Genetic Diversity Analysis and Molecular ID Card Construction of Pinellia ternata Based on Phenotypic Traits and SSR Markers / 中国实验方剂学杂志

ObjectiveTo study the genetic diversity and genetic relationship of Pinellia ternata germplasm resources and provide the basis for germplasm identification， variety breeding， and resource conservation. MethodIn this study， 27 P. ternata were used as experimental materials to determine seven phenotypic characters， such as plant height， leaf length， and leaf width. Simple sequence repeats （SSR） primers were designed based on P. ternata transcriptome data， and polymerase chain reaction （PCR） amplification was performed on 27 P. ternata samples. The genetic diversity of P. ternata germplasm was analyzed by POPGENE32， PowerMarker V3.25， and NTSYS-PC 2.10e software. ResultA total of 10 pairs of highly polymorphic primers （PIC>0.5） and four pairs of moderately polymorphic primers （0.25<PIC<0.5） were selected. The average number of alleles detected was 3.928 6， and the average Nei's diversity index （H） and Shannon's index （I） were 0.557 8 and 1.002 9， respectively， indicating a high level of genetic diversity. Cluster analysis divided the Pinellia ternata into seven categories， and P. ternata in the same province were in the same categories. The SSR molecular ID cards of 27 P. ternata germplasm were constructed with 14 pairs of primers， and the rapid identification of P. ternata in each region was realized. ConclusionThe results of this study can lay a foundation for the genetic diversity and population structure of P. ternata and provide a scientific basis for the identification of P. ternata germplasm resources， map construction， and molecular-assisted breeding.

Research on the mechanism of Atractylodes-Pinellia-Poria in the treatment of pancreatic cancer based on network pharmacology and molecular docking / 药学实践杂志

Objective To predict the target of Atractylodes-Panxia-Poria in the treatment of pancreatic cancer, and to explore its potential molecular mechanism by using network pharmacology and molecular docking. Methods Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), PharmMapper, OMIM, GeneCards, STRING, DAVID and Cytoscape software were used to construct a series of network diagrams. The core targets and conduct GO analysis and KEGG pathway enrichment analyses of the target genes were selected. Finally, molecular docking verification of key active ingredients and potential targets were conducted by AutoDock software. Results A total of 35 active ingredients, 190 related targets, 1566 targets of pancreatic cancer and 76 intersection targets were screened for the treatment of pancreatic cancer with Atractylodes-Panxia-Poria. These intersection targets were mainly involved in several biological processes, including positive regulation of gene expression, cytokine-mediated signaling pathway and regulation of apoptotic process, etc, which were also related to pathways in cancer, hepatitis B, colorectal cancer, chemical carcinogenesis-receptor activation, pancreatic cancer, and MAPK signaling pathway, etc. Molecular docking results showed that the main active components of Atractylodes-Panxia-Poria had certain affinity with the potential targets of pancreatic cancer. Conclusion Atractylodes-Panxia-Poria mainly exerts a therapeutic effect on pancreatic cancer through multi-component, multi-target and multi-pathway, which provides a certain theoretical basis for the clinical application of Atractylodes -Panxia-Poria in the treatment of pancreatic cancer.

Analysis of active components in aboveground and underground parts of Pinellia ternata and comparison of antitussive effect / 中国药房

OBJECTIVE To study active components and antitussive effect of aboveground part of Pinellia ternata （non- medicinal stems and leaves）， and compare them with the underground part of P. ternata （medicinal underground tubers）， providing scientific basis for the comprehensive utilization and product development of P. ternata. METHODS TLC， GC， HPLC and UPLC- MS/MS were used for qualitative and quantitative analysis of amino acids， volatile oil， total flavonoids and succinic acid from the aboveground and underground parts of P. ternata. The antitussive effects of the aboveground and underground parts of P. ternata were compared and studied through cough inducing experiment with concentrated ammonia water. RESULTS Results of TLC showed that at the corresponding positions on the chromatograms of the reference substances of P. ternata， and arginine， alanine， valine， leucine and rutin control， the aboveground and underground parts of P. ternata showed spots of the same color. Results of GC showed that the similarity among characteristic chromatograms of volatile oil from aboveground and underground parts of P. ternata was 0.767； results of HPLC and UPLC-MS/MS showed that compared with underground parts of P. ternata， the contents of succinic acid， quercetin， kaempferol and isorhamnetin increased by 0.15%， 0.15%， 0.09% and 0.03%， and aspartate content decreased by 2.5 mg/g. Pharmacodynamics results showed that compared with model control group， the cough incubation period of rats was prolonged significantly in administration groups （P＜0.05）， and the cough frequency within 3 min was significantly decreased （P＜0.05）； there was no statistical significance in the cough frequency within 3 min among administration groups （P＞0.05）. CONCLUSIONS The composition of amino acids， volatile oils and total flavonoids in aboveground part of P. ternata are similar to underground part of P. ternata， while the content of aspartic acid is lower than that in underground part. The aboveground part of P. ternata can prolong the cough incubation period of rats and reduce the number of coughs， which has a certain antitussive effect， but the effect is slightly weaker than that of the underground part.

Comprehensive evaluation of Pinellia ternata germplasm resources based on phenotypic trait classification / 中国中药杂志

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 μg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.

Identification and Expression Analysis of AP2/ERF Family in Stress Responses of Pinellia ternata Based on Transcriptome Data / 中国实验方剂学杂志

ObjectiveTo identify the functions of the AP2/ERF family members in Pinellia ternata and promote the genetic improvement of P. ternata varieties. MethodWe identified and conducted a systematic bioinformatics analysis of the AP2/ERF family member genes in P. ternata based on the three generations of transcriptome data. Real-time polymerase Chain reaction　（Real-time） PCR was employed to determine the expression pattern of AP2/ERF genes in different tissues and under different stress conditions. ResultA total of eight full-length AP2/ERF family members were identified from the transcriptome data， which were classified into three sub-gene families： AP2， ERF， and DREB. The deduced AP2/ERF proteins in P. ternata had the length of 251-512 aa， the theoretical pI of 5.29-11.72， the instability index of 45.90-82.41， subcellular localization in the nucleus， and conserved domains and motifs. AP2/ERF genes were expressed in different tissues of P. ternata， with high expression levels in the leaf. The stress response experiments showed that PtERF1 mainly responded to NaCl stress. The expression of PtERF2 and PtERF4 was significantly up-regulated under low temperature and polyethylene glycol （PEG）-simulated stress. PtERF3 responded to both low temperature and NaCl stress. The expression of PtERF5 was induced by high temperature， low temperature， NaCl and PEG stress. The expression of PtERF7 was up-regulated under high temperature， while that of PtERF8 under low temperature. ConclusionThe AP2/ERF genes in P. ternata can respond to stress and have the potential functions of regulating photosynthesis and improving root stress resistance.

Physiological and transcriptional responses to heat stress in a typical phenotype of Pinellia ternata / 中国天然药物

Pinellia ternata is an important medicinal plant, and its growth and development are easily threatened by high temperature. In this study, comprehensive research on physiological, cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P. ternata. First, P. ternata exhibited tolerance to the increased temperature, which was supported by normal growing leaves, as well as decreased and sustained photosynthetic parameters. Severe stress aggravated the damages, and P. ternata displayed an obvious leaf senescence phenotype, with significantly increased SOD and POD activities (46% and 213%). In addition, mesophyll cells were seriously damaged, chloroplast thylakoid was fuzzy, grana lamellae and stroma lamellae were obviously broken, and grana thylakoids were stacked, resulting in a dramatically declined photosynthetic rate (74.6%). Moreover, a total of 16 808 genes were significantly differential expressed during this process, most of which were involved in photosynthesis, transmembrane transporter activity and plastid metabolism. The number of differentially expressed transcription factors in MYB and bHLH families was the largest, indicating that these genes might participate in heat stress response in P. ternata. These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P. ternata.

Identification, biological characteristics, and control of pathogen causing Pinellia ternata soft rot in Hubei province / 中国中药杂志

This study was designed to identify the pathogen causing soft rot of Pinellia ternata in Qianjiang of Hubei province and screen out the effective bactericides, so as to provide a theoretical basis for the control of soft rot of P. ternata. In this study, the pathogen was identified based on molecular biology and physiological biochemistry, followed by the detection of pathogenicity and pathogenicity spectrum via plant tissue inoculation in vitro and the indoor toxicity determination using the inhibition zone method to screen out bactericide with good antibacterial effects. The control effect of the bactericide against P. ternata soft rot was verified by the leave and tuber inoculation in vitro. The phylogenetic tree was constructed based on the 16 S rDNA, dnaX gene, and recA gene sequences, respectively, and the result showed that the pathogen belonged to the same branch as the type strain Dickeya fangzhongdai JS5. The physiological and biochemical tests showed that the pathogen was identical to D. fangzhongdai, which proved that the pathogen was D. fangzhongdai. The pathogenicity test indicated that the pathogen could obviously infect leaves at 24 h and tubers in 3 d. As revealed by the indoor toxicity test, 0.3% tetramycin, 5% allicin, and 80% ethylicin had good antibacterial activities, with EC_(50) values all less than 50 mg·L~(-1). Tests in tissues in vitro showed that 5% allicin exhibited the best control effect, followed by 0.3% tetramycin and 10% zhongshengmycin oligosaccharide, and their preventive effects were better than curative effects. Therefore, 5% allicin can be used as the preferred agent for the control of P. ternata soft rot, and 0.3% tetramycin and 10% zhongshengmycin oligosaccharide as the alternatives. This study has provided a certain theoretical basis for the control of P. ternata soft rot.

Method Establishment for Content Determination of 8 Nucleosides in Wild and Cultivated Pinellia ternata and the Difference Analysis / 中国药房

OBJECTIVE：To establish the metho d for the content determination of 8 nucleosides in wild and cultivated Pinellia ternate，and to conduct the difference analysis. METHODS ：The contents of uracil ，uridine，inosine，xanthine，adenine， guanosine，β-thymidine and adenosine in 20 batches of P. ternate （wild product YS 1-YS8，cultivated product ZP 1-ZP12）were determined by HPLC method. Based on the contents of the above 8 nucleosides，cluster analysis ，principal component analysis （PCA），partial least squares analysis （OPLS-DA）were used to comprehensively evaluate the wild and cultivated P. ternata . RESULTS：The contents of uracil ，uridine，inosine，xanthine，adenine，guanosine，β-thymidine and adenosine in 20 batches of P. ternate were 0.02-0.24，0.01-0.24，0.06-0.37，0.02-0.14，0.04-0.22，0.14-0.42，0.01-0.09，0-0.32 mg/g，respectively. Cluster analysis，PCA and OPLS-DA showed that 8 batches of wild P. ternate （YS1-YS8）were clustered into one category ，and 12 batches of cultivated P. ternate （ZP1-ZP12）were clustered into one category. Main characteristic markers of wild P. ternate were guanosine，uridine，adenosine and adenine ，while the main characteristic markers of cultivated P. ternate were urinine ，xanthine， inosine，and β-thymidine. CONCLUSIONS：The method for the content determination of 8 nucleosides in P. ternate is established. Nucleosides as quality markers can effectively distinguish wild and cultivated P. ternata ，and the quality of the wild P. ternate was better than that of cultivated P. ternate .

Secondary metabolites of the endophytic fungus Myrothecium sp. from Pinellia ternata / 中国药科大学学报

@#Using a series of purification methods including silica gel， Sephadex LH-20 and preparative high performance liquid chromatography， secondary metabolites of Myrothecium sp. were purified from the ethyl acetate extract of the solid fermentation product. Based on structure characterization and investigation on the physical and chemical properties a， twelve monomeric compounds were identified as 3''-hydroxyverrucarin A (1), verrucarin A (2), verrucarin L acetate (3), verrucarin J (4), verrucarin K (5), roridin A (6), roridin D (7), roridin H (8), roridin J (9), verrol 4-acetate (10), (3S, 3aS, 6α, 6aR)-dihydrosporothrioride (11) and 4,6-dihydroxy-1(3H)-isobenzofuranone (12). Compounds 1, 5 and 9 -12 were isolated from Myrothecium sp. for the first time.Compounds 2, 3, 4, 7 and 8 exhibited strong inhibitory effects on Candida albicans, with a minimal inhibitory concentration （MIC50） of 0.318, 0.218, 0.047, 0.569 and 0.558 μg/mL, respectively.

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.

Research on rules of syndrome differentiation and medication of coronary heart disease based on data mining / 中草药

Objective: To analyze the rules of coronary heart disease syndrome differentiation based on the data mining technology. Methods: First of all, the famous medical records and prescriptions were collected and normalized to construct the database of coronary heart disease prescriptions. Then, IBM SPSS Statistics 20.0, a statistical software, was used to conduct statistics on the distribution of drug frequency, disease site syndrome frequency and disease syndrome frequency. Finally, the Apriori algorithm was applied to carry out data mining and analyze the underlying law of drug compatibility. Results: A total of 145 prescriptions were selected and analyzed, including 216 Chinese medicines, eight syndromes of disease location and 12 syndromes of disease nature. Forty-six herbs with higher frequency were found, and the top five TCM herbs were Salvia miltiorrhiza, Glycyrrhiza uralensis, Trichosanthes kirilowii, Pinellia ternata and Ligusticum chuanxiong. The main syndromes of disease location were heart, kidney, spleen and liver, while the main syndromes of disease nature were blood stasis, qi deficiency, phlegm, deficiency of yang, qi stagnation, and deficiency of yin. When the minimum support was 15% and the minimum confidence coefficient was 70%, the association rule analysis results showed that a total of 15 association rules for two-herb-combinations and 26 association rules for the trigeminy medications were obtained. The most frequency two-herb-combinations were Allium macrostemon→T. kirilowii", "Schisandra chinensis→Ophiopogon japonicus", "Curcumae Radix→S. miltiorrhiza. The most frequently used trigeminy medications were A. macrostemon + P. ternata→T. kirilowii, S. miltiorrhiza + A. macrostemon→T. kirilowii and A. macrostemon + G. uralensis→T. kirilowii. Conclusion: The syndrome differentiation and medication of TCM in the treatment of coronary heart disease mainly focus on invigorating the circulation of blood and resolving stasis, moving qi, nourishing and replenishing qi, clearing up phlegms. It is consistent with the main syndromes of the disease nature, such as blood stasis and qi deficiency, and the herbs mostly return to the heart meridian, which is consistent with the main syndromes of the disease location. The rules of syndrome differentiation and medication of based on the data mining technique have great value for clinical medication guidance and application to analyze the prescription for coronary heart disease.

Transcriptome analysis of stems of Pinellia ternata under drought stress simulated by PEG / 中草药

Objective: In this study, the transcriptome profile of the stems from Pinellia ternata treated by 30% polyethylene glycol simulated drought stress was analyzed, the key enzyme genes responded to the drought stress were explored, so as to explore the molecular mechanism of P. ternata sprout tumble responded to the drought stress. Methods: The stem segments of P. ternata under drought stress were used as research materials. The Illumina Hiseq 2500 platform was applied for transcriptome sequencing, and bioinformatics analysis was performed on the transcriptome data. Results: A total of 23.00 Gb clean data was obtained, 37 467 952 and 39 903 362 clean reads were gained from the control groups and the drought stress treatment, respectively, and 100 274 uingenes were assembled by Trinity software. A total of 41 132 unigenes were finally obtained with functional annotations against the Nr, eggNOG, Pfam, Swiss-Prot, KOG, GO, KEGG, and COG databases. Furthermore, 4 052 differentially expressed genes (DEGs) were obtained, in details, 2 080 DEGs were up-regulated while 1 972 DEGs were with down-regulated expression. KEGG Pathway enrichment analysis showed that DEGs were mostly involved in ribosome, carbon metabolism, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of amino acids process. The expression levels of the genes encoded plant hormone metabolism and signal transduction, redox-related enzymes, heat shock proteins and vacuolar processing enzymes were significantly promoted after drought stress treatment, while most of the DEGs encoded aquaporins were down-regulated. Conclusion: By analyzing the transcriptome profiles of P. ternata under drought stress simulated by PEG, the candidate genes associated with water metabolism, plant hormone and signal transduction, as well as the related responsive proteins were obtained, which could provide abundant genetic resources and theoretical basis for understanding the mechanism of P. ternata sprout tumble caused by drought stress.

Comparative Study on the Fingerprints and Content of 5 Kinds of Nucleosides in Raw Products and Different Processed Products of Pinellia pedatisecta / 中国药房

OBJECTIVE：To establish the fingerprint of raw produc ts and different processed products of Pinellia pedatisecta ， to determine the contents of 5 kinds of nucleosides ，and to compare the differences of components between the raw products and processed products. METHODS ：P. pedatisecta raw products ，processed products by Processing Standard of Chinese Medicine in Henan Province （called“Standard processed product ”for short ）and processed products by new integrated processing technology in the production area （called“new integrated processed product ”for short ）were collected as investigation objects （12 batches of each）. The determination was performed on SymmetryShield RP 18 column with mobile phase consisted of acetonitrile （A）-0.1％ acetic acid aqueous water solution （B）（gradient elution ）at the flow rate of 0.8 mL/min，with the column temperature of 30 ℃， the detection wavelength of 270 nm，and the injection volume of 15 µL. HPLC fingerprints of 3 kinds of P. pedatisecta samples were established by using Similarity Evaluation System of TCM Chromatographic Fingerprints （2004 A version ） ，and the similarity of fingerprints was evaluated. The chromatographic peaks were identified by comparing with the reference chromatogram. Five nucleosides （adenine，hypoxanthine，uridine，xanthine，inosine）were quantitatively analyzed. SPSS 21.0 software was used for cluster analysis of 36 batches of samples. RESULTS ：The results of fingerprint and content determination met the relevant requirements. The similarity of 3 kinds of sample with their control fingerprint were all greater than 0.990. There were 22 common peaks in the raw products of P. pedatisecta ，and 16 common peaks were identified in the 2 kinds of processed products （the same 6 peaks disappeared from 2 kinds of processed products ）. Fivecomponents were identified in 3 kinds of samples ，such as adenine（peak 3），hypoxanthine（peak 7），uridine（peak 8）， 1064056472@qq.com xanthine（peak 9）and inosine （peak 11）. Results of content determination showed that total contents of 5 kinds of nucleosides in 2 kinds of proc essed products were all· decreased；the contents of them in descending order w as raw product ＞new i ntegrated processed products ＞Standard processed products. Results of cluster analysis showed that 36 batches of samples could be clustered into 2 categories，i.e. raw product was clustered into one category and 2 kinds of processed products into other one . CONCLUSIONS ：Established method is stable ， feasible and suitable for the quality evaluation of raw products and different processed products of P. pedatisecta . Fingerprints have changed significantly and the total content of 5 kinds of nucleosides in P. pedatisecta are all decreased after processing ，but that of new integrated processed products is slightly higher than that of Standard processed products .

Analysis of shading on DNA methylation by MSAP in Pinellia ternata / 中国中药杂志

Pinellia ternata is a medicinal herb of Araceae, and its tubers are used as medicines. It is a common Chinese herbal medicine in China and has a large market demand. When exposing to strong light intensity and high temperature during the growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade can effectively delay sprout tumble formation and increase its yield, however the relevant regulation mechanism is unclear. DNA methylation, as a self-modifying response to environmental changes, is often involved in the regulation of plant growth and development. In this study, P. ternata grown under natural light and 90% shading were selected as the control group and the experimental group for genomic DNA methylation analysis by using methylate sensitive amplification polymorphism(MSAP). The results showed that a total of 617 loci were detected with 20 pairs of primers, of which 311 were in the natural light group and 306 in the shading group. The methylation sites in the light and shading groups accounted for 58.2% and 71.57%, respectively, and the methylation ratios in the methylation sites were 27.65% and 29.41%, respectively, indicating that shading significantly induced the genome DNA methylation of P. ternata. Compared to the natural light group, shading promoted 32.51% of the genes methylation, while inducing 16.25% gene demethylation. This study reveals the DNA methylation variation of P. ternata under shading conditions, which lays a preliminary theoretical foundation for further analysis of the mechanism of shading regulation of P. ternata growth from epigenetic level.

Variation analysis of genomic methylation induced by high temperature stress in Pinellia ternata / 中国中药杂志

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.

Construction and characterization of an infectious clone of Soybean mosaic virus isolate from Pinellia ternata / 生物工程学报

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.

Discrimination of toxic ingredient between raw and processed Pinellia ternata by UPLC/Q-TOF-MS/MS with principal component analysis and T-test / 中草药·英文版

Objective: To investigate the toxicity difference between raw and processed Pinelliae Rhizoma (Banxia in Chinese, BX), the rhizoma of Pinellia ternata, from the view of chemical composition. Methods: Sixteen samples of raw and processed BX were prepared and analyzed by UPLC/Q-TOF-MS/MS. The discrimination (chemical marker)between the two group was investigated by principal component analysis (PCA)and T-test analysis. According to the accurate charge-to-mass ratio, MS/MS fragments, and comparison of corresponding data with the reference or database, the chemical markers were identified preliminarily. Results: Liquiritin, liquiritigenin, and lysophosphatidylcholine (LPC)were identified as the characteristic markers. The reducing of LPC in processed BX was one of the main reasons for detoxification because LPC could induce the inflammatory response; Liquiritin and liquiritigenin showed the anti-inflammatory effect and reduced liver injury, therefore the appearance of them in processed BX was an another reason for detoxification. Conclusion: An approach to explain the mechanisms of reducing the toxicity in medicinal plants by processing was proposed. Moreover, the chemical markers of toxicity could be used to differentiate the raw material from processed herbs for the quality control and safety application in clinical practice.

Cloning and expression analysis of calmodulin gene from Pinellia ternate / 中国中药杂志

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.

Programmed cell death induced by drought stress in sprout tumble of Pinellia ternata / 中国中药杂志

To further study the mechanism of sprout tumble caused by drought,drought stress was simulating with 30% PEG 6000,physiological,and then the morphological changes of Pinellia ternata cells at different treatment time were detected. The results indicated that,along with the period of drought stress continued,the contents of chlorophyll and water potential were decreased,relative electrical conductivity,contents of soluble sugar and MDA increased. Sprout tumble of P. ternata first occurred on the fourth day during drought stress,large scale of sprout tumble appeared on the eighth day with about 73% of tumble rate. The nuclei exposed to drought stress for 2 days were flattened,lobed,invalidated or irregular in shape and significant showed the apoptotic morphological characteristics. Adenylate transferase( ANT) gene expressions were inhibited by drought,with the rapid increase of Caspase-3 enzyme activity,the cell death rate increased. All this proves that the essence of sprout tumble caused by drought is programmed cell death,which may be a self dormancy protection mechanism of P. ternata against adverse environment.

Therapeutic observation on tuina at head and abdomen for headache due to phlegm turbidity / 针灸推拿医学（英文版）

Objective: To observe the clinical efficacy of tuina at head and abdomen for headache due to phlegm turbidity. Methods:A total of 56 patients with headache due to phlegm turbidity were randomized into a tuina group and a Chinese medicine group by the random number table, with 28 cases in each group. The tuina group was treated mainly with tuina at the head and abdomen, while the Chinese medicine group was treated with oral administration of Ban Xia Bai Zhu Tian Ma Tang(Pinellia, Atractylodes Macrocephala and GastrodiaDecoction). The course of treatment was 30 d. The scores of headache index, traditional Chinese medicine syndrome scale, and the therapeutic efficacy were observed. Results:There were 2 dropouts in each group during treatment. The total effective rate was 92.3％ in the tuina group, significantly higher than 76.9％ in the Chinese medicine group (P<0.05). The scores of headache index and traditional Chinese medicine syndrome scale in both groups decreased after treatment (bothP<0.05), and scores in the tuina group were lower than those in the Chinese medicine group (bothP<0.05). Conclusion:Tuina mainly at head and abdomen is effective in treating headache due to phlegm turbidity, and has a better effect than Ban Xia Bai Zhu Tian Ma Tang (Pinellia, Atractylodes Macrocephala and GastrodiaDecoction).