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The key factors for producing the best quality Chinese herbal medicines are high-quality germplasm, suitable cultivation area and the proper processing methods for herbal raw materials. Gentiana crassicaulis in Gentiana (Sect. Cruciata), Gentianaceae is one of the original plants of the Chinese herb Qinjiao (Gentianae Macrophyllae Radix), and its type specimen was collected in Lijiang, Yunnan. There is a long planting history of the herb in this area. In this study a sampling plot was designated in these traditional planting areas. G. crassicaulis was planted and herbal raw materials were harvested from the plot. The raw materials were prepared locally and at a pharmaceutical factory in Shanghai using processing methods such as "sweating" or "no sweating", "slicing" or "no slicing" (whole root), and "stoving" or "no stoving" (air drying). The quality of all processed samples was evaluated. In addition, molecular markers were determined for identifying cultivated and wild samples from Lijiang, Yunnan. The results are as follows: ① Samples from the sampling plot and the field are taxonomically identified as Gentiana crassicaulis. ② A total of 270 sequences of trnC-GCA-petN, atpB-rbcL, psbN, ndhB-rps7 and ycf1 were obtained, and three genotypes were determined from the cultivated samples; the type III was shared by both cultivated and wild plants. Based on the molecular markers, a DNA barcoding method to identify cultivated and wild samples of G. crassicaulis from Lijiang, Yunnan was established. ③ Total content of loganic acid and gentiopicroside in all samples was ≥ 2.5%, and above the Chinese Pharmacopoeia (2020) limit. ④ In HPLC fingerprinting, 9 common peaks were assigned and similarity between all samples was > 0.999; and ⑤ In a PCA score plot all slice samples were clustered, while whole root samples were scattered. Therefore, our studies could provide basic data for optimizing the processing method, producing best quality Gentianae Macrophyllae Radix, and evaluating the quality of different ecotype varieties and the multiple origin of herbal medicines.
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italic>Gentiana crassicaulis Duthie ex Burk. is one of the plant sources of Gentianae Macrophyllae Radix (QinJiao). Gentiana tibetica King ex Hook. f. and Gentiana robusta King ex Hook. f. are relative species of G. crassicaulis. Due to the large intraspecific morphological variation, G. crassicaulis showed high morphological similarity with G. tibetica and G. robusta. And the distribution area of the three species overlaps to some extent, which makes it difficult to identify them. On the basis of morphological identification, the method of molecular identification of the three species was constructed in this study based on chloroplast genomes. The chloroplast genome of Gentiana tibetica is 148 765bp long, with LSC, SSC and IR 81 163 bp, 17 070 bp and 25 266 bp, respectively. The structure of the three is consistent. The chloroplast genome sequences of G. tibetica and G. crassicaulis are highly similar, and the number of variable sites is 9 (149 267 bp in total). Diagnostic SNP that could effectively identify the three species was screened and verified, and a dual-peak SNP detection method was established for the effective identification of each species and mixed samples. Our study provides basic data for the molecular identification of G. crassicaulis and its related species, and the arrangement of related Tibetan medicine.
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As the main chemical constituents, iridoids are widely distributed within Gentiana, Gentianaceae, with promising bioactivities. Based on the previous work, the transcriptome of G. lhassica, an original plant of Tibetan herb "Jieji Nabao", was sequenced and analyzed in this study, and the transcriptome databases of roots, stems, leaves, and flowers were constructed so as to explore unigenes that may encode the key enzymes in the biosynthetic pathway of iridoids. Then, qRT-PCR was used to validate the relative expression levels of 11 genes named AACT, DXS, MCS, HDS, IDI, GPPS, GES, G10H, 7-DLNGT, 7-DLGT, and SLS in roots, stems, leaves, and flowers. Also, the total contents of gentiopicroside and loganic acid were determined by HPLC, respectively. The results are as follows:(1)a total of 76 486 unigenes with an average length of 852 bp were obtained;(2)335 unigenes were involved in 19 stan-dard secondary metabolism pathways in KEGG database, with phenylpropanoid biosynthesis having the maximum number(75 unigenes), and no isoflavone biosynthetic pathway was annotated;(3)171 unigenes participatedin 27 key enzymes encoding in the biosynthetic pathway of iridoids, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) gene was highly expressed;(4)qRT-PCR results were approximately consistent with RNA-Seq data and the relative expression levels of the 11 genes were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root);(5)the total contents of gentiopicroside and loganic acid were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root), and the difference was significant. This study provides basic scientific data for accurate species identification, evaluation of germplasm resources, research on secondary pro-duct accumulation of medicinal plants within Gentianaceae, and protection of endangered alpine species.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , Gentiana/genetics , Iridoids , TranscriptomeABSTRACT
italic>Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.
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As two original plants of Tibetan herb Jieji, Gentiana waltonii Burk. and Gentiana lhassica Burk. belong to Section Cruciata of Gentiana, Gentianaceae. Here, we report on whole chloroplast genome sequences in the alpine species, respectively, and the features of plastomes were investigated. The plastome of G. waltonii is 148 705 bp long (148 652 bp in G. lhassica) and encodes 112 genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Two pseudogenes, namely ψrps16 and ψinfA, were found in plastomes. In addition, two novel loci were detected, and a species-specific polymerase chain reaction assay was developed for differentiating G. waltonii and G. lhassica from 10 alpine species in Section Cruciata. Gentiana. Our study provides basic data for identifying Tibetan herbs, alpine species conservation and molecular phylogenetic studies of Gentiana and Gentianaceae.
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Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches: one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.
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The roots and flowers of Gentiana waltonii and Gentiana robusta are used as Tibetan herb Jie-Ji in traditional Tibetan medicine, with iridoids as the main active ingredient and index components. To study the pathway of iridoid biosynthesis, roots, stems, leaves and flowers of G. waltonii and G. robusta were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiseqXTen. After removing insignificant reads and de novo splicing, 79 455 and 78 466 unigenes were obtained from G. waltonii and G. robusta respectively, with average length as 834 bp and 862 bp. The unigene GO functions could be divided into three categories of 65 branches. The unigenes were aligned in KOG database and were classified into 25 classes according to function. In KEGG database, 315 and 340 unigenes of G. waltonii and G. robusta were implicated in 20 standard secondary metabolic pathways, respectively. Furthermore, 80 and 57 unigenes of the two species were analyzed to encode 24 key enzymes in the pathway related to iridoid biosynthesis. There were differences in gene expression among different organs. Based on sequence data, significant amounts of SSRs, SNPs and InDels were detected in each dataset. This study provides a platform for further development of molecular markers, excavation of functional genes, and research into metabolic pathways and their regulatory mechanism within G. waltonii and G. robusta.
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italic>Gentiana section Cruciata (Gentianaceae) is a medicinally important section of herbs, including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji. Here, we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data. A total of 144 nad1/b-c and nad5/d-e sequences from 11 species within Gentianaceae were obtained, including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica (outgroup). The results showed that mtDNA nad1/b-c has species- level resolution within the section of Cruciata, i.e. the variable in the position 45 “C” could be used as a stable marker locus to distinguish G. robusta from other taxa; the variable in the position 352 and 353 “GA” could distinguish G. crassicaulis and G. tibetica from other taxa within the section. Intraspecies genotype variability was detected in nad1/b-c sequences of G. officinalis and G. siphonantha, respectively. These genotypes could be used as potential DNA barcode. In addition, intraspecies genotype variability was detected in nad5/d-e sequences of G. macrophylla, G. officinalis and G. siphonantha, respectively. Based on the stable marker locus, a species-specific PCR protocol was developed using the primer PF to identifying G. robusta in the section. This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana, and provide the essence for the species identification within Gentiana section Cruciata.
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As a common Tibetan herb, Bawo Sebo was mainly used in the treatment of rheumatoid arthritis and urarthritis in Traditional Tibetan medicine. Based on our ethnobotanical survey, the origin of the herb was determined as Swertia verticillifolia T. N. Ho et S. W. Liu (Gentianaceae), endemic to the region of the Qinghai-Tibet Plateau. The diagnostic characters:perennial; stem leaves in whorls; corolla campanulate, yellow-green, 4-lobed; nectary 1 per corolla lobe, naked. Also, its complete chloroplast (cp) genome was sequenced. It is 151 682 bp in length, including a large single copy (LSC) region of 82 623 bp, a small single copy (SSC) region of 18 335 bp and a pair of inverted repeats (IRs) of 25 362 bp. It contains 129 unique genes, including 84 protein-coding genes, 37 tRNAs and 8 rRNAs. This study provides information for understanding the diversity of Swertia cp genomes, and the alpine species identification, conservation and molecular phylogenetic researches of Swertia and Gentianaceae.
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Located in the transition zone between the Qinghai-Tibet Plateau and the Loess Plateau, Gansu province is one of the distribution centers of Sect. Cruciata, Gentiana (Gentianaceae) in China. Six species in the section, G. crassicaulis, G. straminea, G. siphonantha, G. officinalis, G. dahurica and G. macrophylla, are native to Gansu. In this paper, samples of 6 species and Halenia elliptica (outgroup) were collected. Nuclear DNA ITS, chloroplast DNA matK, rbcL, rpoC1, trnL (UAA) intron, psbA-trnH, atpB-rbcL, trnS (GCU)-trnG (UCC), rpl20-rps12 and trnL (UAA)-trnF (GAA) were sequenced from these samples. Based on the sequence analyses, high intragenomic polymorphisms were detected in ITS regions of G. crassicaulis, G. straminea, G. siphonantha, G. officinalis and G. dahurica, and they showed incomplete concerted evolution. A methodological study to identifying such close-related species as G. macrophylla, G. officinalis and G. dahurica was carried out based on the special genotypes. The results showed that 7 cp DNA sequence fragments could be used to identify G. crassicaulis, G. straminea and G. siphonantha. With nr ITS genotype II, III and IV of G. dahurica, the species can be distinguished from the close-related G. officinalis using 12 cloned sequences in a sample (with statistical significance). The cp DNA sequences of G. macrophylla were classified into two genotypes, and with genotype II, the species can be distinguished from the close-related G. officinalis and G. dahurica using 6 test samples each (with statistical significance). Furthermore, DNA barcode sequences were determined for all 6 species in Gansu. Also, the studies provide some basic data for analyses of genetic diversity and identification of Gentiana species.
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Objective: To construct the DNA barcoding of medicinal plants in genus Lycium L. from China based on sequences of ITS, nine chloroplast DNA segments, and two mitochondrial DNA segments. Methods: Specimens and samples of Lycium ruthenicum, L. barbarum var. auranticarpum, L. barbarum, L. chinense, and L. dasystemum were collected and nuclear DNA ITS, chloroplast DNA matK, rbcL, rpoC1, trnL (UAA) intron, psbA-trnH, atpB-rbcL, trnS (GCU)-trnG (UCC), rpl20-rps12, trnL (UAA)-trnF (GAA), and mitochondrial DNA nad1/b-c and nad5/d-e were sequenced from these samples. Gentiana lhassica was used as the external group to make statistical analysis. Results: One hundred and eight sequences were obtained. The mutation site of ITS region was the most abundant; The six chloroplast DNA fragments were selected; Two mitochondrial DNA fragments could be used as the bar code recommendation sequences of the three species from the genus, and the multiple genomic and multi fragment combination DNA bar code of each species was constructed. Conclusion: ITS region and the screening of six chloroplast DNA fragments have the significance of species identification; The two mitochondrial DNA fragments have a certain significance. The study may provide the reference for the species identification and genetic background of the plants in Lycium L. from China.
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The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.
Subject(s)
Genetic Variation , Gentiana , Classification , Genetics , Phylogeny , Plant Proteins , Genetics , Plants, Medicinal , Classification , Genetics , Scrophularia , Classification , Genetics , Swertia , Classification , Genetics , TibetABSTRACT
The alpine plant Gentiana robusta is an endemic species to the Sino-Himalayan subregion. Also, it is one of the original plants used as traditional Tibetan medicine Jie-Ji. We sequence the nuclear ribosomal internal transcribed spacer (ITS) regions, matK, rbcL, rpoC1, trnL (UAA), psbA-trnH, atpB-rbcL, trnS( GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF( GAA) fragments of cp DNA in both G. robusta and such relative species as G. straminea, G. crassicaulis and G. waltonii. With Halenia elliptica as the outgroup, molecular systematic analysis reveals that G. robusta is a natural hybrid. G. straminea is the mother of hybrids, but the father is not very clear. In addition, the molecular markers for distinguishing G. robusta from the parental species or closely related species are identified, respectively. Our studies may provide valuable reference for the species identifications of medicinal plants with complex genetic backgrounds.
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DNA Barcoding, Taxonomic , DNA, Plant , Genetics , Gentiana , Classification , Genetics , Molecular Sequence Data , Phylogeny , Plant Proteins , Genetics , Plants, Medicinal , Classification , GeneticsABSTRACT
This paper is aimed to microscopic identification of traditional Chinese medicines (TCMs) using an in situ imaging method. In this study, two kinds of Zingiberaceae seeds, Amomi Rotundus Fructus and Alpiniae Katsumadai Semen, were investigated by synchrotron radiation in-line X-ray phase-contrast computed tomography (IXPCT) imaging method. The results showed that the microstructures of these Zingiberaceae seeds could be clearly obtained from the virtual slices information in different observing angles. It proves that IXPCT is an effective imaging method, which can provide the imaging information for the microscopic identification of the intact TCMs in situ and non-destructively.
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Amomum , Cell Biology , Imaging, Three-Dimensional , Methods , Medicine, Chinese Traditional , Seeds , Cell Biology , Tomography, X-Ray ComputedABSTRACT
Objective: To identify the common Tibetan traditional medicine of Meconopsis Vig. using nuclear gene internal transcribed spacer (ITS) and chloroplast psbA-trnH sequence. Methods: Ethnopharmacological study was carried out, three species of Ou-Bei (M. torquata, M. punicea, and M. integrifolia) and two species of Cai-Wen (M. racemosa and M. horridula) were collected. The ribosomal DNA ITS and chloroplast psbA-trnH noncoding region sequences were determined and analyzed. Results: The ITS sequences of M. racemosa and M. horridula were completely the same, while variable site could be detected in each pairwise comparison of ITS sequences in other species; The psbA-trnH sequence analysis showed that the variable sites could be detected in each pairwise comparison of the sequences. The combination of them could be used to identify all the five species. Conclusion: The combination of ITS and psbA-trnH sequence could be used to identify the Tibetan traditional medicine Ou-Bei and Cai-Wen in Meconopsis Vig.
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<p><b>OBJECTIVE</b>To identify the common Tibetan herb Chuan-Bu.</p><p><b>METHOD</b>Local herbalists were visited to observe which plants were being used as Chuan-Bu. Samples of the indigenous plants were collected at the same time. Leaf materials were collected from field surveys. Total genomic DNA was extracted from silica gel-dried leaf samples. The PCR products were purified and directly sequenced.</p><p><b>RESULT</b>As the origin of Chuan-Bu in Tibet autonomous region was authenticated, two species were determined, i. e. Euphorbia stracheyiand E. wallichii. Also, based on our earlier research, the origin of Chuan-Bu in Gansu province, is from E. kansuensis. The sequences of ITS1 for E. stracheyi and E. wallichii were 261 bp in size, and 221 bp in ITS2, respectively. The size of the 5.8S coding region was 164 bp for all species examined in the genus. Especially, there was a heterozygous locus in ITS1 (C/G; position 72) for E. stracheyi. The nucleotide divergence between sequences of the 6 species in pairwise comparisons was calculated and the result showed that the variable site could be detected in each pairwise comparison of sequences. Also, there were 8 point mutations in the 5.8S coding region.</p><p><b>CONCLUSION</b>nrDNA ITS sequences can be used as the molecular markers to identify the Tibetan herb Chuan-Bu and such Traditional Chinese Medicines from the same genus Euphorbia as E. lathyris, E. humifusa and E. pekinensis.</p>
Subject(s)
DNA, Plant , Genetics , DNA, Ribosomal Spacer , Genetics , Euphorbia , Classification , Genetics , Genetic Markers , Molecular Sequence Data , Phylogeny , TibetABSTRACT
Objective: To study the genetic diversity of 17 samples of Gentiana straminea from different habitats of Tibet. Methods: Sampling in populations was carried out, and the related species G. crassicaulis from the same area was as the out group; nr DNA ITS sequences were amplified and analyzed; UPGMA systematic tree was constructed. Results: One polymorphic site of G. straminea from Tibet was found; the six samples of out group have the identical DNA ITS sequences. Conclusion: The difference between G. straminea and the out group is obvious; seventeen samples of G. straminea from different habitats are divided into two groups; genetic diversity is more abundant; the result could provide the basic reference for the quality evaluation and the construction of core germplasm of G. straminea in Tibet.
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Objective To study the expression of VEGF-C and its receptor in breast carcinoma tissue and in peritumoral tissue,as well as their clinic significance.Methods Immunohistochemistry SP method was used to examine the expression of VEGF-C and VEGFR3 in 70 cases of breast cancer and in its peritu- moral tissue.Results In all 70 cases of breast cancer,the positive expression rate of VEGF-C in breast car- cinoma tissue was 78.6 %,and its rate in peritumoral tissue was 54.3 %.There was a significant stastistic dif- ference between the two groups(P