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.

Inoculation Effects of Dendrobium officinale Mycorrhizal Fungi on Their Plantlets

[Objective] This study aimed to explore the inoculation effects of Dendrobium officinale mycorrhizal fungi on their plantlets. [Method] Endophytic strains Tj1, Tj2 and Tj3 were obtained by isolation and purification from mycorrhiza of wild Dendrobium officinale and inoculated on the root system of Dendrobium officinale for inoculation test. [Result] Under tissue-culture conditions, at early stage, Tj1 strain hadn＇t shown promotion effect on Dendrobium officinale, Tj2 strain had shown relatively strong promotion effects, and Tj3 strain had promoted the growth of roots; at late stage, Tj1 strain had shown relatively strong promotion effects, Tj2 strain had shown the best inoculation effects and the strongest promotion effects, while Tj3 strain had caused root and seedling rot problems of the plantlets; under outdoor conditions, after inoculation with Tj2 strain, the number of leaves and lateral buds were increased, the growth of lateral root and the increase of plant height were significant, the leaves of Dendrobium officinale plantlets were large and dark green and an obvious root enlargement phenomenon was observed. [Conclusion] The two inoculation methods both indicate that Tj2 strain has relatively strong promotion effects on the growth of Dendrobium officinale roots and shoots, the increase of plant number and plant height, and the germination of new shoots and roots, which proved the effective establishment of symbiotic relationship between Tj2 strain and Dendrobium officinale. Therefore, T2 strain has practical application values on the successful cultivation of Dendrobium officinale plantlets.

The Interplanting Technology of Pleurotus ostreatus and Dendrobium officinale

In summer, Cha No. 39 of 292 bags and 205 of 244 bags were inter- planted with Dendrobium officinale on cultivation bed, respectively, whose yields and biological efficiencies reached 102.46 kg and 103.98%, and 96.77 kg and 113.32%, accordingly. The seedlings of Dendrobium officinale interplanted with Pleurotus os- treatus grew by 2.78 cm during 10 weeks on cultivation beds, and the seedlings grew by 2.68 cm in the cultivation beds without interplanting arrangement. These demonstrated that the interplanting of Dendrobium officinale and Pleurotus ostreatus has none adverse effects on growth of Dendrobium officinale.

Research Progress of Medicinal Secondary Metabolites and Gene Cloning of Dendrobium officinale

Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.

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.

Evaluation of Heavy Metals and Pesticide Residues in Dendrobium officinale Produced in Guangxi

[Objectives]To establish a method for the determination of heavy metals and pesticide residues in Dendrobium officinale produced in Guangxi and its culture substrate.[Methods]The contents of five heavy metals(Pb,Cd,As,Hg and Cu)were determined by the atomic absorption spectrophotometric(AAS)method;residues of 9 organochlorine pesticides(α-BHC,β-BHC,γ-BHC,δ-BHC,p,p′-DDT,p,p′-DDE,p,p′-DDD,o,p′-DDT and pentachloronitrobenzene)were determined by gas chromatography(GC).[Results]The contents of five heavy metals in the medicinal materials of D.officinale produced in Guangxi were 0.96,0.21,0.021 mg/kg,and not detected,and 0.35 mg/kg.The contents of five heavy metals in the culture substrate were 0.03,0.19,0.21,0.004 and 1.80 mg/kg,respectively;the content of 9 organochlorine pesticide residues in the sample was not detected.[Conclusions]The detection results of heavy metal content and organochlorine pesticide residues in D.officinale produced in Guangxi and its substrate are consistent with the limit requirements of the Chinese Pharmacopoeia.This experiment is expected to provide reliable data reference for ensuring the safety of the drug resources of D.officinale in Guangxi.

The protective effect of Dendrobium officinale polysaccharides on photoaging fibroblasts by scavenging reactive oxygen species and promoting the expression of TGF-β1

The purpose of this study was to explore the protective effect of Dendrobium officinale polysaccharides （DOP） onphotoaging human skin fibroblasts and its specific mechanism of action. The photoaging fibroblast model wasestablished by ultraviolet B （UVB） irradiation. The toxic effects of different concentrations of DOP were detected usingMTT. Senescent cells were detected using a β-galactosidase kit. Reactive oxygen species （ROS） in cells were detectedusing a flow cytometer. The expression of matrix metalloproteinase-1 （MMP-1）, type I collagen C-terminal peptide（CICP）, and transforming growth factor β-1 （TGF-β1） in spent culture medium was detected by ELISA. The resultsshowed that the low concentration of DOP （20, 40, 80 μg/mL） had no cytotoxicity on fibroblasts. After 60 mJ/cm2UVBirradiation, the number of aging β-gal-positive cells increased, the levels of CICP and TGF-β1 in spent culture mediumdecreased, while the levels of MMP-1 and ROS increased. After administration of DOP on photoaging fibroblasts, thenumber of aging β-gal-positive cells decreased, the levels of ROS and MMP-1 decreased, and the levels of TGF-β1 andCICP increased. This experiment suggests that DOP has the effect of removing ROS induced by UVB, regulating thebalance of collagen production and degradation, and protecting photoaging human skin fibroblasts.

Optimization of Rooting Medium for Tissue Culture Seedlings of Dendrobium officinale by Response Surface Methodology

[Objectives] To increase the survival rate of tissue culture seedlings of Dendrobium officinale,and optimize the conditions of rooting medium by the response surface methodology( RSM). [Methods]The effects of 6-BA concentration,NAA concentration,potato amount and the amount of mashed banana on the growth of seedlings were determined by single factor experiment and were analyzed by Box-Behnken design and response surface methodology. [Results]The optimal culture conditions: rooting medium 1/2 MS + 6-BA 0. 24 mg/L + mashed banana 87. 63 g/L + potato 89. 30 g/L + NAA 0. 52 mg/L + sucrose 20. 0 g/L + activated carbon 4. 0 g/L + agar 7. 0 g/L,p H 5. 8,and light intensity 2 000 Lx. [Conclusions]The model established by response surface methodology has a good predictability and could be used to optimize the conditions of tissue culture and rooting medium 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.

Process Optimization of Extraction Technique of Polysaccharide from Leaves of Dendrobium officinale

[Objectives] To optimize the extraction process of polysaccharides from leaves of Dendrobium officinale. [Methods]The polysaccharides from D. officinale were extracted by internal boiling method. The extraction rate of polysaccharides from D. officinale was studied.Based on the single factor experiment,the L_9( 3~3) orthogonal experiment was designed to optimize the extraction process of polysaccharides from D. officinale. [Results]The optimum process for extracting polysaccharides from D. officinale by internal boiling method was as follows:1. 00 g of D. officinale powder was desorbed with 6. 0 mL of 20% ethanol for 20 min,35. 0 mL of 90℃ water was rapidly added,and the extraction time was 5 min. [Conclusions] Under this condition,the extraction rate of polysaccharides from D. officinale was 12. 31%,which was 3. 75 percentage points higher than that of traditional reflux extraction.

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.

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.

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.

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.