Identification and Molecular Characterization of the Alkaloid Biosynthesis Gene Family in Dendrobium catenatum

As one of the main active components of Dendrobium catenatum, alkaloids have high medicinal value. The physicochemicalproperties, conserved domains and motifs, phylogenetic analysis, and cis-acting elements of the genefamily members in the alkaloid biosynthesis pathway of D. catenatum were analyzed by bioinformatics, and theexpression of the genes in different years and tissues was analyzed by qRT-PCR. There are 16 gene families,including 25 genes, in the D. catenatum alkaloid biosynthesis pathway. The analysis of conserved domains andmotifs showed that the types, quantities, and orders of domains and motifs were similar among members ofthe same family, but there were significant differences among families. Phylogenetic analysis indicated that thegene family members showed some evolutionary conservation. Cis-acting element analysis revealed that therewere a large number of light-responsive elements and MYB (v-myb avian myeloblastosis viral oncogene homolog)-related elements in these genes. qRT-PCR showed that expressions of gene family members involved in alkaloidsynthesis were different in different years and tissues of D. catenatum. This study provides a theoretical basisfor further exploration of the regulatory mechanisms of these genes in the alkaloid biosynthesis of D. catenatum.

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.

Dendrobium species regulate energy homeostasis in neurodegenerative diseases: a review

Brain energy homeostasis is a vital physiological function in maintaining a balanced internal metabolic environment.The impairment of energy homeostasis is recognized as a key pathophysiological basis for brain metabolic disorders and related neurodegenerative diseases.Dendrobium species(‘Shihu’in Chinese)such as D.officinale,D.huoshanense,D.nobile,D.chrysanthum,D.loddigesii,D.moniliforme,D.gratiosissimum,D.candidum and D.caulis are widely used as traditional Chinese medicines/nutraceuticals to control and treat neurodegenerative disorders.These dietary herbs and their derived compounds possess a variety of biological properties,such as suppression of oxidative stress and neuroinflammation,regulation of energy homeostasis mainly through improving brain mitochondria function,insulin signaling and lipid metabolism.Furthermore,they reduce neurotoxicity,alleviate brain injury and neuropathy,and prevent neurodegenerative conditions including stroke,Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease in humans and/or rodents.Moreover,the nutraceuticals from Dendrobium species promote gut health and aid digestion,which appear to be associated with beneficial effects on brain energy homeostasis.Based on the above-mentioned health benefits associated with Dendrobium species,this work reviews their nutraceutical role in neurodegenerative disorders and further suggests the need to elucidate mechanisms of the underlying molecular actions.

Metabolic and transcriptomic analyses elucidate a novel insight into the network for biosynthesis of carbohydrate and secondary metabolites in the stems of a medicinal orchid Dendrobium nobile

Dendrobium nobile is an important medicinal and nutraceutical herb.Although the ingredients of D.nobile have been identified as polysaccharides,alkaloids,amino acids,flavonoids and bibenzyls,our understanding of the metabolic pathways that regulate the synthesis of these compounds is limited.Here,we used transcriptomic and metabolic analyses to elucidate the genes and metabolites involved in the biosynthesis of carbohydrate and several secondary metabolites in the stems of D.nobile.A total of 1005 metabolites and 31,745 genes were detected in the stems of D.nobile.The majority of these metabolites and genes were involved in the metabolism of carbohydrates(fructose,mannose,glucose,xylulose and starch),while some were involved in the metabolism of secondary metabolites(alkaloids,β-tyrosine,ferulic acid,4-hydroxybenzoate and chrysin).Our predicted regulatory network indicated that five genes(AROG,PYK,DXS,ACEE and HMGCR) might play vital roles in the transition from carbohydrate to alkaloid synthesis.Correlation analysis identified that six genes(ALDO,PMM,BGLX,EGLC,XYLB and GLGA) were involved in carbohydrate metabolism,and two genes(ADT and CYP73A) were involved in secondary metabolite biosynthesis.Our analyses also indicated that phosphoenol-pyruvate(PEP) was a crucial bridge that connected carbohydrate to alkaloid biosynthesis.The regulatory network between carbohydrate and secondary metabolite biosynthesis established will provide important insights into the regulation of metabolites and biological systems in Dendrobium species.

Pollination Biology of the Endangered Herbal Medicines Dendrobium chrysotoxum(Orchidaceae)

Pollination biology studies of the endangered herbal medicines Dendrobium chrysotoxum were conducted in natural pollination conditions using flower observation,pollinator observation and artificial pollination experiments.Populations of D.chrysotoxum with fragrance and nectar were pollinated by Ctenoplectra davidi Valhalla(Hymenoptera:Apidae)species.The floral structure of D.chrysotoxum adapted precisely to its pollinators.Flowers had a low capsule setting(0.17%)under natural conditions.However,compared to open pollination,artificial pollination experiments showed a significant increase in capsule setting,and D.chrysotoxum was cross-compatible and self-compatible,but there was pollinator limitation also.This study will provide important information for the preservation of this endangered species.

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.

Anti-oxidation Activity in Vitro of Polysaccharides of Dendrobium Huoshanense and Dendrobium moniliforme

[ Objective ] The aim was to study the effects of polysaccharides of Dendrobium Huoshanense and Dendrobium moniliforme on oxygen free radicals and lipid peroxidation in vitro. [ Method] The scavenging action on .OH of polysaccharides, the inhibition action on O2^- of polysaccharides, MDA production of lipid peroxidation were measured by Fenton reaction, self oxidation system of C6H3（ OH）3 and TBA method respectively. [ Result] The half scavenging concentration on ·OH of potysaccharides of Dendrobium Huoshanense and Dendrobium moniliforme was 6.79 and 6.75 mg/ml respectively. As to their half inhibition concentration on O2 , the data was 3, 04 and 3.44 mg/ml respectively. Two kinds of Dendrobium polysaccharides bothhad inhibitory actions on lipid peroxidation of liver homogenate induced by auto-oxidation and inducedoxidation, and they could alleviate oxidation damages of mice liver mitochondria induced by Vc-Fe2＋ system. [Conclusion] Two kinds of Dendrobium polysacchaddes both had remarkable anti-oxidation activity in vitro.

Comparison of Stomatal Characteristics between Dendrobium loddigesii and Dendrobium candidum

[Objective]This study was to reveal the physiological and ecological mechanism of growth and development of test-tube seedlings.[Methods]Leaf stomata of test-tube seedlings of D.loddigesii and D.candidum at different growth and development stages were observed under scanning electron microscope,and their fluorescence induction response curve was assayed by using modulate fluorometer.[Results] At each growth and development stage,D.loddigesii test-tube seedling has a higher leaf stomatal density over D.candidum,while a lower stomata opening rate.Along with the growth,the size of stomata of both D.loddigesii and D.candidum did not change obviously,but their stoma density increased by 83% and 17.6% respectively.Leaf stomata opening rate reached the highest at the age of 240-day-old.Under the conditions of three settled light intensities,stomatal opening degree of D.loddigesii was increased by the intensity of illumination,the maximum aperture occurred at the light intensity of 54 μmol/m2·s.Given the same culture condition,both the maximum electron transport rate（14 μmol/m2·s）and light saturation point（318 μmol/m2·s）of D.loddigesii test-tube seedling are lower than that of D.candidum（20 μmol/m2·s,483 μmol/m2·s）.Moreover,the potential photosynthetic capacity of D.loddigesii is relatively poor.[Conclusion]The photosynthetic rate varies among different species of Dendrobium,which could be attributed to their different stomatal characteristics.Usually,the light intensity of tissue culture chamber is set up as 27 μmol/（m2·s）,where both D.loddigesii and D.candidum do not acquire an optimal state for their leaf stomatal opening.Considering the characteristics to select suitable culture condition,the stomatal aperture and photosynthetic rate could be enhanced,which can promote the growth and development of test-tube seedlings.

春石斛品种Dendrobium Second Love‘Tokimeki’与金钗石斛多糖免疫活性的比较研究

为比较春石斛栽培品种Dendrobium Second Love‘Tokimeki’与金钗石斛的粗多糖、精多糖(除蛋白多糖)和多糖水洗组分(过层析柱分离的水洗组分)的免疫活性差异,分别对昆明小鼠灌胃上述6种不同纯度的多糖,观察其对小鼠脏体指数、迟发性变态反应、抗体积数和小鼠腹腔巨噬细胞吞噬百分比与吞噬指数等免疫学指标的影响。结果表明:2种石斛的免疫活性随多糖纯度的提高而增强,纯度最高的多糖水洗组分免疫活性最强,且各指标均显著高于对照。D.Second Love‘Tokimeki’和金钗石斛同等纯度的多糖在胸腺指数、脾脏指数、小鼠耳肿胀率及小鼠抗体积数上均无显著差异,而D.Second Love‘Tokimeki’的3种不同纯度多糖的吞噬指数均达到了与金钗石斛多糖水洗组分无显著差异的水平。综合来看,栽培品种D.Second Love‘Tokimeki’的多糖免疫活性与金钗石斛多糖相近,具有良好的药用开发价值。

铁皮石斛(Dendrobium catenatum)快速繁殖的研究进展——兼论其学名与中名的正误

铁皮石斛是中国长期使用的名贵药用植物。近20年来,该药用植物的研究和生产已取得了显著的进展。本文系根据文献信息和在中国兰科植物种质资源保护中心的多年实验,对铁皮石斛快速繁殖研究的进展做一简要总结和评价。鉴于该种植物的学名和中名在不少文献中存在使用混乱,本文将在命名上和分类上予以澄清。笔者认为铁皮石斛的学名应当用其最早的合法名称Dendrobium catenatum Lindl.,而中名则应当用其最为常用的"铁皮石斛"。

Antioxidant phenanthrenes and lignans from Dendrobium nobile

To study the antioxidant constituents from the stems of Dendrobium nobile, and to discuss theft structure-activity relationship. Compounds were isolated from a 60% ethanolic extract by various chromatographic techniques and were identified by spectral analysis. The antioxidant activities of compounds were evaluated by DPPH free radical scavenging assay. Five phenanthrenes and four lignans were obtained from the active fractions ofD. nobile. Their structures were identified as fimbriatone （1）, confusarin （2）, flavanthrinin （3）, 2,5-dihydroxy-4,9-dimethoxyphenanthrene （4）, 3,7-dihydroxy-2,4-dimethoxyphenanthrene （5）, syringaresinol （6）, pinoresinol （7）, medioresinol （8） and lirioresinol-A （9）, respectively. Compounds 2 and 6 exhibited more potent DPPH scavenging activities than vitamin C. All the above compounds were reported from this plant for the first time, and compounds 3, 4 and 9 were reported for the first time from the genus of Dendrobiurn. For all phenanthrenes and lignans, an electron-donating methoxyl group in the ortho position to the phenolic hydroxyl group exhibits enhanced antioxidant activities.

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.

Effects of Hormone Factors on the in vitro Culture Flowering Induction of Dendrobium officinate Kimura et Migo

[Objective]The aim was to select the suitable hormone factors for flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.[Method]The test-tube plantlets from the stems of Dendrobium officinate Kimura et Migo were used as the experimental materials and MS medium as the basic medium.Comparative tests have been done between single-factor hormone treatments（different concentrations of PP333 or TDZ） and multi-factor hormone treatments（different combinations of PP333,TDZ,6-BA and NAA） to research the effects of hormone factors on the flowering induction of the plantlets.[Result]Among the single-factor hormone treatments,the suitable concentration and the rate of flower buds formation of PP333 treatment were 0.2 mg/L and 8.5%,the that of TDZ treatment were 0.06 mg/L and 15.5%;the effects of multi-factor hormone treatments on the flowering induction were ordered as follow：（PP333 ＋ 6-BA ＋ NAA ＋ TDZ）〉 （PP333 ＋ 6-BA ＋ NAA）〉 （PP333 ＋ 6-BA） and（PP333 ＋ NAA） ;the most suitable treatment was PP333 0.3 mg/L ＋ 6-BA 0.5 mg/L ＋ NAA 0.5 mg/L ＋ TDZ 0.06 mg/L,the rate of flower bud formation and the rate of the blossomed flower were respectly reached to 80.4% and 90.3%.[Conclusion]PP333 and TDZ showed the important effect on the flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.The effect of TDZ was better than that of PP333.It is much more conducive to the flower bud formation,when using appropriate concentration of TDZ combined with other hormones properly.

Determination of Heavy Metals in Dendrobium candidium Wall.ex Lindl.by ICP-MS

[Objective] This research aimed to establish an inductively coupled plasma mass spectrometry （ICP-MS） method for determination of five kinds of heavy metals （Pb, Cd, Cu, As and Hg） in Dendrobium candidium Wall. ex Lindl. [Method] The samples were digested in HNO3-H202 acids system by closed vessel microwave di- gestion. At the same time, internal standard was added to avoid the matrix effect. [Result] The five kinds of heavy meals all showed good linear relationships, and the correlation coefficients ranged from 0.998 2 to 0.999 9. The detection limits of the five kinds of heavy metals were in the range of 0.008-0.750 μg/L, while the stan- dard recovery rates were in the range of 90.4%-96.9%. The relative standard devi- ations ranged from 1.6% to 8.2%. The heavy metals in the Ginseng and Astragalus national standard samples were determined by the established ICP-MS method, and the results were in conformity with the standard values. [Conclusion] The established method has simple and convenient operation and accurate and reliable results, and it meets the requirements by determination of heavy metals in Dendrobium candididium Wall. ex Lindl.

Optimization of Culture Conditions for Chromium-enriched Protocorm of Dendrobium candidum

[Objective] To optimize the techniques for culturing Cr-enriched Dendrobi-um candidum protocorm. [Method] By adopting Plackett-Burman test, the effects of time, temperature, Cr-content in medium, NAA content, KT content and light intensi-ty on Cr-enriched protocorm were researched. Three factors of time, Cr-content in medium and light intensity had statistical y significant effects; then steepest ascent procedures were applied to define optimal response region of these three factors; fi-nal y the optimal factors were determined by RSM analysis. [Result] The results showed that the optimal conditions for Cr-enrichment were chromium concentration at 0.37 mg/L, culture time of 59 d and il umination intensity of 1 822.22 lx. The predicted value of Cr-content in protocorm was 5.08 mg/kg. [Conclusion] RSM can optimize the techniques for culturing Cr-enriched protocorm of Dendrobium can-didum, provide the optimum process parameters and lay the foundation for further research.

Analysis of Genetic Diversity of Dendrobium by RSAP Marker

[Objective] This study aimed to analyse the genetic diversity of and genet- ic relationship between 33 Dendrobium species by using RSAP （Restriction Site Amplification Polymorphism） marker technique. [Method] A PCR-reaction system was established, by which 33 pairs of primers screened from 45 pairs were used to con- duct the PCR amplification. [Results] The 33 pairs of primers produced 2 047 ampli- fied fragments, of which 2 044 were polymorphic, with a polymorphism rate of 99.8%, and each pair of primers produced 61.9 polymorphic fragments on average. The genetic similarity coefficients between species ranged from 0.081 to 0.442, sug- gesting that the experimental materials used in this study possess a wide genetic background. According to the result of RSAP, the 33 Dendrobium species were di- vided into 6 groups by the cluster analysis （UPGMA）. The result showed that the genetic relationship between D. acinaciforme and D. guangxiense was the closest, and that between D. sinense and either D. lituiflorum or D. hancockii was the fur- thest. [Conclusion] The result suggested that Dendrobium germplasms have a great genetic diversity. This study provides a molecular basis for the classification and breeding of Dendrobium plants, as well as the selection of hybridization parents in the future.

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.

Effects of Six Kinds of Fungal Elicitors on Growth of Dendrobium hybrida cultivar ‘088' Tissue Culture Seedlings

To optimize the technique of rapid propagation of Dendrobium hybrida seedlings and to explore a hormone-free tissue culture method for D.hybrida,six kinds of mycorrhizal fungi which were isolated from the wild orchids were made into fungal elicitors.These fungal elicitors were added into the DE medium with concentrations of 40,60 and 80 ml/L,respectively.After a 90-d culturing,the effects of fungal elicitors on the growth of D.hybrida cultivar ‘088＇ tissue culture seedlings were studied.The results showed that treatment 13（T13） extremely significantly increased the fresh weight,but other treatment groups had no significant effects.In addition,T1,T5,T9,T11 and T13 extremely significantly influenced the contents of chlorophyll a and chlorophyll a ＋ b.However,T1 and T11 had extremely significantly effect on the content of chlorophyll b.Combining the effects on fresh weight and chlorophyll content,it could be concluded T13（40 ml/L of Y05） has promoting effects on the growth of D.hybrida tissue culture seedlings.

Evaluation of Cold Tolerance of Different Dendrobium Species in North China

T o investigate cold tolerance of different cultivars and species in Dendrobium Sw. in North China, 8 strains of Dendrobium officinal and other 5 Dendrobium species were used to compare their cold-hardiness index in this study. The content of electrolyte leakage was increasing in Dendrobium Sw. as the temperature decreasing, while the content of peroxidase(POD) activity and the soluble sugar showed a rising trend before a downward trend. Besides, external morphology was another evaluation standard of cold tolerance. Thus, there was a significant difference between Dendrobium officinal and other Dendrobium species in the cold tolerance, and their cold tolerance capacity was as follows: s1 > s100 >s13 > 1301 > s300 > Dendrobium nobile > s126 > s145 > s95 > Dendrobium lindleyi > Dendrobium chrysotoxum > Dendrobium thyrsiflorum > Dendrobium lohohense.

A New Fluorenone Derivative from Dendrobium Chrysotoxum

A new fluorenone derivative, named chrysotoxone(3) was isolated from the ethanolic extract of the stems of Dendrobium chrysotoxum (Orchidaceae), along with three known compounds. The structure of the new fluorenone was elucidated as 2,5,8trihydroxy1,4dimethoxy9fluorenone.