[Screening of an Armillaria gallica strain for Gastrodia elata cultivation].

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

[Application of strigolactone analogs in storage of Gastrodia elata].

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

[Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata].

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

[Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius].

Baby Boom(BBM) gene is a key regulatory factor in embryonic development and regeneration, cell proliferation, callus growth, and differentiation promotion. Since the genetic transformation system of Panax quinquefolius is unstable with low efficiency and long period, this study attempted to transfer BBM gene of Zea mays to P. quinquefolius callus by gene gunship to investigate its effect on the callus growth and ginsenoside content, laying a foundation for establishing efficient genetic transformation system of P. quinquefolius. Four transgenic callus of P. quinquefolius with different transformation events were obtained by screening for glufosinate ammonium resistance and molecular identification by PCR. The growth state and growth rate of wild-type and transgenic callus were compared in the same growth period. The content of ginsenoside in transgenic callus was determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The results showed that transgenic callus growth rate was significantly higher than that of wild-type callus. In addition, the content of ginsenoside Rb_1, Rg_1, Ro, and Re was significantly higher than that in wild-type callus. The paper preliminarily proved the function of BBM gene in promoting growth rate and increasing ginsenoside content, which provided a scientific basis to establish a stable and efficient genetic transformation system for Panax plants in the future.

[Processing technology of rice-steamed Rehmanniae Radix unearthed from tomb of Haihunhou in the Western Han Dynasty].

With the rice-steamed Rehmanniae Radix unearthed from the tomb of Haihunhou in the Western Han Dynasty as the re-ference, the present study evaluated the quality of Rehmanniae Radix and investigated the processing technology of rice-steamed Rehmanniae Radix to lay the foundation for the research on rice-steamed Rehmanniae Radix products. With catalpol and rehmannioside D as the investigation indexes, the quality and grade of Rehmanniae Radix from different producing areas were evaluated with the methods in 2020 edition of Chinese Pharmacopoeia. UPLC method was established for the determination of catalpol and rehmannioside D in the rice-steamed Rehmanniae Radix. The effects of steaming time, the amount of supplementary rice, and steaming times in the rice-steamed processing on the quality of products were investigated by L_9(3~4) orthogonal test and multi-index comprehensive balance scoring method combined with the content of catalpol and rehmannioside D and appearance characteristics. At last, the stability of the processing technology was tested. The results showed that the optimal processing technology for rice-steamed Rehmanniae Radix was as follows: Rehmanniae Radix and rice(200 g∶4 g) were steamed twice at atmospheric pressure, four hours each time. The mass fractions of catalpol and rehmannioside D were 0.184% and 0.335%, respectively, and the character score was 6.5. The processing conditions are reaso-nable, stable, and feasible. It can provide a basis for the restoration of the ancient rice-steamed processing technology and references for the development of rice-steamed Rehmanniae Radix products in the future.

[Bioinformatics analysis and function prediction of ginseng TCP transcription factor family].

The longevity mechanism of ginseng(Panax ginseng) is related to its strong meristematic ability. In this paper, this study used bioinformatic methods to identify the members of the ginseng TCP gene family in the whole genome and analyzed their sequence characteristics. Then, quantitative real-time fluorescent PCR was performed to analyze the TCP genes containing elements rela-ted to meristem expression in the taproots, fibrous roots, stems, and leaves. According to the data, this study further explored the expression specificity of TCP genes in ginseng tissues, which facilitated the dissection of the longevity mechanism of ginseng. The ginseng TCP members were identified and analyzed using PlantTFDB, ExPASy, MEME, PLANTCARE, TBtools, MEGA and DNAMAN. The results demonstrated that there were 60 TCP gene family members in ginseng, and they could be divided into two classes: Class Ⅰ and Class Ⅱ, in which the Class Ⅱ possessed two subclasses: CYC-TCP and CIN-TCP. The deduced TCP proteins in ginseng had the length of 128-793 aa, the isoelectric point of 4.49-9.84 and the relative molecular mass of 14.2-89.3 kDa. They all contained the basic helix-loop-helix(bHLH) domain. There are a variety of stress response-related cis-acting elements in the promoter regions of ginseng TCP genes, and PgTCP20-PgTCP24 contained the elements associated with meristematic expression. The transcription levels of PgTCP20-PgTCP24 were high in fibrous roots and leaves, but low in stems, indicating the tissue-specific expression of ginseng TCP genes. The Class Ⅰ TCP members which contained PgTCP20-PgTCP23, may be important regulators for the growth and development of ginseng roots.

[Genetic diversity of protopine-6-hydroxylase in three medicinal Papaver plants].

Sanguinarine is the main active component of the Papaver plants, and protopine-6-hydroxylase(P6 H), involved in the sanguinarine biosynthetic pathway, can oxidize protopine to 6-hydroxyprotopine. The investigation on the diversity of P6 H genes in the medicinal Papaver plants contributes to the acquirement of P6 H with high activity to increase the biosynthesis of sanguinarine. Five P6 H genes in P. somniferum, P. orientale, and P. rhoeas were discovered based on the re-sequencing data of the Papaver species, followed by bioinformatics analysis. With the elongation factor 1α(EF-1α), which exhibits stable expression in the root and stem, as the internal reference gene, the transcription levels of P6H genes in roots and stems of the Papaver plants were detected by real-time fluorescent quantitative PCR. As indicated by the re-sequencing results, there were two genotypes of P6H in P. somniferum and P. orientale, respectively, and only one in P. rhoeas. The bioinformatics analysis showed that the P6 H proteins of the three Papaver plants contained the conserved domain cl12078, which is the characteristic of p450 supergene family, and transmembrane regions. The existence of signal peptide remained verification. Real-time fluorescent quantitative PCR results revealed that the transcription level of P6 H in roots of P. somniferum was about 1.44 times of that in stems(α=0.05). The present study confirmed genetic diversity of P6 H in the three medicinal Papaver plants, which lays a basis for the research on the biosynthesis pathway and mechanism of sanguinarine in Papaver species.

[Analysis of survey data of popularization of traditional Chinese medicine health culture knowledge in 2017].

To continuously track and analyze the popularization and change trend of traditional Chinese medicine(TCM) health culture knowledge, so as to provide scientific basis for formulating relevant policies, strategies and measures for popularizing Chinese medicine health culture knowledge. In this study, PPS combined with random sampling method was used to survey residents aged between 15 and 69 in 328 survey sites in 30 provinces, autonomous regions and municipalities(excluding Tibet Autonomous Region, Hong Kong, Macao and Taiwan). In the study, a standardized questionnaire was used to survey the contact, cognition, trust and use of Chinese medicine health culture knowledge. A total of 89 107 people were respondent in this study, including 87 287 valid questionnaires, with an effective rate of 97.96%. Among them, the urban residents accounted for 51.35%, rural residents accounted for 48.65%; males took up 48.25%, and females took up 51.75%. In 2017, the national Chinese medicine health culture knowledge popularization rate was 91.72%, the reading rate was 89.61%, the trust rate was 89.60%, and the action rate was 55.53%. The study found that TCM health culture knowledge was more popular among young people, high-education residents and non-sickness groups. It is recommended to strengthen the popularization of traditional Chinese medicine in key areas and key populations, provide differentiated Chinese medicine health education to population in different areas, and cooperation with mass media to provide diversified contents and forms.

[Analysis of knowledge acquisition behavior of traditional Chinese medicine based on internet].

The study aims at understanding the situation of Chinese residents' access to Chinese medicine health culture knowledge through the Internet and analyze its influencing factors. A multi-stage PPS sampling method was used to collect 90 720 people for questionnaire survey. The survey found thatthe probability of Chinese residents accessing Chinese medicine health culture knowledge through the Internet was 54.7%. The females(with the males as reference, OR=1.076, 95% CI 1.018-1.137) and central population(with the east as reference, OR=1.235, 95% CI 1.048-1.456), people with Chinese medicine health culture literacy(with the people who do not have Chinese medicine health culture literacy as reference, OR=2.363, 95% CI 1.976-2.827) had a higher probability of acquiring Chinese medicine health culture knowledge through the Internet. Referring to people who were illiterate or less literate,the OR values of people who went to elementary school, junior school, high school/vocational/technical school and junior college/university was 2.396(95% CI 2.062-2.784),4.481(95% CI 3.751-5.352), 6.687(95% CI 5.541-8.07),and 9.109(95% CI 7.385-11.235). The higher the age, the lower the probability of acquiring Chinese medicine health culture knowledge through the Internet. Taking civil servants as a reference, teachers, students, farmers, and workers had a low probability of acquiring Chinese medicine health culture knowledge through the Internet. The OR values was 0.736(95% CI 0.548-0.988),0.609(95% CI 0.449-0.826), 0.424(95% CI 0.325-0.554),and 0.707(95% CI 0.539-0.927). Regions, gender, age, education level, occupation, and possession of Chinese medicine health culture literacy are factors influencing whether residents obtain Chinese medicine health culture knowledge through the Internet.

[Analysis of Chinese citizens' traditional Chinese medicine health culture literacy level and its influence factors in 2017].

To analyze the TCM health culture level and influence factors of Chinese citizens in 2017. PPS sampling combined with random sampling was used to select the residents aged between 15-69 years old in 30 provinces as the respondents,and a questionnaire study was conducted to investigate their TCM health culture level. In 2017,there were 87 287 valid questionnaires for Chinese citizens' TCM health culture level,including 48. 25% male and 51. 75% female,with a sex ratio of 1 ∶ 1. 073. In 2017,the overall TCM health culture level was 13. 39%,specifically 18. 77% for the urban areas and 10. 51% for the rural areas. Compared with people who were illiterate or less literate,people with an educational background of elementary school,junior high school,high school/vocational/technical school and junior college/university had a higher TCM health culture level,and the OR values were 1. 584( 95% CI[1. 166,2. 152]),2. 827( 95%CI[1. 839,4. 345]),5. 651( 95%CI[3. 637,8. 781]),9. 785( 95%CI[6. 187,15. 477]) in order. With civil servants as the reference,medical workers had a higher TCM health culture level( OR = 1. 829,95%CI[1. 279,2. 616]),while farmers had the lowest TCM health culture level( OR = 0. 493,95% CI[0. 349,0. 697]). Compared with people with the annual household income per capita of 20 000 yuan and below,people with the annual household income per capita between 20 000-50 000,50 000-80 000,80 000 yuan or above had a higher TCM health culture level,and the OR values were 1. 176( 95% CI[0. 963,1. 437]),1. 458( 95%CI[1. 168,1. 820]) and 1. 930( 95%CI[1. 509,2. 469]). Based on the differences between urban and rural areas,the influence factors of citizens' TCM health culture level include education,occupation and income.

[Chemical fingerprinting and similarity analysis of Lonicera japonica resources].

LC-MS was used to detect 41 population of Lonicera japonica from different areas. LC-MS chemical chromatographic profile has been established. There were 23 common peaks, seventeen of which were identified according to reference standard and reference; SPSS software was applied to calculate the similarity of chemical fingerprints of 41 batches and the range was from 0.99 to 0.12. On this basis, the L. japonica's metabolites consistency was classified. Combined with comprehensive analysis of genetic identity, we can provide a theoretical basis for the authenticity research of Dao-di herbs and reference information for the breeding of excellent L. japonica.

[Allele-specific PCR to identification of Achyranthis Bidentatae Radix and Cyathulae Radix formula granules].

To establish a robust and accuracy molecular method to identify Achyranthis Bidentatae Radix and Cyathulae Radix formula granules. ITS sequences of Achyranthes bidentata and Cyathula officinalis were aligned, specific SNPs (single nucleotide polymorphisms) were excavated, specific primers were designed and allele-specific PCR method was established. The genomic DNA was successfully extracted from the herbal medicine and its formula granules by using an improved CTAB (cetyltrimethyl ammonium bromide) method and then performed PCR with the designed primers. The 187 bp specific band could be amplified only in the presentation of C. officinalis and its granules when use of C. officinalis specific primers, whereas the 162 bp band could be amplified only in the presentation of A. bidentata and its granules when use of A. bidentata specific primers. This method was also successfully applied in the identification of commercial formula granules.

[Zoological origin survey of commercial hippocampus in Chinese herbal markets by morphological and DNA sequencing identification].

Hippocampus is a precious animal medicine in Chinese herbal medicines. Numerous seahorse species possessing similar morphology were used as commercial hippocampus in herbal markets. Clarifing the zoological of commercial hippocampus in herbal markets is a crucial issue, which contributed to establish authentication and quality control standard. This study investigated 1 156 dried seahorse samples collected from eight main herbal markets using CO Ⅰ fragment DNA sequencing coupling with morphological identification. The results showed that 23 seahorse species were present in the China TCM market. Among them, five species were officially listed in China Pharmacopoeia, seven species namely winged seahorse (Hippocampus alatus), giraffe seahorse (H. camelopardalis), knysna seahorse (H. capensis), beibuwan seahorse (H. casscsio), half-spiny seahorse (H. semispinosus), Europe seahorse (H. hippocampus), zebra seahorse (H. zebra) were found in herbal markets for the first time. The present DNA sequences analysis coupling with morphological identification method could also use to survey the species origin of other Chinese herbal medicines in herbal markets.

[Specific PCR method for identification of Trionycis Carapax and its preparation].

Trionycis Carapax is a commonly used animal medicine in Chinese medicine. It's difficult to identify Trionycis Carapax and its adulterants because of the loss of morphological characteristics after processing. To establish an efficient and stable method to identification Trionycis Carapax, this study combines SDS method with column purification to extract genomic DNA, uses universal primers for polymerase chain reaction (PCR) amplification and sequencing, and designs the specific primers based on the differences in the sequences of Pelodiscus sinensis and their adulterants. When the annealing temperature was 62 °C and the number of cycles was 35, the designed primer Biejia-272.F/R was used for PCR amplification and got optimum results. The crude drug and preparation of P. sinensis were all amplified to obtain a specific band of approximately 300 bp, while the adulterants showed no such a band. This method can be used as a rapid and accurate method to identify the authenticate of P. sinensis.

[Multiplex PCR to simultaneous identification of five traditional Chinese medicinal seahorses].

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.

[Study on calibration standard for reference drug of Lonicera japonica cultivated in Xinmi].

The calibration technical specification for reference drug of Lonicera japonica cultivated in Xinmi (Mi Yin Hua) established in this study can be used as the theoretical basis and technical standard for internal quality control and evaluation of reference drug of Mi Yin Hua and for modern research and characteristics identification of Dao-di herbs. Based on the quality standard of L. japonica in Chinese Pharmacopoeia (version 2015), 10 batches samples of Mi Yin Hua also conformed to the stipulation. The LC-MS fingerprint common mode of Mi Yin Hua was obtained, and a total of 23 characteristic peaks were selected as the common fingerprint peaks, and eight common chromatographic peaks in the fingerprint was identified based on standard substances and references. The similarities of 10 batches samples were greater than 0.95. Seven core primers were used to construct SSR fingerprint and the ten batches samples were only 0 or 1 site disaccord with the standard diagram of Jianshan Damaohua germplasm from Xinmi. This paper constructed the first calibration standard of Dao-di herbals by combination of macroscopical identification, SSR fingerprint and LC-MS fingerprint, and the calibration standard provide theoretical basis and technical standard for evaluation system of Dao-di herbals.

[Application of rapid PCR to authenticate Ranae Oviductus].

Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.

[Research strategies and application of reference Dao-di herbs].

Establishing evaluation system of Dao-di herbs has been a problem to be solved in the field of traditional Chinese medicine (TCM), and is also a difficult problem in restricting the realization of TCM quality. On the basis of national drug standard substance requirements, this paper puts forward to set up reference Dao-di herbs in the first step of the evaluation system of Dao-di herbs, and discusses the properties, evaluation index system and its development requirements of reference Dao-di herbs, aiming at supporting the modern research and characteristics identification of Dao-di herbs in the future.

[Identification of Panax ginseng, P. notoginseng and P. quinquefolius admixture by multiplex allele-specific polymerase chain reaction].

To achieve a molecular method to identify Panax ginseng, P. notoginseng,P. quinquefolius and their admixture. The ITS,18S and matK sequences of Panax genus were analyzed to develop species-specific SNP marker. Three pairs of species-specific primers were designed to establish a multiplex allele-specific polymerase chain reaction (MAS-PCR) and the samples from different region were tested. The results showed that when the annealing temperature was 60 ℃ and the cycle number was 35, approximately 250, 500,1 000 bp specific band were obtained from P. ginseng, P. notoginseng and P. quinquefolius obtain, respectively. This method could also be used to authentificate admixture samples and could detect 0.5% percent of P. notoginseng or P. quinquefolius adulterated in P. ginseng, or 0.5% percent of P. ginseng or P. quinquefolius adulterated in P. notoginseng. The detect limit of P. ginseng in P. quinquefolius was 0.5% and P. notoginseng in P. quinquefolius was 1%. This results showed that the present method could be used as a promise method to identify Panax ginseng, P. notoginseng, P. quinquefolius and their admixture.

[Study on identification of Dendrobium officinale and related species by bidirectional PCR amplification of mismatched and specific alleles].

Based on rDNA ITS sequences of Dendrobium officinale and the other 69 species of Dendrobium, a pair of dismatched allele-specific diagnostic primers, TPSH-AS1F and TPSH-AS1R were designed to authenticate D. officinale from the other species. Thebidirectional PCR amplification were performed using the diagnostic primers with the total DNAs of the original plants or processing products as a template. When the annealing temperature was raised to 60 ℃, only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. Compared with the other authentification methods, the bidirectional PCR amplifications is not only simpler and time-saving but practical and effective.