Inhibitory effect of three strains of biocontrol microbes on pathogens causing rhizome rot of Polygonatum cyrtonema / 中国中药杂志

Rhizome rot is one of the main disease in the cultivation of Polygonatum cyrtonema, and it is also a global disease which seriously occurs on the perennial medicinal plants such as Panax notoginseng and P. ginseng. There is no effective control method at present. To identify the effects of three biocontrol microbes(Penicillium oxalicum QZ8, Trichoderma asperellum QZ2, and Brevibacillus amyloliquefaciens WK1) on the pathogens causing rhizome rot of P. cyrtonema, this study verified six suspected pathogens for their pathogenicity on P. cyrtonema. The result showed that Fusarium sp. HJ4, Colletotrichum sp. HJ4-1, and Phomopsis sp. HJ15 were the pathogens of rhizome rot of P. cyrtonema, and it was found for the first time that Phomopsis sp. could cause rhizome rot P. cyrtonema. Furthermore, the inhibitory effects of biocontrol microbes and their secondary metabolites on three pathogens were determined by confrontation culture. The results showed that the three tested biocontrol microbes significantly inhibited the growth of three pathogens. Moreover, the secondary metabolites of T. asperellum QZ2 and B. amyloliquefaciens WK1 showed significant inhibition against the three pathogens(P<0.05), and the effect of B. amyloliquefaciens WK1 sterile filtrate was significantly higher than that of high tempe-rature sterilized filtrate(P<0.05). B. amyloliquefaciens WK1 produced antibacterial metabolites to inhibit the growth of pathogens, and the growth inhibition rate of its sterile filtrate against three pathogens ranged from 87.84% to 93.14%. T. asperellum QZ2 inhibited the growth of pathogens through competition and antagonism, and P. oxalicum QZ8 exerted the inhibitory effect through competition. The research provides new ideas for the prevention and treatment of rhizome rot of P. cyrtonema and provides a basis for the di-sease control in other crops.

Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema / 中国中药杂志

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

Effect of Wine Processing on Odour Formation of Polygonatum cyrtonema Rhizoma by GC-MS / 中国实验方剂学杂志

ObjectiveBy exploring the volatile components， polysaccharide composition and changes in the contents of five carbohydrate components of Polygonatum cyrtonema rhizoma before and after processing， and then the effect of yellow rice wine on the odour formation of P. cyrtonema rhizoma was investigated. MethodThe volatile components of P. cyrtonema rhizoma before and after processing were detected by headspace gas chromatography-mass spectrometry（HS-GC-MS）， and sample data were subjected to principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） using SIMCA 14.1， then the differences between these components of P. cyrtonema rhizoma before and after processing were screened according to the principle of variable importance in the projection（VIP） value>1. Crude carbohydrate components in raw and wine-processed P. cyrtonema rhizoma were subjected to oxime and silylation， the carbohydrate components were analyzed by gas chromatography-mass spectrometry（GC-MS/MS）， and the relative contents of various components were calculated by peak area normalization， then quantitative analysis of four carbohydrate components was also carried out. ResultA total of 23 volatile components were identified from the raw products and the wine-processed products， including 15 components in raw products and 20 components in wine-processed products. Among them， 2-methylbutyraldehyde and isovaleraldehyde had a sweet odor and their contents increased after processing， but the contents of hexanal and caproic acid decreased， new components such as 2-acetylfuran and 5-methylfuranal were produced after processing. PCA and OPLS-DA results showed that there were significant differences between raw products and the wine-processed products， a total of 13 differential compounds were screened out， of which 7 showed an upward trend in relative content and 6 showed a downward trend. A total of 7 carbohydrate components， including 5 monosaccharides and 2 disaccharides， were identified in raw products and the wine-processed products. The results of determination showed that the contents of fructose， glucose， mannose and sucrose in P. cyrtonema rhizoma increased after wine-processing， and their increases were 4.54， 1.51， 2.93， 3.66 times， respectively. ConclusionAfter processing， the increase of aromatic flavor of P. cyrtonema rhizoma may be related to the increase of the contents of aldehydes such as 2-methylbutyraldehyde and isovaleraldehyde， while the decrease of raw flavor may be related to the decrease of the contents of volatile components such as hexanal and hexanoic acid， the increase of sweet flavor may be related to the increase of the contents of monosaccharides and oligosaccharides such as fructose and sucrose.

Origin identification of Polygonatum cyrtonema based on hyperspectral data / 中国中药杂志

In this study, visual-near infrared(VNIR), short-wave infrared(SWIR), and VNIR + SWIR fusion hyperspectral data of Polygonatum cyrtonema from different geographical origins were collected and preprocessed by first derivative(FD), second derivative(SD), Savitzky-Golay smoothing(S-G), standard normalized variate(SNV), multiplicative scatter correction(MSC), FD+S-G, and SD+S-G. Three algorithms, namely random forest(RF), linear support vector classification(LinearSVC), and partial least squares discriminant analysis(PLS-DA), were used to establish the identification models of P. cyrtonema origin from three spatial scales, i.e., province, county, and township, respectively. Successive projection algorithm(SPA) and competitive adaptive reweighted sampling(CARS) were used to screen the characteristic bands, and the P. cyrtonema origin identification models were established according to the selected characteristic bands. The results showed that(1)after FD preprocessing of VNIR+SWIR fusion hyperspectral data, the accuracy of recognition models established using LinearSVC was the highest, reaching 99.97% and 99.82% in the province origin identification model, 100.00% and 99.46% in the county origin identification model, and 99.62% and 98.39% in the township origin identification model. The accuracy of province, county, and township origin identification models reached more than 98.00%.(2)Among the 26 characteristic bands selected by CARS, after FD pretreatment, the accuracy of origin identification models of different spatial scales was the highest using LinearSVC, reaching 98.59% and 97.05% in the province origin identification model, 97.79% and 94.75% in the county origin identification model, and 90.13% and 87.95% in the township origin identification model. The accuracy of identification models of different spatial scales established by 26 characteristic bands reached more than 87.00%. The results show that hyperspectral imaging technology can realize accurate identification of P. cyrtonema origin from different spatial scales.

Comparison of Determination Methods of Saccharides in Polygonatum cyrtonema and Optimization of Its Wine- steaming Technology / 中国药房

OBJECTIVE：To compare the methods for the con tent determination of polysaccharide and reducing sugar in Polygonatum cyrtonema， and to optimize the wine-steaming technology of P. cyrtonema . METHODS ： The contents of polysaccharide in P. cyrtonema were determined by anthrone-sulfuric acid method and phenol-sulfuric acid method. The contents of reducing sugar in P. cyrtonema were determined by anthrone-sulfuric acid method ， phenol-sulfuric acid method and 3， 5-dinitrosalicylic acid （DNS）method，respectively. Taking appearance and property scores of processed products ，the contents of polysaccharide，reducing sugar and total sugar as indicators ，the amount of alcohol added ，steaming time and moistening time as factors，the wine-steaming technology of P. cyrtonema was optimized by Latin square design. The contents of polysaccharide ， reducing sugar and total sugar were compared before and after steaming. RESULTS ：The linear ranges of polysaccharide and reducing sugar obtained by anthrone-sulfuric acid method were both 0.006 6-0.033 mg/mL（R2＝0.999 9）. RSDs of precision ， stability（90 min）and reproducibility tests were all lower than 3％ and 2％，respectively. Average recoveries were 99.75％（RSD＝ 0.48％，n＝6）and 103.40％（RSD＝1.25％，n＝6），respectively. The linear ranges of polysaccharide and reducing sugar obtained by phenol-sulfuric acid method were both 0.002 5-0.025 mg/mL（R2＝0.999 2）. RSDs of precision ，stability （90 min） and reproducibility tests were all lower than 5％ and 6％，respectively. Average recoveries were 112.80％（RSD＝2.36％，n＝6）and 99.20％（RSD＝3.47％，n＝6）. The linear range of reducing sugar obtained by DNS method was 0.01-0.18 mg/mL（R2＝0.999 9）. RSDs of precision ，stability（90 min）and reproducibility tests were all lower than 2％. Average recoveries was 96.95％（RSD＝ 1.19％，n＝6）. The optimal wine-steaming technology of P. cyrtonema included the amount of alcohol added of 20％，moistening time of 2 h and steaming time of 7 h. RSDs of average contents of polysaccharide ，reducing sugar and total sugar in wine-steamed products were all lower than 3％ in 3 times of validation tests （n＝3）. The average contents of polysaccharide ，reducing sugar and total sugar in 4 batches of P. cyrtonema were 16.3％，11.2％ and 27.4％；those of 4 batches of wine-steamed products were 3.4％， 61.0％ and 64.4％，respectively. CONCLUSIONS ：The anthrone- ） sulfuric acid method is the best for the determination of poly- saccharide in P. cyrtonema ；DNS method is the best for the pandongmei1228@126.com determination of reducing sugar in P. cyrtonema. The content ofpolysaccharide in wine-steamed products is decreased signifi- cantly，while the contents of reducing sugar and total sugar are increased significantly.

Analysis of Chemical Constitutions of Polygonatum cyrtonema Dried Rhizomes Before and After Processing with Wine Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

Objective:To quickly analyze and identify the components in raw and wine-processed products of <italic>Polygonatum cyrtonema</italic> （PC） dried rhizomes by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， and then find out the differential components before and after processing. Method:The ACQUITY UPLC BEH C₁₈ column （2.1 mm×100 mm， 1.8 μm） was used with 0.1% formic acid aqueous solution-acetonitrile as the mobile phase for gradient elution at a flow rate of 0.25 mL·min^-1. Electrospray ionization was selected for collection and detection in positive and negative ion modes， and the data were analyzed by PeakView 1.2.0.3. According to the retention time， accurate relative molecular weight and fragmentation ion information provided by MS， and combined with the reference substance and literature， the components were identified. After normalized treatment， the MS data of each sample were analyzed with principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA）， and then the differential components before and after processing were screened according to the principle that variable importance in the projection （VIP） value was >1. Result:A total of 38 components were identified from raw and wine-processed products of PC dried rhizomes， including 15 steroidal saponins， 6 alkaloids， 3 flavonoids， 2 amino acids， 2 organic acids and 10 others. The results of PCA and OPLS-DA showed that there were significant differences in the contents of components in PC dried rhizomes before and after processing， and 16 differential components such as kingianoside Z， disporopsin and linoleic acid were screened. Conclusion:UPLC-Q-TOF-MS technique can accurately and comprehensively identify the components in PC dried rhizomes， these components are mainly steroidal saponins， flavonoids and alkaloids. It takes a great difference in the contents of components before and after processing， and transformation of the same category components is the main reason for the differences of raw and wine-processed products， which will provide reference for the researches on material basis and processing chemistry of PC dried rhizomes.

Effects of different storage conditions on edible quality and antioxidant activity of Polygonatum cyrtonema flowers / 中国中药杂志

The flower of Polygonatum cyrtonema has good edible and medicinal values. In this study, four samples of P. cyrtonema flowers from different regions were selected as test materials. The contents, composition and antioxidant activities of lipid-soluble pigments and alcohol-soluble components were determined under different light and temperature conditions, which help to reveal the discoloration reason and the composition variation patterns during storage. The results showed that light and temperature had different effects on the lipid-soluble pigments and alcohol-soluble components in the dried flowers during storage. After storage for 4 weeks, the contents of total chlorophyll, carotenoids, phenols and saponins in the samples exposed to light respectively decreased by 62.62%, 66.4%, 68.7% and 43.4% compared with those in the dark. The decreases in the contents of chlorophyll a, chlorophyll b, lutein, β-carotene and zeaxanthin were 64.64%, 56.74%, 59.2%, 77.7% and 45.4%, respectively. The contents of pigments and components in the samples stored at-20 ℃ were significantly higher than those at room temperature and 4 ℃, indicating that low temperature was conductive to the stability of lipid-soluble pigments and alcohol-soluble components. The samples stored at low temperature and in the dark had the strongest free radical scavenging activity. The results suggest that P. cyrtonema dried flowers should be stored in low temperature environment without light, which can slow down the degradation of internal components. The study provides a theoretical basis for the production, processing and storage of P. cyrtonema flowers.

Quality evaluation of Polygonatum cyrtonema based on HPLC fingerprint and multi-component quantitative analysis / 中国中药杂志

The medicinal and edible Polygonatum cyrtonema is one of the original species of Polygonati Rhizoma. In this study,HPLC fingerprints for 25 batches of P. cyrtonema from 6 provinces were established. A total of 14 common peaks were identified and the similarities of the fingerprints were in the range of 0. 939-0. 999. In additon, the partial least squares-discriminant analysis(PLSDA) demonstrated that the samples had low discriminability except for JX-1 and most components of them had no significant correlation with environmental factors such as longitude, latitude, and altitude. Thus, chemical composition specificity of P. cyrtonema in natural distribution areas had no obvious regularity and their variation might be induced by the local environment. This conclusion explained the lack of records about Dao-di area of Polygonati Rhizoma. However, JX-1 boasted significantly higher content of 5-hydroxymethylfurfural(HMF) and 4',5,7-trihydroxy-6,8-dimethylhomoisoflavone( HIF), thick and long inflorescence and rhizome, and extremely high yield. Therefore, excellent variety of P. cyrtonema might have great potential to improve the quality and yield of Polygonati Rhizoma. Moreover, three components of HMF, polygonalline A(PA), and HIF were identified in the fingerprint. Among them, HMF has the activities of blood rheology improvement, antioxidation, and anti-myocardial ischemia and PA is an indolizine alkaloid with potential anti-inflammatory activity. HIF, the characteristic homoisoflavone in Polygonatum, has the pharmacological activities of regulating blood glucose and anti-tumor. A quantitative analysis method can provide a theoretical basis for the improvement of the quality evaluation of Polygonati Rhizoma.

Investigation on reproductive characteristics of Polygonatum cyrtonema / 中国中药杂志

The study is aimed to investigate the reproductive biology characteristics of Polygonatum cyrtonema, especially including phenology, flower bud differentiation, flowering timing, floral traits, pollen vigor and stigma receptivity. The results showed that P. cyrtonema forms inflorescence before the leaves spread. In the wild, P. cyrtonema is mainly pollinated by insects such as bumblebees, with a seed setting rate of 65.12%. The seed setting rate of indoor single plant isolation or self-pollination enclosed by parchment paper bag is 0, indicating that it is self-incompatible. In Lin'an city, seedlings begin to emerge from mid-March to early April(the temperature is higher than 7.5 ℃), buds begin to emerge from the end of March to mid-April, and then undergo the full bloom stage from mid-to-late April, and the final flowering stage from the end of April to mid-May. The whole flowering period lasts 36 to 45 days. There are obvious differences in the phenology of different provenances. The flowers come into bloom from the base to the top along the aboveground main axis, which usually contain 4-22 inflorescences with(2-) 4-10(-21) flowers per inflorescence. The flowering pe-riod for a single plant is 26-38 days. The single flower lasts about 20-25 days from budding to opening and withers 2 days after pollination, and then the ovary will gradually expand. If unpollinated, it will continue to bloom for 3-5 days and then wither. Flower development period is significantly related to pollen vigor and stigma remittance. The pollen viability is the highest when the flower is fully opened with anthers gathering on the stigma, and the receptivity is the strongest when the stigma protrudes out of the perianth and secretes mucus. The fruits and seeds ripen in October, and proper shading can ensure the smooth development and maturity of the seeds. This study provides a basis for the hybrid breeding and seed production of P. cyrtonema.

Contrastive analysis of extraction of Polygonatum cyrtonema planting area based on data of "Resource 3" / 中国中药杂志

Polygonatum cyrtonema is a famous bulk medicinal material which is the medicinal and edible homologous. With the implementation of the traditional Chinese medicine industry to promote precise poverty alleviation, the planting area of P. cyrtonema in Jinzhai is becoming larger and larger in recent years. Jinzhai is located in the Dabie Mountainous area, which is the largest mountain area and county in Anhui Province. The cultivation of P. cyrtonema is scattered, and the traditional Chinese medicine resources investigation is not only inefficient and accurate. In this study,the "Resource 3"(ZY-3) remote sensing image was used as the best observation phase,and the method of support vector machine classification was used. The method of parallelepiped, minimum distance, mahalanob is distance, maximum likelihood classification and neural net were used to classify and recognize the P. cyrtonema in the whole region. In order to determine the accuracy and reliability of classification results, the accuracy of six supervised classification results was evaluated by confusion matrix method, and the advantages and disadvantages of six supervised classification methods for extracting P. cyrtonema field planting area were compared and analyzed. The results showed that the method of support vector machine classification was more appropriate than that using other classification methods. It provides a scientific basis for monitoring the planting area of P. cyrtonemain field.

Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema / 中国中药杂志

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

Ecology suitability study of Polygonatum cyrtonema / 中国中药杂志

Using the 260 geographical distribution records of Polygonatum cyrtonema in China, combined with 53 environmental factors, the maximum entropy modeling(MaxEnt) was used to study the ecological factors affecting the suitability distribution of P. cyrtonema. The ArcGIS software was used to predict the potential distribution of the population of P. cyrtonema. The dominant factors were chosen by using the Jackknife test and the Receiver Operating Characteristic(ROC) curve was used to evaluate the simulation. The results showed that high value of area under curve(AUC) denoted good results, which significantly differed from random predictions. Based on the evaluation criterion, the accuracies of the predictions of P. cyrtonema potential distribution in the current periods were excellent. The main environmental factors affecting the suitable growth of P. cyrtonema were the monthly precipitation, the wettest monthly precipitation, the annual average temperature range and the precipitation of November, March, February, April, May and October. There are 9 environmental factors in soil type. The potential fitness of P. cyrtonema in China is high, mainly concentra-ted in Hunan, western Hubei, Guangdong, northeastern Guangxi, southeastern Guizhou, Jiangxi, southwestern Anhui, Fujian, Zhejiang, Shaanxi, southwestern Henan and Chongqing. The growth distribution of the potential distribution area of P. cyrtonema was divided, and the zoning map of the growth suitability of P. cyrtonema was formed. Through the comparative analysis of the potential distribution range based on MaxEnt and the distribution range of literature records, the understanding of the distribution range of P. cyrtonema was expanded.

Main nutrients and functional ingredients in flowers of Polygonatum cyrtonema and P. filipes / 中国中药杂志

To reveal the main nutrients and functional ingredients in the flowers of Polygonatum cyrtonema and P. filipes, the content of the polysaccharides, saponins, amino acids, total phenols, mineral elements, and the DPPH free radical scavenging rates were determined. The flowers and rhizomes of P. cyrtonema were collected from Qingyang in Anhui and Qingyuan in Zhejiang, while the flowers and rhizomes of P. filipes were collected from Longyou in Zhejiang, respectively. The results showed that the polysaccharides content in flowers varied from 60.88 to 97.00 mg·g~(-1), about half of that in rhizomes. The saponins content in flowers varied from 32.55 to 40.93 mg·g~(-1), which was close to the content in rhizomes. The content of total phenols ranged from 40.79 to 50.95 mg·g~(-1), approximately 4.5 times of that in rhizomes. The total amino acids content in flowers was 111.85 to 131.03 mg·g~(-1), about 2.3 times of the content in rhizomes. The essential trace element content was abundant in flowers. The contents of heavy metal elements were all within the limits set by the standards. The DPPH free radical scavenging rate IC_(50) varied from 1.77 to 3.25 mg·mL~(-1), less than one-fifth of that in rhizomes, showing a significant superiority of antioxidant activity compared to rhizomes. The results initially revealed the fundamental of "the flowers exceed the rhizomes in effect", the common saying about the traditional Chinese medicinal herbs over the years, indicating a great developing potential of the flowers. Besides, as polysaccharides, saponins, amino acids, total phenols and other nutritive substances in flowers differ widely among species and provenances, it's important to develop variety breeding to improve the quality and yield of flowers.

Main nutrients and functional ingredients in shoots of Polygonatum cyrtonema / 中国中药杂志

In order to reveal the main nutrients and functional ingredients in the shoots of Polygonatum cyrtonema, the polysaccharides, proteins, amino acids, and total phenols were determined. The tested samples cultured in Ma'nijiaonong, Hengtang village, Tianmushan town, Lin'an, Zhejiang, which were collected from three provenances(Pan'an and Longquan in Zhejiang and Qingyang in Anhui). The results showed that the polysaccharide content of the shoots varied from 2.34% to 12.73%, roughly one-third of rhizomes. The protein content varied from 107.75 to 192.49 mg·g~(-1), nearly 5.50 times more than rhizomes. Moreover, the average of total amino acid content was 193.13-248.74 mg·g~(-1), approximately 4.16 times of rhizomes. And the essential amino acids account for 35.57%-39.44% of the total amino acids content, which was close to the standard of the ideal protein proposed by FAO/WHO(the essential amino acid/total amino acid is about 40%). In addition, the taste amino acids(TaAA) changed from 160.12 to 208.29 mg·g~(-1), revealing the material basis of "shoots were extremely delicious" in Chinese ancient herbal medicine. Additionally, the total phenols varied from 51.21-58.76 mg·g~(-1), about 2.96 times of rhizomes. The DPPH free radical scavenging rate of tested shoots was over 95%, which obviously superior to rhizomes. Therefore, the shoots of P. cyrtonema is a very high-quality vegetable and functional food with good development potential. Furthermore, the main nutrients and functional substances in P. cyrtonema shoots are closely related to the provenances and harvesting seasons. It is important to improve the quality and yield of the shoots by strengthening the variety of breeding and cultivation techniques.

Biosynthetic pathways of Polygonatum cyrtonema polysaccharide and diosgenin based on its transcriptomic data / 中国中药杂志

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.

Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress / 中国中药杂志

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

Analysis on SSR loci in transcriptome and development of molecular markers in Polygonatum cyrtonema / 中草药

Objective: EST-SSR loci were identified and analyzed based on the transcriptome sequencing data in Polygonatum cyrtonema, in order to develop SSR markers suitable for evaluation and application of germplasm resources on P. cyrtonema. Methods: SSR loci were identified and analyzed in all of 126 544 Unigenes by using MISA tool. SSR primers were designed by using Primer 3.0 software and 50 pairs of SSR primers were randomly selected for validation test. Results: A total of 12 317 SSR loci, including the types of 2-6 nucleotide repeats with occurring frequency of 1/5.91 kb, were identified from 9 982 Unigenes in P. cyrtonema transcriptome. The distribution frequency of SSRs was 9.73%. Dinucleotide repeat was the main type, accounting for as much as 53.14% of all SSRs, followed by trinucleotide repeat (33.31%). The validation test on 50 pairs of SSR primers showed that 29 of them (58%) generated fragments with expected molecular size from P. cyrtonema. The capillary electrophoresis using fluorescence-labeled SSR markers showed that nine genotypes were identified at seven SSR loci in P. cyrtonema, which further demonstrated the validity of these SSR primers. Conclusion: There are numerous SSRs in P. cyrtonema transcriptome with high frequency, rich repeat types and relatively high polymorphic, which will provide abundant valuable candidate markers for genetic diversity analysis and genetic mapping construction in P. cyrtonema, also can be used as a technical tool for molecular identification among Polygonatum species and for molecular marker assistant breeding in superior cultivars of P. cyrtonema.

Study on genetic diversity and genetic variation law of Polygonatum cyrtonema / 中草药

Objective: To study the genetic diversity and geographical distribution of Polygonatum cyrtonema resources. Methods: ISSR technique was applied to analyze 118 individuals from 20 P. cyrtonema provenances in six provinces, including Anhui, Jiangxi, Fujian, Hunan, Hubei, and Zhejiang. Results: The results showed that 130 clear bands were amplified by 16 primers with 123 polymorphic bands and the average percentage of polymorphic loci (PPL) was 94.62%, PPL within provenances was 33.85%-60.00%. Nei's genetic diversity index (He) was 0.183 8, Shannon's information index (I) was 0.267 4 and gene differentiation index (Gst) was 0.529 3. There were abundant genetic diversities existing in wild resources of P. cyrtonema. The UPGMA clustering analysis revealed that individuals from the same provenance were almost clustered together firstly, explaining that the genetic differentiation among different provenances was higher than those within provenances. When genetic similarity (GS) was 0.61, 118 germplasms can be divided into four categories, including Wuyi Mountains, Wuling and Luoxiao Mountains, Dabie Mountains, Donggong, and Tianmu Mountains. Conclusion: P. cyrtonema has high genetic diversity, genetic variation was closely related to mountains, and the isolation of plains and water areas between mountains was one of the main causes of genetic differentiation among groups. This study had essentially theoretical value and practical significance for the protection of the germplasm resources and the breeding of the species.

Phylogenetic and mutation point analysis of DNA barcoding sequences in Polygonatum cyrtonema / 中草药

Objective: In this work, phylogenetic analysis was used to compare the ITS2 and psbA-trnH sequences of Polygonatum cyrtonema samples from different geographical sources, so as to explore the genetic diversity and genetic relationship of these resources. Methods: PCR method was used to amplify the regions of ITS and psbA-trnH, and the sequences of ITS2 and psbA-trnH were obtained after the amplified fragment sequences were blasted in NCBI database. The neighbor joining (NJ) and maximum parsimony (MP) methods were used to construct phylogenetic trees and Kimura two-parameter (K2-P) model was used to calculate the genetic distance of different samples. Mega and DNAman softwares were applied for mutiple alignment of ITS2 and psbA-trnH sequences of 25 samples of P. cyrtonema. Results: The lengths of ITS2 and psbA-trnH sequences of Anhui Qingyang and Fujian Taining samples of P. cyrtonema were 224 bp and 620 bp, respectively. The lengths of ITS2 and psbA-trnH of the remaining 24 samples were 225 bp and 621 bp, respectively. ITS2 and psbA-trnH had seven and four mutation points, respectively. These 25 samples were clustered into two groups based on ITS2 sequences. Five samples in Hunan and Guizhou were clustered into one group, while the other 20 samples were clustered into another group. The genetic distance showed that the samples from Huaxi and Jianhe in Guizhou Province and Jianyang in Fujian Province had the largest genetic distance. Phylogenetic tree constructed by psbA-trnH sequences were unable to distinguish 25 samples from different geographical sources. Conclusion: Phylogenetic and mutation analysis will provide the theoretic foundation to utilize the resources of P. cyrtonema, investigate their evolution, and evaluate their genuineness. The results of mutation point will also be used in the identification of related P. cyrtonema resources.

Research progress on Polygonatum cyrtonema processed by nine times steaming and nine times shining / 中草药

The nine times steaming and nine times shining processing method of Polygonatum cyrtonema was originated in ancient China and developed in modern times. The historical evolution of its processing technology includes raw-used, single-steamed, re-steamed, nine times steaming and nine times shining. It can eliminate toxicity, enhance curative effect, change meridians, facilitate storage and eliminate bacteria and so on. After nine times steaming and nine times shining, the chemical composition and drug efficacy of P. cyrtonema changed significantly, and it is widely used in medicine, health care products, common food, cosmetics and other fields. The historical evolution, processing mechanism, composition change, and clinical application of P. cyrtonema are reviewed in this paper, which provide a scientific basis for optimizing the nine times steaming and nine times shining technology of P. cyrtonema, lay the foundation for establishing the quality control standard control system, and provide a reference for the study of observing the ancient processing.