Functional analysis of flavanone 3-hydroxylase(F3H)from Dendrobium officinale,which confers abiotic stress tolerance

Flavonoids are important bioactive components in Dendrobium officinale,a medicinal orchid.They are involved in many biological activities,including protecting plants against biotic and abiotic stresses.Research on the key genes related to flavonoid biosynthesis in D.officinale is limited.In this study,one of the key flavonoid biosynthesis genes,flavanone 3-hydroxylase(F3H),was characterized from D.officinale.The open reading frame of DoF3H was 1134 bp long and it encoded a 377-amino acid protein.The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs.DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers,followed by roots,stems and leaves,a pattern that was similar to the content of flavonoids.Recombinant DoF3H protein,which was localized in the cytosol,could convert naringenin to dihydrokaempferol.The mRNA levels of DoF3H were significantly induced by salt and cold stresses.Furthermore,the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses.These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids,and provide a new application for improvement of abiotic tolerance in D.officinale.

Study on Mechanism of Dendrobium officinale Kimura et Migo in Preventing and Treating Exercise-induced Muscle Damage(EIMD)in Rats

[Objectives]This study was conducted to observe the mechanism of Dendrobium officinale Kimura et Migo on gastrocnemius muscle in rats with exercise-induced muscle damage(EIMD).[Methods]The micro-injury model of skeletal muscle was established by treadmill training.Forty two SD rats were randomly divided into a control group,1,12 and 24 h exercise groups,D.officinale 2 ml+1 h exercise group,D.officinale 2 ml+12 h exercise group,and D.officinale 2 ml+24 h exercise group,with 6 rats in each group.Various D.officinale groups were given the drug once in the morning and once in the evening at a dose of 2 ml/time,a week in advance.Except for the quiet group,the samples were collected from the 1,12 and 24 h exercise groups after anesthesia following 1,12 and 24 h of exercise for the last time,respectively,and the D.officinale 2 ml+1 h exercise group,D.officinale 2 ml+12 h exercise group and D.officinale 2 ml+24 h exercise group were also sampled after anesthesia following 1,12 and 24 h of exercise for the last time,respectively.The contents of ATP,CK-MM and CK in rat serum were determined by enzyme-linked immunosorbent assay(ELISA).The histopathological changes of gastrocnemius muscle were observed by HE staining.PCR and Western-blot detection were carried out to analyze the effects of D.officinale on IGF-1 mRNA and protein expression in gastrocnemius muscle.[Results]Compared with the quiet group,the ATP contents in the serum of rats in the 1,12 and 24 h exercise groups significantly decreased(P<0.01),while the CK and CK-MM contents significantly increased(P<0.01).The expression of IGF-1 mRNA and protein in the gastrocnemius muscle tissue significantly increased(P<0.01).Compared with the 1 h exercise group,the ATP content and IGF-1 protein expression in the gastrocnemius muscle tissue of the D.officinale liquid+1 h exercise group significantly increased(P<0.05),while the CK and CK-MM contents significantly decreased(P<0.01).Compared with the 12 h exercise group,the D.officinale liquid+12 h exercise group showed a significant increase in ATP content(P<0.01),significant increases in IGF-1 mRNA and protein expression in the gastrocnemius muscle tissue(P<0.01),and significant decreases in CK and CK-MM contents(P<0.01).Compared with the 24 h exercise group,the ATP content and IGF-1 mRNA and protein expression in the gastrocnemius muscle tissue of the D.officinale liquid+24 h exercise group significantly increased(P<0.01),while the CK and CK-MM contents significantly decreased(P<0.01).From the pathological tissue morphology of the gastrocnemius muscle in rats with EIMD treated with D.officinale,it could be concluded that the gastrocnemius muscle of each exercise group was significantly damaged,and the damage was significantly alleviated after administration of D.officinale liquid.[Conclusions]The effects and mechanism of D.officinale on prevention and treatment of EIMD in rats might be related to the promotion of IGF-1 mRNA and protein expression in injured tissues by reducing ATP energy consumption,CK-MM and CK activity.

Investigation on the Morphological Characteristics of Dendrobium officinale Plantlets Propagated from Different Explants

[ Objective] This study aimed to investigate the differences in morphological characteristics of Dendrobium officinale plantlets propagated from different explants. [ Method] Randomly 1 000 D. offtcinale plantlets propagated via different regeneration pathways were selected for morphological investigation and classification. [ Result] D. officinale plantlets propagated from stem segment explants exhibited highly consistent morphological characteristics, while those propagated from seed explants exhibited a variety of morphological characteristics. [ Conclusion] Therefore, using seed explants for regeneration can effectively broaden the germplasms resources of D. officinale.

The bibenzyl derivatives of Dendrobium officinale prevent UV-B irradiation induced photoaging via SIRT3

Dendrobium officinale is a valuable medicinal herb that is widely used in traditional Chinese medicine.The chemical constituents of D.officinale have attracted much attention and a large number of compounds have been reported including many bibenzyl derivatives.13 bibenzyl derivatives from D.officinale were sent for molecular docking,sur-face plasmon resonance(SPR)assay and after detection of Mn-SOD and SIRT3 activities in or not in HaCaT cells,it was concluded that bibenzyl derivatives did not directly activate Mn-SOD but promoted SIRT3 proteins.In addition,HaCaT cells were irradiated with UV-B to induce an oxidative stress model in vitro to further verify the effect of bibenzyl derivatives.The results show that bibenzyl derivatives could directly bind to SIRT3,enhance the deacetylation and then activate Mn-SOD,so as to protect UV-B induced skin photoaging.

Physiological Responses of Dendrobium officinale under Exposure to Cold Stress with Two Cultivars

This study aimed to explore the cold tolerance of two cultivars of Dendrobium officinale(MG1,MG2)grown in different regions of China.Under-2℃ incubation,cultivar MG1 remained active after 3 d,and continued to grow after returning to room temperature.However,MG2 could only maintain its activity after 2 d treatment at−2℃,and the seedlings died with the low temperature treatment time.Investigation of the characteristics of the plants grown in the south(Hangzhou)or north(Zhengzhou)of China indicated that the leaves of MG1 also had reduced stomatal density,the highest thickness,and a compact microstructure.The contents of proline and soluble sugars were higher in MG1 than those in MG2.The cultivar MG1 had higher SOD enzyme activity than MG2,while CAT and POD activities in samples from Zhengzhou were higher than those from Hangzhou.The contents of polysaccharides and alkaloids in stems of in MG1 were higher than those in MG2,while the content of flavonoids in the Zhengzhou samples was higher than that in the Hangzhou samples.In addition,plant heights,stem diameters,and chlorophyll content were higher in MG1.Overall,MG1 had better cold resistance than MG2.MG1 is a cold tolerant cultivar with thick leaves and reduced stomatal density,higher contents of soluble sugars,proline,CAT,POD,polysaccharides,flavonoids and alkaloids,which together make it more adaptable to low temperatures.Thus,the cultivar MG1,with its demonstrated cold tolerance,can accordingly be grown on a large scale in cold regions,thereby expanding the available planting area for this important traditional medicinal plant to meet the increasing commercial demand for it.

Pathogen Identification of Stem Rot in Dendrobium officinale of China

For the control of stem rot in Dendrobium officinale, symptoms were observed and the pathogen was isolated and identified from April 2013 to August 2015. The pathogen only infected the stem of D. officinale, resulting in death of stem above the lesion. Strain TP-1 accounted for 65.7% of isolated colonies. After inoculation of TP-1 for 14 d, water-soaked necrotic lesions about 3 mm in diameter were observed, which were similar to natural infections, and the original strain was obtained after re-isolation. Morphology and sequencing identification confirmed that TP-1 was Lasiodiplodia theobromae. This is the first report of stem rot in D. officinale caused by L. theobromae in China.

Effects of Planting Methods on Levels of Common Pesticides and Heavy Metals in Dendrobium officinale

The levels of common pesticides and heavy metals in Dendrobium officinale grown from 2012 to 2014 were measured, and three planting methods were used in 2014. The quality of plant husbandry improved during the study period. Thirteen types of pesticides were detected in D. officinale grown in 2014 and antimicrobials, including carbendazim, difenoconazole and azoxystrobin, were evident in 61.53% of plants. These three pesticides constituted 72.33% of all the antimicrobials detected. Planting methods affected the levels of pesticides and heavy metals. Trellising in plastic sheds was optimal, being associated with the lowest amounts of both pesticides and heavy metals. Both cadmium（Cd） and lead（Pb） levels were high in treed plants, whereas Cr, As and Cu levels were the highest in potted plants.

Field Efficacy Evaluation of 68% Metalaxyl-M·Mancozeb WDG against Dendrobium officinale Blight

The control effect of 68% Metalaxyl-M·Mancozeb WDG against Dendrobium officinale blight was studied. The results showed that the control effect of 68% Metalaxyl-M·Mancozeb WDG at the dose of 1 133-1 360 mg/kg against D. officinale blight was over 78.4% at 14 d post spraying, which was equivalent to that of control agent 72% Cymoxanil·Mancozeb WP(1 200 mg/kg) and 80% Mancozeb WP(1 143 mg/kg).Therefore, 68% Metalaxyl-M·Mancozeb WDG sprayed at the dose of 1 133-1 360 mg/kg could effectively control the damage of D. officinale blight.

Functions of Plant Growth Substances in the Growth of Dendrobium officinale Kimura et Migo

[Objectives] To study the effects of plant growth substance on the growth and development of Dendrobium officinale Kimura et Migo. [Methods] Comparative methods were applied to study the effects of plant growth substance on the growth and development of D. officinale Kimura et Migo. [Results] Plant growth regulators play an important role in the process of tissue culture rapid propagation of D. officinale Kimura et Migo,and the main regulatory pathways are as follows.( i) Plant growth regulators promote the growth and development of D. officinale Kimura et Migo through regulating the changes of its endogenous hormones.( ii) Plant growth regulators affect the accumulation of polysaccharide content in D. officinale Kimura et Migo through regulating the changes in chlorophyll content and enzymes involved in sucrose metabolism.( iii) Plant growth regulators can increase the activity of antioxidant enzymes of D. officinale Kimura et Migo,to increase its stress resistance. [Conclusions]Using molecular biology techniques,combined with the changes of plant hormones in D. officinale Kimura et Migo,and analysis on key enzyme in the synthesis of plant hormones and gene expression,it is feasible to study the rules o changes in the content of active content of D. officinale Kimura et Migo.

Efficacy Evaluation of 16% Validamycin·thifluzamide SC against Selerotium rolfsii in Dendrobium officinale Kimura et Migo

[Objective] The paper was to understand the control efficacy and the optimal dosage of 16% validamycin？thifluzamide SC on controlling Selerotium rolfsii in Dendrobium officinale Kimura et Migo. [Method] The control efficacy of 16% validamycin·thifluzamide SC against S.rolfsii was determined by spraying agents on the whole plant of D.officinale. The significance of differences was analyzed by Duncan＇s new multiple range test （DMRT）. [Result] The control efficacy of 16% validamycin.thifluzamide SC sprayed at the doses of 80-160 mg a.i./kg was 85.33% at 14 d post spraying, higher than that of control agent 4% validamycin AS sprayed at the dose of 80 mg a.i./kg （78.36%）, and equivalent to that of control agent 240 g/L thifluzamide SC sprayed at the dose of 120 mg a.i./kg （90.60%）. [Conclusion] 16% Validamycin·thifluzamide SC sprayed at the doses of 80-160 mg a.i./kg could effectively control the damage of S.rolfsii in D.officinale, and could be popularized in the production.

Correlation Analysis of Quantitative Microscopic Characteristics and Polysaccharide Content of Dendrobium officinale from Guangxi

[Objectives]The paper was to investigate the correlation between the microscopic characteristic constants and the index component polysaccharide content of Dendrobium officinale from Guangxi.[Methods]The paraffin sections of D.officinale were made,and the microscopic characteristic constants of D.officinale stem,including cross section area,vascular bundle area and phloem area,were determined by NIS-Elements D microscopic image processing software.The contents of polysaccharides in D.officinale were determined by UV-Vis spectrophotometry,and the quantitative values of microscopic characteristics of D.officinale with different growth years were analyzed by SPSS 23.0(IBM SPSS Statistics 23.0)data statistical software.The correlation between quantitative microscopic characteristics of D.officinale and its polysaccharide content was analyzed.[Results]According to the correlation between the index component of D.officinale and its quantitative microscopic characteristics,the measurement indexes,the percentage of vascular bundle in the cross section area and the percentage of hard structure in the cross section area,were strongly negatively correlated with polysaccharide content.[Conclusions]The microscopic characteristic constant values of vascular bundles of D.officinale can be used as a method to verify polysaccharide content.

Problems in Storage of Dendrobium officinale and Study on Its Preservation Technology

Due to extensive harvesting,the ecological environment of Dendrobium officinale has been destroyed,and there are very few wild D.officinale today.As the tissue culture technology of D.officinale continues to mature,there are more and more imitation,wild cultivation,greenhouse cultivation and other large-scale production of D.officinale to meet the market demand,but the storage and preservation of fresh products is a big problem.The existing problems in storage of D.officinale and its preservation technology are discussed,so as to provide some reference for the storage and preservation of D.officinale fresh products.

Research Progress of Dendrobium officinale Kimura et Migo

The effects of light on the quality and related genes of Dendrobium officinale were summarized, and the main chemical components and pharmacological effects of D. officinale were analyzed. The biomass accumulation of D. officinale is related to light quality and light intensity. Monochromatic light quality can make polysaccharide accumulate, and mixed light quality can promote cell proliferation and component accumulation of D. officinale. Under different light intensity, the polysaccharide content of D. officinale is different, and it changes with the change of light intensity. When a certain intensity is exceeded, the polysaccharide content decreases with the increase of light intensity. The chemical components of D. officinale are polysaccharides, alkaloids, amino acids, volatile components, etc. The analysis of the growth and components of D. officinale provides reference and basis for improving the quality and chemical components of D. officinale and promotes the research and development of related products of D. officinale.

New Utilization Model of Karst Rocky Desertified Land in Southwest China:Ecological Planting of Dendrobium officinale Kimura et Migo on Bare Rocky Mountains

The Yunnan-Guangxi-Guizhou rocky desertified area in southwestern China is one of the contiguous extremely poor areas identified in the Outline of Poverty Alleviation and Development in the Rural Areas of China(2011-2020).In rocky desertified areas,due to long-term severe soil erosion,large areas of bedrock are exposed or gravels are accumulated.This bare rock and gravel-mulched land has become the main land type in the rocky mountains.Under normal circumstances,it cannot be directly used for agriculture,forestry,animal husbandry,and fishery production,and is classified as land that is difficult to be utilized.In recent years,in Debao County,Baise City,Guangxi Zhuang Autonomous Region,as a deeply impoverished county in the Yunnan-Guangxi-Guizhou rocky desertified area,a new land use model of karst rocky desertified land,that is,planting Dendrobium officinale Kimura et Migo on bare rocky ground,has emerged,and certain poverty alleviation benefits have been achieved initially.In this article,on the basis of analyzing the suitability of planting D.officinale Kimura et Migo on karst rocky desertified land,the practice of planning D.officinale Kimura et Migo on the rocky desertified land in Debao County was elucidated,and then suggestions for reasonable promotion of ecological planting of D.officinale Kimura et Migo in karst rocky desertified areas were put forward.

Research progress on polysaccharide compositions of Dendrobium officinale

More and more attention has been paid to the polysaccharide components of Dendrobium officinale (D. officinale). They are rich in active components and have extensive pharmacological effects. This paper systematically summarized the extraction, separation, purification process, content determination, chemical compositions and pharmacological activities of D. officinale polysaccharides, which provided the basis for the preparation, quality control and further development and utilization of the medicinal materials.

Field Efficacy of Imidacloprid against Dendrobium officinale Aphid

[Objective]The paper was to investigate the control effect of imidacloprid against Dendrobium officinale aphid. [Method]The control effects of 70%imidacloprid WP 4 000,5 000 and 6 000 times dilution and 10% imidacloprid 750 times dilution on D. officinale aphid were studied,respectively. [Result]70%Imidacloprid WP had quick and durable control effects on D. officinale aphid,and the control effect improved with the increasing concentration. The control efficacy at 7 d post secondary administration was up to 95. 4%-97. 8%. [Conclusion]70% Imidacloprid WP is safe to crops within the test dosage,and can be used for prevention and control of D. officinale aphid.

The Genome of Dendrobium officinale Illuminates the Biology of the Important Traditional Chinese Orchid Herb

Dendrobium officinale Kimura et Migo is a traditional Chinese orchid herb that has both ornamental value and a broad range of therapeutic effects. Here, we report the first de novo assembled 1.35 Gb genome se- quences for D. officinale by combining the second-generation Illumina Hiseq 2000 and third-generation PacBio sequencing technologies. We found that orchids have a complete inflorescence gene set and have some specific inflorescence genes. We observed gene expansion in gene families related to fungus symbiosis and drought resistance. We analyzed biosynthesis pathways of medicinal components of D. officinale and found extensive duplication of SPS and SuSy genes, which are related to polysaccharide generation, and that the pathway of D. officinale alkaloid synthesis could be extended to generate 16- epivellosimine. The D. officinale genome assembly demonstrates a new approach to deciphering large complex genomes and, as an important orchid species and a traditional Chinese medicine, the D. officinale genome will facilitate future research on the evolution of orchid plants, as well as the study of medicinal components and potential genetic breeding of the dendrobe.

The chromosome-level reference genome assembly for Dendrobium officinale and its utility of functional genomics research and molecular breeding study

Dendrobium officinale,an important medicinal plant of the genus Dendrobium in Orchidaceae family,has been used as traditional Chinese medicine(TCM)for nearly thousands of years.Here,we report the first chromosome-level reference genome of D.officinale,based on Pac Bio long-reads,Illumina short-reads and HiC data.The high-quality assembled genome is 1.23 Gb long,with contig N50 of 1.44 Mb.A total of 93.53%genome sequences were assembled into 19 pseudochromosomes with a super scaffold N50 of 63.07 Mb.Through comparative genomic analysis,we explored the expanded gene families of D.officinale,and also their impact on environmental adaptation and biosynthesis of secondary metabolites.We further performed detailed transcriptional analysis of D.officinale,and identified the candidate genes involved in the biosynthesis of three main active ingredients,including polysaccharides,alkaloids and flavonoids.In addition,the MODIFYING WALL LIGNIN-1(MWL1)gene,which inferred from Genome-Wide Association Studies(GWAS)based on the resequencing date from D.officinale and five related species and their morphologic features,may contribute to the plant production(yield of stems)of D.officinale.Therefore,the high-quality reference genome reported in this study could benefits functional genomics research and molecular breeding of D.officinale.

Dendrobium officinale Kimura et Migo and American ginseng mixture: A Chinese herbal formulation for gut microbiota modulation

Dendrobium officinale Kimura et Migo(D. officinale) is a famous traditional Chinese medicine(TCM). A mixture of D. officinale and American ginseng has been shown to enhance cell-mediated immunity, humoral immunity, and monocyte/macrophage functions in mice. Here, the effects of a D. officinale and American ginseng mixture on the structure of gut microbial community in dogs were examined using high-throughput 16S rRNA gene amplicon sequencing. The data revealed that while the mixture did not change the diversity of gut microbial community significantly, differences among individuals were significantly reduced. Furthermore, the mixture-responsive operational taxonomic units(OTUs) exhibited a phase-dependent expression pattern. Fifty-five OTUs were found to exhibit a mixture-induced expression pattern, among which one third were short-chain fatty acid(SCFA)-producing genera and the others were probiotic genera included Lactobacillus spp., Sutterella, Alistipes, Anaerovorax, Bilophila, Coprococcus, Gordonibacter, Oscillibacter, among others. By contrast, 36% of the OTUs exhibiting a mixture-repressed expression pattern were disease-associated microorganisms, and six genera, namely Actinomyces, Escherichia/Shigella, Fusobacterium, Slackia, Streptococcus and Solobacterium, were associated with cancer. In addition, five genera were closely associated with diabetes, namely Collinsella,Rothia, Howardella, Slackia and Intestinibacter. Our results indicate that this D. officinale and American ginseng mixture may be used as a prebiotic agent to enhance SCFA-producing genera and prevent gut dysbiosis.

A transcriptome-based analysis reveals functional differences among Dendrobium officinale Kimura&Migo species from different growing regions and with different quality levels

Dendrobium officinale Kimura&Migo(D.officinale)is a common,high medicinal value food and herbal medicine used in the treatment of yin-deficient diseases.Adulterants obtained with similar inexpensive species of Dendrobium plants are available in the market.However,Dendrobium species with different quality levels or from different regions as well as analogous species have a similar appearance,which makes the discrimination of Dendrobium species difficult.Reliable objective methods for authenticating D.officinale are lacking.Here,samples of Dendrobium officinale Kimura&Migo(D.officinale)from two regions with three quality levels and sample of the analogous species Dendrobium compactum Rolfe ex W.Hackett(D.compactum)were subjected to genome-wide biological response fingerprinting(BioReF)in RAW264.7 cells by RNA sequencing.The biological pathways affected by these samples could distinguish D.officinale from D.compactum.The high-quality D.officinale samples from Yunnan exhibited the strongest inhibition of lipopolysaccharide(LPS)induced inflammatory genes and pathways,followed by samples from Myanmar.The medium-and low-quality samples from Yunnan exhibited weaker effects,and D.compactum exerted mild effects.The RNA sequencing results were strengthened by the results from analyses of the expression of the inflammation-related genes Il6,Ccl5,and Cox2 and the production of nitric oxide.Thus,transcriptomic profiling can be used to evaluate the quality of D.officinale and distinguish its origin.