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Objective To clone IRX9 gene from Dendrobium huoshanense and perform the bioinformatics and codon bias analysis. Methods The full-length cDNA sequence was cloned from the IRX9 gene based on transcriptome sequencing data of D. huoshanense generated in our previous study by using RT-PCR and further analyzed by bioinformatic methods. Results The cloned IRX9 gene was 1 533 bp, containing a 1 047 bp open reading frame (ORF) which encoded 348 amino acids. The theoretical molecular weight was 39 350 and its isoelectric point was 7.26. IRX9 protein was a hydrophilic protein and had no signal peptide, which had multiple phosphorylation sites and glycosylation sites and might be located in the plasma membrane. Phylogenetic analysis indicated that sequence of amino acids had the highest homology with D. candidum and the conserved region “DXD” of the GT-A superfamily. Codon bias analysis showed that IRX9 gene prefered to use A/T ending codon, with 27 skewed codons. Nicotiana is the most suitable host for exogenous expression of IRX9 gene. Conclusion The IRX9 gene was cloned from Dendrobium huoshanense successfully and it provided a theoretical basis for regulating the growth and development of D. huoshanense and improving the yield and quality of D. huoshanense.
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Objective: To establish a HPLC fingerprint method for assessing the quality of Dendrobium huoshanense, in addition to determining concentrations of syringic acid, rutin, dendrophenol and naringenin in this crude drug. Methods: Agilent C18 column (250 mm × 4.6 mm, 5 μm) was utilized with the mobile phase comprising methanol-0.1% phosphoric acid with the flow rate of 1 mL/min in a gradient elution manner. The detection wavelength was set at 240 nm and the column temperature was 30 ℃. The resultant chromatograms were imported to Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012.1) to obtain retention time and peak area of samples. Similarity for 39 sets of samples were analyzed. The peak area data were processed by SPSS software for cluster analysis and the clustering effect was discussed. Results: The line relationship of this way was good (R > 0.999), with high precision regarding instrument used (RSD < 3.00%), the method showed good reproducibility (RSD < 3.00%), standard recovery was between 99.26% and 100.32% (RSD of 0.35%-1.67%). Different growth period and different planting patterns of D. huoshanense were distinct regarding the concentration of four compounds. Conclusion: The method is useful to evaluate and discriminate D. huoshanense at different growth period for the purpose of providing scientific reference on harvest, development and evaluation of D. huoshanense.
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Objective To study the secondary metabolites of endophytic fungus Fusarium lactis in Dendrobium huoshanense. Methods Compounds were isolated from the EtOAc extract by chromatography technology and their structures were elucidated on the basis of comprehensive spectroscopic analysis. Results Twenty-one compounds were isolated and identified as N-phenethylacetamide (1), 1H-indole-3-carbaldehyde (2), thymidine (3), uracil (4), lignoren (5), uridine (6), hexahydrate (7), adenosine (8), cyclo-glycine-(L)-proline (9), cyclo (D)-proline-(L)-phenylalanine (10), cyclo (L)-proline-(L)-phenylalanine (11), 2-pyrrolidinone (12), N-methyl-2-pyrolidinone (13), cyclo-(L)-4-OH-proline-(L)-phenylalanine (14), brevianamide F (15), 3-methyl- piperazine-2,5-dione (16), 7,8-dimethylbenzo[g]pteridine-2,4 (1H,3H)-dione (17), cyclo (L)-tyrosine-(L)-phenylalanine (18), beer sterols (19), daidzein (20), and erythritol (21). Conclusion All compounds are isolated from Fusarium lactis for the first time.
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Object To study the effects of different growth regulators and organic additives on rooting of regenerated shoots from Dendrobium huoshanense C Z Tang et S J Cheng protocorm like bodies Methods In order to induce root development, the regenerated shoots were cultured on MS medium containing 3% sucrose and 0 8% agar and supplemented with NAA, IBA, BA, KT, banana mud or amino acids at different concentrations During rooting induction, the development of roots was observed and the frequency of rooted shoots was scored Results The frequency and the process of root initiation were significantly influenced by different growth regulators or organic additives For initiation and development of roots, the suitable concentration of NAA, IBA, BA, KT, banana mud or amino acids was 0 2 , 0 1, 0 8, 0 8 mg/L, 20% and 0 1%, respectively Conclusion In all media tested, NAA, IBA or banana mud display greater effects on rooting of regenerated shoots of D huoshanense than others and KT enables shoots to keep green
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Objective To optimize the best subculture cycle through studying the influence of subculture time on tube plantlet growth of Dendrobium huoshanense and the change of medium composition.Methods Height,tiller,fresh weight,and chlorophyll content of the tube plantlet and pH value,water content,and sugar content of the medium were measured after different cycles of the subculture,the cost of culture medium for subculture was calculated as well.Results The height of the tube plantlet increase 282.86%,the tiller increase by 3.5 times,fresh weight reaches its maximum,chlorophyll content of the tube plantlet almost reaches its maximum after 40 d subculture;while water content and sugar content of the medium are decreased to the lowest point,pH value of medium(
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Objective To design a pair of allele-specific diagnostic primer for authenticating Dendrobium huoshanense from other species of Dendrobium Sw.only by PCR.Methods Based on rDNA ITS sequences database of D.huoshanense and other species of Dendrobium Sw.,an allele-specific diagnostic primer pairs was designed.Diagnostic PCRs were performed using the primer with the total DNAs of 19(original) plants from Dendrobium Sw.as templates.The positive was the genuine.Results When the(annealing) temperature was raised to 60 ℃, the template DNA of D.huoshanense could be amplified whereas the diagnostic PCR of the rest species were all negative.Conclusion The diagnostic PCRs can authenticate D.huoshanense from other species of Dendrobium Sw.efficiently.Compared to the authentication method by sequencing DNA fragments and morphological traits etc.,the allele-specific diagnostic PCR is not only simple with time-saving but also practical and effective.