ABSTRACT
Objective To compare and analyze the transcriptome of rhizome and leaves of Dioscorea zingiberensis, and excavate the key enzyme genes related to the saponin biosynthetic pathway in D. zingiberensis. Methods The transcriptome of rhizome and leaves of D. zingiberensis were sequenced by Illumina HiSeq2000 high-throughput sequencing technique. According to sequence annotate results to find the differentially expressed genes. Then the key enzyme genes related to the biosynthesis of diosgenin were identified according to the content of saponins in rhizomes and leaves of D. zingiberensis. The expression levels of some candidate genes were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). Results A total of 81 660 Unigenes were gained and 64.33% of them were annotated in NT, NR, Swiss-Prot, KOG, GO, and KEGG databases. Based on their expression and KEGG annotation, totally 227 catalytic enzyme genes of 29 kinds that may participate in D. zingiberensis saponin biosynthetic pathway were screened. The expression pattern of some catalytic enzymes was correlated with the content of saponin. Also were found five D. zingiberensis endophyte genes. Conclusion This experiment obtained candidate key enzyme genes tentatively that involved in the biosynthesis of saponin. Some candidate enzyme genes may participate in the post-modification process of steroidal saponins in D. zingiberensis. In additon, it was found that D. zingiberensis endotrophic bacteria may be involved in the saponin biosynthesis. The results laid a foundation to further elucidate the molecular mechanism of sapogenin synthesis pathway.
ABSTRACT
Objective: To identify WRKY genes from the rhizomes of Dioscorea zingiberensis and analyze the protein characteristics and expression level of these genes. Methods: The transcriptional EST database of the rhizomes of D. zingiberensis was used to search the analogs of AtWRKY genes by BLASTn, the full-length open reading frames (ORF) of DzWRKY and its protein characteristics were studied using bioinformatic method, and the expression levels of DzWRKY genes in rhizomes and leaves were detected from transcriptional data of the rhizomes of D. zingiberensis. Results: Twenty-seven DzWRKY transcription factors family genes with full length ORF were isolated from the rhizomes of D. zingiberensis, and two WRKY domains were confirmed in six WRKY genes. All the DzWRKY proteins were predicted as hydrophilic proteins and nucleoproteins with highly conserved WRKY domains. The 27 DzWRKY and 19 AtWRKY proteins were divided into three groups by phylogenetic analysis. All DzWRKY genes showed higher expression level in the leaves compared to the rhizomes, and highly expressed genes were mainly in groups I and IId. DzWRKY proteins exhibited lower sequence identity. Conclusion: The DzWRKY genes are successfully isolated for the first time, which would provide a reference for the study on the roles of WRKY in development and active components biosynthesis of the rhizomes of D. zingiberensis.
ABSTRACT
OBJECTIVE: To preliminarily establish the technology of fermentation pretreatment followed by two-phase combination acid hydrolysis for the extraction of diosgenin from the dry tubers of Dioscorea zingiberensis C. H. Wright. METHODS: Orthogonal experiment design was used with the yield of diosgenin as the monitoring index to optimize the conditions for fermentation pretreatment and two-phase combination acid hydrolysis. Two types of conventional methods were compared with the newly developed method. RESULTS: Fine powder of the tubers of Dioscorea zingiberensis C. H. Wright was mixed with water and fermented at 30°C for 72 h under stirring at 500 r · min-1, After that, sulfuric acid and methanol were added into the suspension to reach their final concentration of 10%, and petroleum ether was added to form a two-phase immiscible reaction system which was extracted in oil bath at 120°C for 4 h. As a consequence, the average yield of diosgenin achieved 4.28%, significantly higher than the 3.01% and 3.28% of the two conventional methods. CONCLUSION: This newly developed method of fermentation pretreatment followed by two-phase combination acid hydrolysis is proved to be more convenient than the traditional methods with a higher yield.
ABSTRACT
Objective The main economic qualities of natural triploid were evaluated and the result laid a foundation to culture new triploid variety of Dioscorea zingiberensis. Methods The ploidy level of series of D. zingiberensis from some areas of Yunnan Province were identified and the qualities of outputs per plantlet,propagation ratio,and diosgenin content etc. were measured. Results Five natural triploids were found and the qualities of the triploids all displayed diversity,output per plantlet 1 090.00—628.57 g,propagation ratio (ploidy) 72.43—29.43,dry substance ratio 36.95%—24.06%,and diosgenin content 4.40%—1.50%. The outputs of series 1 and series 3 exceed 1 000 g,the diosgenin content of series 2 reached to 4.40%,and series 3 displayed excellent quality of outputs per plantlet,propagation ratio,and diosgenin content. The synthetical index is maximum. Conclusion The series comprising good quality are obtained by the discovery of triploid and evaluation of qualities. The triploid variety is to be created with judgment and selection of progeny.
ABSTRACT
Objective To study the chemical constituents of Dioscorea zingiberensis.Methods The fresh rhizome of D.zingiberensis was extracted three times,3 h once with EtOH-H2O at 80 ℃.The EtOH was evaporated under reduced pressure to give a residue which was suspended in water and then was exacted with petroleum ether,ethyl acetate,and n-butanol fraction.The water fraction was isolated by the reversed-phase ODS column chromatography.The chemical structures were elucidated by means of 1H-NMR,13C-NMR,135DEPT,HMQC,and HMBC spectroscopic analyses.Results Three steroidal saponins were isolated from the fresh rhizome of D.zingiberensis.The compounds were identified as:diosgenin-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅰ),(25R)-26-O-?-D-glucopyranosyl-furost-5-en-3?,22?-diol-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅱ),and(25R)-26-O-?-D-glucopyranosyl-furost-5-en-3?,22?-diol-7-carbonyl-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅲ).Conclusion Compound Ⅲ is a novel compound named as zingiberenin H.
ABSTRACT
Objective To study water-soluble constituents of Dioscorea zingiberensis in order to seek active components. Methods Various chromatographic techniques were used to isolate the constituents. Structures of compounds were elucidated by spectroscopic analyses of 1H-NMR, 13C-NMR, 135DEPT, HMQC, HMBC, and TOCSY. Results One new steroidal saponin was isolated and identified as 26-O-(?-D-glucopyranosyl)-(25R)-furost-5-en-3?, 26-diol-22-OMe-3-O-｛?-L-rhamnopyranosyl-(1→4)-[?-D-glucopyranosl-(1→3)-?-D-glucopyranosl-(1→2)]-?-D-glucopyranoside｝. Conclusion The compound is a novel compound named as zingierenin E. The other two compounds are reported for the first time from D. zingiberensis.
ABSTRACT
Objective By comparing the physiological index and diosgenin content of rhizome between the artificial tetraploid and the wild type diploid of Dioscorea zingiberensis, it is aimed at revealing the potential utility of polyploid breeding in medicinal D. zingiberensis. Methods Three clones of the natural diploid and the artificially induced tetraploid of D. zingiberensis were used as materials in this study. The content of SOD, PPO, and soluble sugar of leaves was determined by spectrophotometry, CAT content was measured by using the method of KMnO_4 titration. And the diosgenin content in rhizome was analyzed by HPLC. Results The tetraploid plants showed higher level of SOD, PPO, CAT, soluble sugar content in leaves, and diosgenin content in rhizome than those of the diploid origins. The diosgenin content in the three clones of tetraploid plants increased to 27% as compared to wild type. Conclusion Artificially induced tetraploid presents high content of diosgenin and great potential in stress resistance, which would be available in good seed breeding for high yield of diosgenin.
ABSTRACT
Objective To study the genetic relationship among Dioscorea zingiberensis with different diosgenin content at DNA level.Methods DNA of all types was amplified by 12 random primers from 40 random primers,the polymorphic bands of RAPD were counted.And the results of types were analysed by POPGENE 1.31(Tool for Population Genetic Analysis 1.31) software.Results A total of 73 bands was obtained,64 of total bands(87.67%) was polymorphic.The results indicated that there were apparent and abundant genetic variations in D.zingiberensis. Conclusion The formation and accumulation of diosgenin could be affected significantly by the genetic basis of D.zingiberensis.
ABSTRACT
Objective To create new tetraploid resource of Dioscorea zingiberensis in the hope of potential breeding materials with high yield and high level of diosgenin. Methods Callus with tiny green buds were directly soaked in colchicine solution or cultured in medium plus with colchicine to prohibit the isolation of chromosomes and induce tetraploid mutants. Results Tetraploids were achieved successfully. The most efficient treatment for inducing tetraploid was soaking the tiny buds in 1?103 mg/L colchicine solution for 24 h, the inducing rate could be up to 35. 2%. The induced tetraploids exhibited notable difference with common wild type (diploids) in morphology, physiology, and microscopic structure. ConclusionThe tetraploid plants show the advantages of gigantic size and vigorous growth. Thus, the established technique system to induce tetraploid from tissue cultured callus would provide an efficient alternative pathway for medicinal plants of Dioscorea L. breeding.
ABSTRACT
ObjectTo study the accumulation and distribution of dioscin in the different part of rhizome of Dioscorea zingiberensis C. H. Wright and the differences of its diosgenin. Methods Histochemistry has been used to study the accumulation and distribution of dioscin in the different part of rhizome and diosgenin was determined by HPLC. Results Dioscin distributed mainly in ground tissue. The amount of dioscin accumulated in the plant part from the region distributed with small vascular bundle (SVB) is the most abundant, and the diosgenin is also the highest in the same region; both of them in the region with no vascular bundle (NVB) are next to that of SVB; the amount of dioscin accumulated in the region distributed with big vascular bundle (BVB) is the lowest, and the diosgenin is the lowest in the same region too. Conclusion Diosgenin in every region of the three of male rhizome is higher than that of female rhizome and diosgenin in every region of the three of monoecism is between those of male and female rhizome.
ABSTRACT
AIM:To study the application of ultrasonic extraction in process of extracting diosgenin from Dioscorea zingiberensis C.H.Wright,and to evaluate its merits.METHODS:A new process of ethanol extracting and hydrochloric acid hydrolysis was provided to extraction diosgenin,and five different extraction methods were studied and compared,and the reaction mechanism was discussed.RESULTS:The ultrasonic extraction method was more efficient.Compared with the traditional water-hydrolylis,extraction rate was 15 percent higher,dosage of hydrochloric acid was registered 90 percent decrease,and the production was purer.CONCLUSION:The grinding pretreatment of Dioscorea zingiberensis is help to enhance the effects of ultrasonic extraction,the method has some advantages in efficiency,energy-saving and low pollution.