-Q-TOF-MS/MS and metabolomics / 药学学报

italic>Dendrobium officinale Kimura et Migo is a rare Chinese herbal medicine, while Dendrobium crepidatum Lindl is a local medicine in Yunnan, both of which have the function of nourishing yin and stomach. To reveal the differences in chemical composition between the two species, ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) was used to analyze the chemical composition of stems and leaves of D. officinale and D. crepidatum. Principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) were used to determine the differences in metabolites between species and parts of Dendrobium. Fifty-eight chemical compounds were identified in the two species. Analysis indicated that the side ring of alkaloids connected with nitrogen was readily cleaved during analysis. The results of PCA analysis showed that the stems and leaves of D. officinale and D. crepidatum could be easily differentiated, and the chemical constituents of D. officinale and D. crepidatum were significantly different. OPLS-DA analysis showed that there were 16 metabolite differences between the stems and 22 differences in metabolites between the leaves of D. officinale and D. crepidatum. The main metabolite differences in components between the two Dendrobium species were dendrocrepidine B, dendrocrepidine C and dendrocrepine. There were 14 differences in metabolites between the stems and leaves of D. crepidatum. In conclusion, the chemical compositions of D. officinale and D. crepidatum are quite different; the small molecular compounds of D. officinale are mainly terpenoids and flavonoids, and the content of alkaloids is low. There is no significant difference between stem and leaf. In contrast, D. crepidatum is mainly composed of alkaloids and terpenoids, with crepidamine and dendrocrepine as its unique components, and there are great differences in the components between stems and leaves. This study provides a theoretical basis for the development and utilization of Dendrobium resources.

Pesticide multi-residues in Dendrobium officinale Kimura et Migo: Method validation, residue levels and dietary exposure risk assessment.

Dendrobium officinale Kimura et Migo (D. officinale) is a dual-use plant with both botanical medicine and food applications, drawing increasing attentions. Pesticides are inevitably applied on D. officinale in commercial artificial-sheltered cultivation, yet little is known about pesticide residue levels in D. officinale. A modified high through-put QuEChERS method coupled with HPLC-MS/MS was developed and validated to detect 76 pesticides in D. officinale. Graphitized multi-wall carbon nanotubes (g-MWCNTs) was selected as the clean-up sorbent, showing relative weak affinity to triazole fungicide having planar structure in their molecular and low matrix effects of pesticides in D. officinale samples compared to MWCNTs and pesticarb. The validated method was applied to analyze pesticide residues in 86 real D. officinale samples collected from three main producing provinces. 43 different pesticides were detected with highest residue of 6.11 mg/kg for dimethomorph. Given possible health risks related to pesticide residues, accordingly, risk assessment of human exposure to pesticides via intake of D. officinale was thus performed, indicating that the pesticide residue in fresh or dry D. officinale would not cause potential risk to human health either in the long-term or short-term. This work improved our understanding of potential exposure risk of pesticide multi-residues in D. officinale.

Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography.

An on-line cyclodextrin assisted sweeping-micellar electrokinetic chromatography (CD assisted sweeping-MEKC) was developed for the simultaneous separation and concentration of four neutral analytes (erianin, dendrophenol, naringenin and scoparone) in Dendrobium officinale Kimura et Migo (D. officinale). The D. officinale was directly determined by this on-line stacking method after simple extraction and dilution. The optimized background solution (BGS) was 50 mM phosphoric acid (PA) containing 100 mM SDS and 30% (v/v) methanol. The best separation and concentration performance of analytes dissolved in 90 mM CD and 100 mM PA was achieved in a short analysis time when injected at 50 mbar for 100 s. Compared with conventional sweeping-MEKC and MEKC method, significant improvement in enrichment efficiency was achieved by using this proposed method. A series of validation studies of the present method was performed under the optimal conditions. Good linearities were obtained with the correlation coefficients in the range of 0.994-0.999, the detection limits were ranged from 13 to 40 ng/mL. Sensitivity enhancement factors (SEFs) were in the range of 28.5-46.8 compared with traditional injection (injection time 3 s). Therefore, the proposed method was successfully applied for the separation and concentration of neutral analytes in real samples.

Advances and prospects of pharmacological activities of Dendrobium officinale Kimura et Migo polysaccharides / 药学学报

italic>Dendrobium officinale Kimura et Migo (D. officinale) has been used as a valuable traditional Chinese medicine for more than 2 000 years in China. Modern research has confirmed a wide range of pharmacological activities, such as regulating blood sugar, improving gastrointestinal inflammation, and regulating immunity. Polysaccharides are the main active ingredients of D. officinale. With the intensive studies of the pharmacological activities of D. officinale, evidence for the pharmacological effects and potential mechanisms of D. officinale polysaccharides has increased dramatically. In this review, we summarized the latest progress in the pharmacological and mechanical studies of D. officinale polysaccharides, and based on the pharmacological efficacy and oral absorption and utilization characteristics of D. officinale polysaccharides, it is proposed that regulating the gut microbiota may be one of the key mechanisms for D. officinale to exert its beneficial effects. Research on the mechanism of D. officinale polysaccharides puts forward new research directions and prospects.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

Molecular cloning and characterization of a novel DoRLK gene from Dendrobium officinale / 中草药

Objective: To identifiy a receptor-like kinase (RLK) encoding gene named as DoRLK (GenBank accession No. ANC68272.1) from Dendrobium officinale. Methods: RT-PCR and RACE technologies were used to isolate the full length cDNA of DoRLK. Characteristics of physiochemical properties, conserved domains, and subcellular localization of the deduced DoRLK protein were determined by a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 7.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. Results: The results showed that the full length cDNA of DoRLK was 1 715 bp in length and encoded a 423 aa protein with a molecular weight of 47 800.88 and an isoelectric point (pI) of 9.47. The deduced DoRLK protein, like other RLK proteins, constituted one conserved domains (85—370) and one transmembrane motifs (250—270). Multiple sequence alignment and phylogenetic analyses demonstrated that DoRLK had high identity (43.62%—63.35%) to a number of RLK genes from various plants and was closely related to Phalaenopsis equestris, Phoenix dactylifera and Asparagus officinalis. Real time quantitative PCR (qPCR) analysis revealed that DoRLK was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.22 fold over that in the leaves,followed by that in the stems with 2.75 fold. Conclusion: Molecular characterization of DoRLK will be useful for further functional elucidation of the gene involving in D. officinale responses to environmental factors.

Advances in immunoregulatory effects and bioactive compounds of polysaccharides from Dendrobium officinale / 中草药

Based on the previous studies in recent years, this advances made a summary of immunoregulatory effects from Dendrobium officinale. And the research advances on related bioactive compounds of polysaccharides in extraction, separation and purification, structural composition and content determination were systemically reviewed. This paper intended to provide a theoretical basis for reasonable research and exploitation of related health foods of D. Officinale.

Research progress on genomics, transcriptomics and functional genes of Dendrobium officinale / 中草药

Dendrobium officinale is one of the most precious medicinal plants in the family of Orchidaceae, and rich in polysaccharides, astragalus, bibenzyls and alkaloids. It has effects such as antioxidant, anti-cancer, immuno-enhancing, lowering blood sugar, and alleviating liver injury. Its huge medicinal, scientific and commercial value has raised a research upsurge, especially in the past five years, nucleic acid molecular biology of Dendrobium officinale has made more and more exciting results. In order to provide scientific guidance for further research on functional genes, this paper reviews the recent progress in research on genomics, transcriptomics and functional genes of D. officinale.

Cloning and expression analysis of an F-box protein gene DoSKP2A in Dendrobium officinale / 中草药

Objective: To clone an F-box protein namely S phase kinase-associated protein gene DoSKP2A with full length cDNA, in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern analysis. Methods: RACE technology were used for gene identification. Characteristics of physiochemical properties, conserved domains and subcellular localization of the protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 7.0, respectively. Real time quantitative PCR was used for gene expression analysis. Results: DoSKP2A gene was cloned (GenBank accession KU160472). The full length cDNA of DoSKP2A was 1 507 bp in length, and ORF was 1 101 bp, encoding a 366-aa protein with a molecular weight of 39 590 and an isoelectric point of 7.9. The deduced DoSKP2A protein, without transmembrane or signal peptide residues, contained an F-box core domain (26-88), a leucine-rich repeat (202-226), and multiple conserved motifs. DoSKP2A had high identities (64.6%-72.4%) with SKP2As proteins from various plants. DoSKP2A belonged to the monocotyledons subgroup of the SKP2As evolutionary tree. DoSKP2A gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots, with 6.16 fold, then the stems and the lowest in the leaves. Conclusion: The novel full-length F-box protein gene DoSKP2A was obtained, along with bioinformatics and expression characteristics, which provided molecular basis for the growth and development, signal transduction, and stress resistance of D. officinale.

Cloning and expression analysis of gene DoSRK2E in Dendrobium officinale / 中草药

Objective To clone the SnRK2 gene in Dendrobium officinale and investigate its characteristics and expression pattern. Methods RT-PCR and RACE techniques were used to clone the full-length cDNA of DoSRK2E, with the aids of a series of online bioinformatic software, characteristics including molecular mass, isoelectric point, conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed. Besides, the sequence of the deduced protein was aligned with those of other plant SnRK2 by DNASTAR, and phylogenetic relationships were reconstructed utilizing MEGA. Finally, tissue specific expression pattern of DoSRK2E was tested by quantitative real-time PCR (qRT-PCR). Results The full-length cDNA of DoSRK2E (GenBank accession API65110) is 1 795 bp with a 1 086 bp complete open reading frame (ORF). The predicted molecular mass and isoelectric point of the deduced protein DoSRK2E were 40 850 and 4.80, respectively. No signal peptide nor transmembrane region were detected, this protein contains one protein kinase domain, one ATP binding site, and one Ser/Thr active site, which was predicted most likely to be located in the endoplasmic reticulum membrane. DoSRK2E protein showed high similarity with those from other plant SnRK2, and its phylogenetic location was in Group III of SnRK2 subfamily, and phylogenetically closest to AtSnRK2.6 from Arabidopsis. In addition, qPCR analysis revealed that DoSRK2E showed the highest expression level in root, followed by stem and leaf. Conclusion A SnRK2 family gene DoSRK2E was cloned from the rare and endangered medicinal plant D. officinale for the first time. The Characteristics and expression pattern of this gene were analyzed. This study will provide a basis for further exploration of the regulation mechanisms of DoSRK2E in D. officinale under stress conditions.

Cloning and expression analysis of a novel alkaline/neutral invertase (DoNI2) gene in Dendrobium officinale / 中草药

Objective: To clone novel member of alkaline/neutral invertase (NI) gene in a rare and endangered medicinal plant of Dendrobium officinale, conduct bioinformatic analysis and detect the quantitative expression in different organs. Methods: Primers were designed according to NI gene segment which was selected from leaf transcriptome sequencing results of D. officinale. The full-length cDNA of NI gene was cloned via homology-based cloning and rapid amplification of cDNA ends (RACE) approach. The physical and chemical properties, secondary structure and tertiary structure of NI protein were forecasted and analyzed using related software. The expression levels of NI gene in roots, stems, and leaves of D. officinale were detected using real-time PCR. Results: A novel gene encoding a NI protein was cloned from D. officinale. This gene (named as DoNI2, GenBank accession number: KY794404) had a total length of 2 397 bp with an open reading frame of 1 836 bp, and encoded a predicted polypeptide of 611 amino acids with a molecular weight of 69 050. Bioinformatics predicted that the isoelectric point of DoNI2 gene encoding protein was 6.38, the instability coefficient was 44.95, and the hydrophobic coefficient was −0.232. RT-PCR showed that DoNI2 gene expressed in all organs with highest expression level in stems and the lowest in roots. DoNI2 gene expression was significantly positively correlation with NI enzymatic activities at different growth years of D. officinale. Conclusion: The full length cDNA sequence in a mitochondrial DoNI2 gene was identified, facilitating future functional analysis of the gene involving in the regulation of sugar metabolism in D. officinale.

Cloning and expression analysis on a protein phosphatase encoding gene DoPP2C1 in Dendrobium officinale / 中草药

Objective: To isolate and characterize a protein phosphatase (PP) encoding gene DoPP2C1 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. Methods: qRT-PCR and RACE technologies were used to isolate the full length cDNA of DoPP2C1. Characteristics of physiochemical properties, conserved domains, and subcellular localization of DoPP2C1 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. Results: The full length cDNA of DoPP2C1 (GenBank accession KJ995533) was 1 221 bp in length, and encoded a 285-aa protein with a molecular weight of 31 080 and an isoelectric point of 6.18; The deduced DoPP2C1 protein had one PP2C domain (27-285), which are all conserved among the PP2C proteins. DoPP2C1 protein did not contain a signal peptide or a transmembrane region, and was predicted to locate in cytoplasm; DoPP2C1 had high identities (56.3%-73.7%) with various PP2C proteins in plants; DoPP2C1 was closely related to Oryza sativa OsPP2C62, Sorghum bicolor XP_002462907, Hordeum vulgare BAK00362 and Triticum urartu EMS47641 proteins, and belonged to the Group F1 of the PP2C evolutionary tree; DoPP2C1 gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots and stems, with 7.57 and 1.79 fold, respectively, over that in the leaves. Conclusion: Molecular characterization of DoPP2C1 gene was obtained, which will be useful for further functional determination of the gene involving in the growth and development, physiological stress adaptations, and secondary metabolic regulations of D. officinale.