Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza / 药学学报

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

Characteristics, origin, and processing of Poria in Qing Dynasty Palace:evidence of both historical relics and documents / 中国中药杂志

Poria(Fu Ling) is a bulk traditional Chinese medicine(TCM)with a long history and complex varieties. The royal medical records of the Qing Dynasty include multiple medicinal materials of Fu Ling, such as Bai Fu Ling(white Poria), Chi Fu Ling(rubra Poria), and Zhu Fu Ling(Poria processed with cinnabaris). The Palace Museum preserves 6 kinds of specimens including Fu Ling Ge(dried Poria), Bai Fu Ling, Chi Fu Ling, Zhu Fu Ling, Bai Fu Shen(white Poria cum Radix Pini), and Fu Shen Mu(Poria cum Radix Pini). After trait identification and textual research, we found that Fu Ling Ge was an intact sclerotium, which was processed into Fu Ling Pi(Poriae Cutis), Bai Fu Ling and other medicinal materials in the Palace. The Fu Ling in the Qing Dynasty Pa-lace was mainly from the tribute paid of the officials in Yunnan-Guizhou region. The tribute situation was stable in the whole Qing Dynasty, and changed in the late Qing Dynasty. The cultural relics of Fu Ling in the Qing Dynasty Palace confirm with the archival documents such as the royal medical records and herbal medicine books, providing precious historical materials for understanding Fu Ling in the Qing Dynasty and a basis for the restoration of the processing of the Fu Ling in the Qing Dynasty Palace.

Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea / 药学学报

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK) was one of the key enzymes in the methylerythritol-4-phosphate (MEP) pathway to generate terpenoids. In this study, based on the transcriptome data of Atractylodes lancea, the sequence of the CMK gene was cloned, named AlCMK (GenBank accession number OM283293). The results showed that AlCMK contains a 1 230 bp open reading frame (ORF) encoding 409 amino acids. The deduced protein had a theoretical molecular weight of 44 752.53 and an isoelectric point of 6.67. Transmembrane structure analysis showed that there was no transmembrane structure, and the secondary structure of AlCMK was predicted to be mainly composed of random coil. Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium, Osmanthus fragrans, Eucommia ulmoides, Lonicera japonica and Salvia miltiorrhiza. Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae. The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E. coli BL21 (DE3). The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment. Meanwhile, the enzyme activity was determined by ELISA kit. The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA, and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves. The subcellular localization showed that AlCMK was located in the chloroplast. This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.

Herbal Textual Research on Astragali Radix in Famous Classical Formulas / 中国实验方剂学杂志

Through consulting the ancient herbal medicine， prescription books and medical books， combined with modern relevant literature， standards and other information， this paper made a textual research on the name， origin， producing areas， harvesting and processing methods of Astragali Radix according to different historical development periods， providing a basis for the development of famous classical formulas containing Astragali Radix. According to the textual research， the original name of Astragali Radix is Huangqi， and "Qi" originally refers to the medicinal material Zhimu. Some people began to mistake it for Huangqi in the Ming dynasty， and then gradually used Astragali Radix as a medicinal material. The mainstream basis of Astragali Radix can be determined as the dried roots of Astragalus membranaceus var. mongholicus or A. membranaceus. In different historical periods， A. floridus， A. chrysopterus， A. emestii and other plants of Astragalus or even non-Astragalus were used as local Astragali Radix. The earliest production areas of Astragali Radix were Sichuan， Shaanxi， and Gansu， and then gradually expanded to the northeast. Since the Song dynasty， Mianqi in Shanxi province has been regarded as the genuine variety. In the Qing dynasty， besides Shanxi province， Inner Mongolia was also regarded as a genuine place. In the Republic of China， Huangqi produced in northeast China was praised highly. It is mainly produced in Shanxi， Inner Mongolia， Gansu， northeast and other provinces. The main commodity is cultivated products， and the quality of wild imitation cultivation in Datong and Xinzhou is better than other places. There are many processing methods of Huangqi recorded in the materia medica and prescription books， most of which are raw products， and honey processing is the mainstream of processed products. Based on the current situation of resource cultivation and production， 11 famous classical formulas in The Catalogue of Ancient Famous Classical Formulas （The First Batch） containing Huangqi suggested that all use A. membranaceus var. mongholicus， especially those from Datong and Xinzhou in Shanxi Province. In addition to honey processing of Qingxin Lianziyin， it is suggested to use raw products for other formulas.

Herbal Textual Research on Poria in Famous Classical Formulas / 中国实验方剂学杂志

Through consulting the ancient and modern literature， this paper makes a textual research on the name， origin， producing area， harvesting and processing of Poria， so as to provide a basis for the development of the famous classical formulas containing this medicinal material. The description of Poria and the characteristics of the attached figures in the Chinese herbal literature of the past dynasties are consistent with Poria cocos. The medicinal parts are dried sclerotia or P. cocos peel. Poria was originally produced in Taishan， Shandong province. In the Tang dynasty， along with the change of pine forest resources， producing area of Poria was transferred to Huashan area in Shaanxi province. In the Ming dynasty， the authentic producing area was transferred to Yunnan， and has continued to now. In ancient times， the processing methods of Poria were steaming， boiling， slicing， mashing and other subsequent processing after peeling. It is suggested that Poria in famous classical formulas should be sliced according to the 2020 edition of Chinese Pharmacopoeia.

Herbal Textual Research on Asini Corii Colla in Famous Classical Formulas / 中国实验方剂学杂志

Through consulting the ancient and modern literature， this paper makes a textual research on the name， origin， producing area， harvesting and processing methods of Asini Corii Colla， so as to provide a basis for the development of the famous classical formulas containing the medicinal material. Before the Tang dynasty， cow leather was the main source of Asini Corii Colla， and donkey was rare as an introduced species. From the end of Tang dynasty to Song dynasty， due to the development of doctors' understanding of the properties and effects of medicines， with the increase of the number of donkeys and the limitation of the use of cow leather， the source of Asini Corii Colla changed from cow leather to donkey skin. During the Ming and Qing dynasties， the theory of medicine property was further developed， and all doctors basically agreed that black donkey skin and E-well water were two essential factors for making genuine Asini Corii Colla. Therefore， it is suggested that Asini Corii Colla should take Equus asinus as the authentic origin in the development of the famous classical formulas， attach importance to the quality of water source， take Liaocheng in Shandong province as the authentic producing area， and the processing should be carried out in accordance with the requirements of the 2020 edition of Chinese Pharmacopoeia.

Herbal Textual Research on Magnoliae Officinalis Cortex in Famous Classical Formulas / 中国实验方剂学杂志

Based on the ancient literature of all dynasties， this article makes a systematic textual research on the name， origin， producing area， quality， harvesting and processing of Magnoliae Officinalis Cortex used in the famous classical formulas， and clarifies its information of each link in different historical periods， so as to provide a reference and basis for the development and utilization of the related formulas. The results showed that the main varieties of Magnoliae Officinalis Cortex were Magnolia officinalis or M. officinalis var. biloba. The main production areas are Hubei， Sichuan， Chongqing and other places， forming the famous authentic medicine. The processing methods of the past dynasties are mainly cleansing and processing with ginger. In the formulas clearly marked with ginger processing， ginger-processed products is suggested to choose. If not clearly marked， raw or ginger-processed products can be used as needed.

Herbal Textual Research on Jujubae Fructus in Famous Classical Formulas / 中国实验方剂学杂志

This paper made a systematic textual research on the historical evolution and changes of the name， origin， producing area， harvesting and processing methods of Jujubae Fructus used in famous classical formulas by referring to the ancient literature， so as to provide a basis for the sampling and research of the formulas containing the medicinal materials. According to textual research， there are many names of Jujubae Fructus， most of which are named by characters or producing areas， which are called Dazao. Ziziphus jujuba has always been the mainstream variety in all dynasties， and Z. jujuba var. inemmis has also been used. Considering that the differences between the two are not obvious， we can use Z. jujuba and Z. jujuba var. inemmis as the origins of Dazao. The germplasm resources of Jujubae Fructus are rich， which are distributed all over the country. Qingzhou （now Shandong）， Jinzhou （now Shanxi） Jiangzhou （now Shanxi）， Puzhou （now Shanxi） have been recorded as authentic producing areas of Jujubae Fructus in the past dynasties， especially in Shandong. At the beginning of the 21st century， the planting of Jujubae Fructus in Xinjiang gradually developed， and now has a high market recognition， becoming an emerging production area of high-quality samples. Harvest period of Jujubae Fructus is mostly August in the past dynasties， and this is basically the same as today. The main processing method is simple cleansing and drying. Through textual research， it is suggested that Jujubae Fructus in famous classical formulas should be mainly from Shandong， Shanxi and other traditional high-quality producing areas， the processing method should follow the 2020 edition of Chinese Pharmacopoeia for simple cleansing and drying.

Herbal Textual Research on Coptidis Rhizoma in Famous Classical Formulas / 中国实验方剂学杂志

Through the combing of ancient books of Chinese herbal medicine in the past dynasties， a textual research of Coptidis Rhizoma involved the name， origin， medicinal parts， producing area， quality evaluation， harvesting and processing methods in famous classical formulas was conducted in this paper. After textual research， the mainstream varieties of Coptidis Rhizoma in the Ranunculaceae family before Tang and Song dynasties were Coptis chinensis and C. chinensis var. brevisepala， after the Ming and Qing dynasties， C. deltoidea， C. teeta and C. omeiensis were gradually praised. In ancient times， the authentic producing area of Coptidis Rhizoma has the characteristics of gradually moving to the west. The eastern Coptidis Rhizoma was highly praised in the early stage， while in the later stage， western Coptidis Rhizoma like chicken feet was highly praised. In the early stage， western Coptidis Rhizoma probably originated from C. chinensis and its genus， while Coptidis Rhizoma like chicken feet was cultivated， and no wild species has been found so far. As Coptidis Rhizoma has mixed use of multiple origins in ancient books of past dynasties， based on the current shortage of market resources in C. teeta and C. deltoidea， there are also endangered and protected plants of C. chinensis var. brevisepala and C. omeiensis， combined with the mainstream medicines and resources of past generations， it is recommended to choose C. chinensis as the base of the formulas. In ancient times， there were many processing methods for Coptidis Rhizoma， such as frying and wine-， ginger-， honey-processed. In the process of developing famous classical formulas， the appropriate processing specifications of Coptidis Rhizoma should be selected based on the original source records and the requirements of the medicinal material.

Herbal Textual Research on Chuanxiong Rhizoma in Famous Classical Formulas / 中国实验方剂学杂志

Based on various ancient documents such as materia medica， prescription books， classics and history， combined with relevant research materials in modern times， this paper made a textual research on the name， origin， geoherbalism， harvesting time， processing methods of Chuanxiong Rhizoma， which provides a basis for the development of famous classical formulas containing this herb. According to the textual research， the original name of Chuanxiong is Xiongqiong （芎䓖）， which was first recorded in Shennong Bencaojing ， there are many aliases and trade names in the past dynasties. Since the Song dynasty， doctors all take Xiongqiong produced in Sichuan as the best medicine， so they take Chuanxiong as the rectification of name. In the early stage， the origin of Chuanxiong Rhizoma was relatively complicated， and the main origin was Ligusticum chuanxiong， which was a cultivated and domesticated species of Ligusticum. However， wild related plants of Ligusticum are still used as medicine. After the Ming dynasty， new cultivated varieties appeared in various places， such as Jiangxi L. sinense cv. Fuxiong， which gradually turned to self-production and self-marketing after the Republic of China. After several changes in the authentic producing area of Chuanxiong Rhizoma， Tianshui in Gansu province was highly praised in the Tang dynasty， and Dujiangyan in Sichuan province was the best place in the Song dynasty and later dynasties. Chuanxiong Rhizoma has been widely used in the past dynasties as raw products， and it has also been processed with excipients. For example， wine-processed products can enhance the effect of promoting blood circulation， promoting Qi circulation and relieving pain. There are other processing methods such as stir-frying and vinegar processing. Chuanxiong Rhizoma in the famous classical formulas can be selected according to this research conclusion.

Herbal Textual Research on Bambusae Caulis in Taenias in Famous Classical Formulas / 中国实验方剂学杂志

By consulting the ancient herbal and medical books， combined with modern literature， the name， origin， geoherbalism， harvesting and processing changes of Bambusae Caulis in Taenias in famous classical formulas were sorted out. According to the research， ancient doctors only approved three kinds of bamboo medicinal materials， namely， Jinzhu （䈽竹）， Kuzhu （苦竹） and Danzhu （淡竹）， and took bamboo leaves， made Bambusae Caulis in Taenias and Zhuli （竹沥） for medicine. Bamboo medicinal materials with different origins have different properties， tastes and effects， after clinical optimization， it is gradually considered that Danzhu is the best source of Bambusae Caulis in Taenias and Zhuli. According to the morphological description of the original plants and the attached drawings， it is considered that the Danzhu in ancient Chinese materia medica should be Phyllostachys nigra var. henonis， which has been included in the 2020 edition of Chinese Pharmacopoeia as one of the genuine sources of Bambusae Caulis in Taenias. Therefore， It is suggested that P. nigra var. henonis can be added as the source of Bambusae Caulis in Taenias in famous classical formulas， and the medicinal part is the dry middle layer of its stem. Ginger-processed can increase the anti emetic effect of Bambusae Caulis in Taenias， and the three formulas involving Bambusae Caulis in Taenias from The Catalogue of Ancient Famous Classical Formulas （The First Batch） all contain ginger， and the processing method of Bambusae Caulis in Taenias is not marked in the original formula， so it is suggested to use raw products in the three formulas of Jupi Zhurutang， Wendantang and Zhurutang.

Herbal Textual Research on Alismatis Rhizoma in Famous Classical Formulas / 中国实验方剂学杂志

In this study， name， origin， producing areas， harvesting time and processing methods of ancient Alismatis Rhizoma were systematically researched by consulting the literature of ancient herbs， medical and prescription books， so as to provide a basis for the development of famous classical formula containing this herb. According to textual research， the main base of ancient Alismatis Rhizoma was Alisma plantago-aquatica and A. orientale. A. canaliculatum and A. gramineum and other genera were sometimes used as the source of Alismatis Rhizoma， there was a confusion of medicinal varieties. The earliest producing area of Alismatis Rhizoma was in today's Henan province， and later Hanzhong， Shaanxi province， became the high-quality producing area of Alismatis Rhizoma. Since the Ming dynasty， its production area expanded to Fujian. In the Qing dynasty， Jian'ou in Fujian was the authentic production area of Alismatis Rhizoma. In the period of the Republic of China， Sichuan and Jiangxi were added to the production areas of Alismatis Rhizoma. Based on the research results， it is suggested that the dried tubers of A. orientale from Fujian and Jiangxi or A. plantago-aquatica from Sichuan should be used in the famous classical formulas. In ancient times， Alismatis Rhizoma was processed by wine， but most of the standards and specifications in modern times are no longer included the processing specifications of Alismatis Rhizoma with wine. Although salt-processed Alismatis Rhizoma is commonly used in modern times， it didn't become one of the main processing methods until the Qing dynasty. According to the relevant national documents， it is suggested that Alismatis Rhizoma without clear processing requirements in famous classical formulas should be used as raw products， and the formulas with processing requirements should be selected as processed products such as salt and wine according to the meaning of the formulas.

Herbal Textual Research on Dendrobii Caulis in Famous Classical Formulas / 中国实验方剂学杂志

Through consulting the ancient herbal and medical books， combined with the field investigation， the name， origin， collection and processing of Dendrobium medicinal materials were researched， which provided a basis for the development of famous classical formulas containing this kind of herbs. Due to the wide distribution of D. officinale， the Dendrobium species represented by D. officinale and D. huoshanense， which are short， fleshy and rich in mucus， should be the most mainstream of Dendrobium medicinal materials in previous dynasties. Compared with Shihu， Muhu with loose texture， long and hollow is born on trees. According to the characteristic description， it should be D. nobile， D. fimbriatum and so on， of which D. nobile was the mainstream. The Chinese meaning of Jinchai was confused in the past dynasties， so it was not suitable to be treated as a plant name. The production areas of Dendrobium medicinal materials in the past dynasties have changed with the discovery of varieties， artificial cultivation and other factors. Lu'an， Anhui province， was the earliest recorded in the Han and Wei dynasties. Since the Tang and Song dynasties， it had been extended to Guangdong and Guangxi， and it was considered that "Dendrobii Caulis in Guangnan was the best". In the Ming dynasty， Sichuan and Zhejiang products were highly praised， and in the Qing dynasty， Huoshan products were highly praised. Dendrobium medicinal materials had been used as medicine by stems in all dynasties. The medicinal materials were divided into fresh products and dry products. The fresh products can be used immediately after removing the sediment from the roots. The dry products need further processing， most of them used wine as auxiliary materials for steaming， simmer to paste or decoction into medicine. D. officinale and D. huoshanense have special processing specifications since the middle of Qing dynasty， that is， "Fengdou". According to the research results， in Ganluyin， the effect of Dendrobium medicinal materials is mainly heat clearing， and D. nobile with bitter taste can be selected. The main effect of Dendrobium medicinal materials in Dihuang Yinzi is tonic， D. officinale or D. huoshanense can be selected.

Textual Research on Perillae in Famous Classical Formulas / 中国实验方剂学杂志

Based on the ancient literature of all dynasties， this article makes a systematic textual research on the name， origin， producing area， quality， harvesting and processing of Zisu （Perillae） in the famous classical formulas， so as to clarify the information of the drug in different historical periods and provide a reference for the development and utilization of the related formulas. The main origin of Perillae in the ancient literature was Perilla frutescens var. frutescens （purple leaf type）， followed by P. frutescens var. acuta （purple leaf type）， but not Baisu. Modern chemical composition studies also show that there are obvious differences between Perillae and Baisu， which provides a scientific basis for distinguishing them. Although they are often treated as a species in plant classification， P. frutescens var. frutescens （purple leaf type） is recommended in the development of famous classical formulas， and Baisu should be avoided. Perillae is widely distributed， but its producing area did not record in most of the literature in the past dynasties， or the producing area is described as everywhere today. In the period of the Southern and Northern dynasties， the medicinal parts of Perillae included stems， leaves and seeds， and doctors in the Ming dynasty began to pay attention to the differentiation of different medicinal parts. The harvesting and processing methods of Perillae in the past dynasties are close to that of today. Perillae Fructus is mostly stir-fried and ground into medicine， Perillae Folium and Perillae Caulis are mainly simple cleansing. In production， we can refer to the 2020 edition of Chinese Pharmacopoeia.

Textual Research on Nelumbinis in Famous Classical Formulas / 中国实验方剂学杂志

The name， origin， place of origin， medicinal parts， harvesting and processing of lotus are verified by consulting ancient Chinese herbal medicines and medical books， combined with modern literature， providing a basis for the development of famous classical formulas containing lotus. According to textual research， the original base of lotus is Nelumbo nucifera since ancient times， rhizome （Nelumbinis Rhizomatis Nodus）， leaf （Nelumbinis Folium）， seed （Nelumbinis Semen）， embryo （Nelumbinis Plumula）， receptacle （Nelumbinis Receptaculum）， stamen （Nelumbinis Stamen） and other medicinal parts of N. nucifera can be used as medicine and have different clinical effects. Nelumbinis Semen was originally produced in Henan， and then gradually expanded to Jiangnan. Today， it can be cultivated and planted throughout the country， with Fujian， Hunan， and Jiangxi as the authentic production areas. After combing the medicinal parts of N. nucifera and the historical evolution of its processing， it is suggested that the dried and mature fruits of N. nucifera taking in autumn and removing the shell and Nelumbinis Plumula should be used in Qingxin Lianziyin. Nelumbinis Folium in Erdongtang should be harvested in summer and autumn， and the raw products was used as medicine and processed in accordance with the provision of the 2020 edition of Chinese Pharmacopoeia.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii / 中国天然药物

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Different development phase of transcriptome analysis from Crataegus pinnatifida Bge. and cloning, structure and function prediction of squalene epoxidase involved in triterpenic acid biosynthesis / 药学学报

italic>Crataegus pinnatifida is a traditional Chinese medicine, which contains organic acids, triterpenoid acids and other active components, has important medicinal and edible value. In order to study the difference of gene expression level in different developmental stages of hawthorn and explore the genes of active ingredient biosynthesis in Crataegus pinnatifida, high-throughput Illumina HiSeq 2000 technology were used to conduct transcriptome sequencing and bioinformatics analysis on Crataegus pinnatifida fruits from the same origin at different developmental stages. 78 496 Unigenes with an average length of 941 nt were obtained by Trinity software. Among them, 58 395 Unigenes can be annotated by NR, NT, Swiss prot, KEGG, COG, GO and other public databases. KEGG pathway analysis showed that 52 Unigenes encoding 15 key enzymes involved in the citric acid cycle. There are 62 Unigenes were involved in the triterpene biosynthesis pathway of Crataegus pinnatifida. Two key enzymes SQE of triterpenoid metabolism pathway in Crataegus pinnatifida were cloned and performed bioinformatic analysis. The results showed that ORF of CpSQE1 and CpSQE2 were 1 594 bp and 1 597 bp, respectively, encoding 530 and 531 amino acids. The molecular weight of proteins was 57.6 kDa and 57.5 kDa. Bioinformatics analysis showed that both CpSQE1 and CpSQE2 proteins have a PLN02985 superfamily conserved domain, belonging to the squalene monooxygenase superfamily. The phylogenetic tree shows that CpSQE1 and CpSQE2 are clustered together with SQE with squalene epoxidase function in other plants. This study provides a research basis for further exploring the key genes in the biosynthesis process of hawthorn active ingredients and analyzing the regulation pathway of its active ingredient biosynthesis.

Morphological comparison of glandular and non-glandular trichomes between Artemisia stolonifera and A. argyi / 中国中药杂志

The basic features of glandular and non-glandular trichomes on leaves of Artemisia argyi( germplasms from Qichun,Ningbo,Tangyin,and Anguo,respectively) and related species A. stolonifera were observed by scanning electron microscopy( SEM)and compared. There were significant differences in trichome characteristics of leaves at all parts of A. argyi and A. stolonifera,which were closely related to the difference in chemical components. The length of non-glandular trichomes and size of glandular trichomes on middle leaves were the stablest. A. argyi and A. stolonifera can be distinguished by the density of glandular trichome. Additionally,the four germplasms of A. argyi can be discriminated via the density and curvature of non-glandular trichome. The density of non-glandular trichomes was the highest in A. stolonifera. For A. argyi,the germplasm from Qichun had the highest density of non-glandular trichomes on the abaxial surfaces of upper leaves and that from Ningbo had the largest non-glandular trichome curvature. With regard to the germplasm from Anguo,the T-shaped non-glandular trichomes of long stalks on the adaxial surfaces of the middle leaves were lodging-susceptible,and those with slender heads were wave-like. Statistics results of A. argyi and A. stolonifera are as follows: largest glandular trichomes on the adaxial and abaxial surfaces and highest glandular trichome density on the abaxial surfaces of the lower leaves in A. argyi germplasm from Ningbo,highest density of non-glandular trichomes on the abaxial surfaces of upper leaves in A. stolonifera,and highest density of glandular trichomes and non-glandular trichomes on the adaxial surfaces of the upper leaves in A. argyi germplasm from Qichun. According to the observation result under fluorescence microscope( FM),flavonoids were closely related to the size and density of non-glandular trichomes and size of glandular trichomes. The fluorescence intensity was the strongest and fluorescence area was the largest for flavonoids in A. argyi germplasms from Qichun and Tangyin,while the fluorescence for flavonoids was the weakest in A. stolonifera. It was the first time to observe and analyze the trichome ultrastructure of A. argyi leaves at different positions by SEM and FM. This study clarifies the differences between A. stolonifera and four famous A. argyi germplasms,which provides new evidence for the microscopic identification of A. argyi and its related species and serves as a reference for the study of the relationship of A. argyi structure with its components and functions.

Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea / 中国中药杂志

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid β-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid β-oxidation in A. lancea.

Significance of map labelling method to research on evolution of historical production areas of Dao-di herbs / 中国中药杂志

The research on the historical production areas of Dao-di herbs is of great value to the quality evaluation, production base and protection for geographic indications of Dao-di herbs. Current studies mostly focused on the sorting of written sources, but neglected the excavation of image records such as historical maps and topographic maps. This paper aims at exploreing the geographical scope and evolution mode of historical production area of the Dao-di herbs by combining the method of historical map labelling with the traditional literature and historical research methods. It can be divided into three steps: production area name extraction, historical map labelling and textual research on the historical production area of Dao-di herbs. This method may provide a better way to show the geographical scope and topographic features of the historical production areas of Dao-di herbs through historical maps labelling, which is essential to further explore the evolution of production areas of Dao-di herbs from ancient times to the present by comparing historical maps of different periods, and may be helpful to discover the reasons for the formation and evolution of historical producing areas of Dao-di herbs from different perspectives such as environment, climate, humanities, economy, policy, etc. In addition, the historical map database can be used for map labelling to help establish the relationship between the dynasties, historical names, and change cha-racteristics of the scope of the historical production areas of Dao-di herbs in the following research.