Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

[Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus].

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979 946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

Molecular cloning and expression analysis of SRLK1 gene in self-incompatible Asteraceae species Erigeron breviscapus.

Based on the transcriptome data, using RACE techniques, we cloned the full-length EbSRLK1 gene in a medicinal, self-incompatible Asteraceae species, Erigeron breviscapus. Bioinformatics approaches were used to analyze the DNA and protein sequences, physical and chemical properties, and domains of the encoded protein. The full-length EbSRLK1 cDNA is 2891 base pairs (bp) with an open reading frame (ORF) of 2634 bp, which encodes the EbSRLK1 protein with 878 amino acids and an estimated molecular weight of 98.13 kD. The EbSRLK1 protein has the characteristic domain structure of S-locus receptor-like protein kinases, which contains one transmembrane domain but lacks the signal peptide. Quantitative real-time PCR (qRT-PCR) analysis showed that the EbSRLK1 gene is lowly expressed in roots, stems and leaves, but highly expressed in flowers, especially in flowers one day prior to opening. Western blot analysis showed that the EbSRLK1 protein is expressed in stems, leaves, and flowers, but is almost undetectable in roots. The EbSRLK1 protein expression is induced in self-pollinated but not in cross-pollinated E. breviscapus flowers. Cloning and expression analysis of EbSRLK1 lay a solid foundation for elucidating the role of EbSRLK1 in regulating self-incompatibility in E. breviscapus.

[A semimicroquality evaluation method on Panax notoginseng and its application in analysis of continuous cropping obstacles research samples].

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility-associated genes.

BACKGROUND: Self-incompatibility (SI) is a widespread and important mating system that promotes outcrossing in plants. Erigeron breviscapus, a medicinal herb used widely in traditional Chinese medicine, is a self-incompatible species of Asteraceae. However, the genetic characteristics of SI responses in E. breviscapus remain largely unknown. To understand the possible mechanisms of E. breviscapus in response to SI, we performed a comparative transcriptomic analysis with capitulum of E. breviscapus after self- and cross-pollination, which may provide valuable information for analyzing the candidate SI-associated genes of E. breviscapus. METHODS: Using a high-throughput next-generation sequencing (Illumina) approach, the transcriptionexpression profiling of the different genes of E. breviscapus were obtained, some results were verified by quantitative real time PCR (qRT-PCR). RESULTS: After assembly, 63,485 gene models were obtained (average gene size 882 bp; N50 = 1485 bp), among which 38,540 unigenes (60.70% of total gene models) were annotated by comparisons with four public databases (Nr, Swiss-Prot, KEGG and COG): 38,338 unigenes (60.38% of total gene models) showed high homology with sequences in the Nr database. Differentially expressed genes were identified among the three cDNA libraries (non-, self- and cross-pollinated capitulum of E. breviscapus), and approximately 230 genes might be associated with SI responses. Several these genes were upregulated in self-pollinated capitulum but downregulated in cross-pollinated capitulum, such as SRLK (SRK-like) and its downstream signal factor, MLPK. qRT-PCR confirmed that the expression patterns of EbSRLK1 and EbSRLK3 genes were not closely related to SI of E. breviscapus. CONCLUSIONS: This work represents the first large-scale analysis of gene expression in the self-pollinated and cross-pollinated flowers of E. breviscapus. A larger number of notable genes potentially involved in SI responses showed differential expression, including genes playing crucial roles in cell-cell communication, signal transduction and the pollination process. We thus hypothesized that those genes showing differential expression and encoding critical regulators of SI responses, such as MLPK, ARC1, CaM, Exo70A1, MAP, SF21 and Nod, might affect SI responses in E. breviscapus. Taken together, our study provides a pool of SI-related genes in E. breviscapus and offers a valuable resource for elucidating the mechanisms of SI in Asteraceae.

[Effects of seed priming on physiology of seed germination and seeding growth of Marsdenia tenacissima under NaCl stress].

To offer the reference and method for salt damage in the cultivation of Marsdenia tenacissima, the seeds of M. tenacissima collected from Maguan city ( Yunnan province) were taken as the test materials to study the effects of different priming materials on improving germination and growth under high-level salt stress condition. Four different treatments, which were GA3, KNO3-KH2PO4, PEG-6000, NaCl, combined with ANOVA were applied to test the performance of germination energy, germination percentage, germination index, MDA, SOD, and CAT. The results showed that the seed germination was obviously inhibited under salt stress and the soaked seeds with different priming materials could alleviate the damage of salt stress. Under these treatments, the activities of SOD, CAT the content of soluble protein significantly increased. While the content of MDA significantly decreased. The maximum index was obtained when treated with 1.20% KNO3-KH2PO4, the germination percentage increased from 52.67% to 87.33% and the activity of SOD increased from 138.01 to 219.44 respectively. Comparing with the treatment of 1.20% KNO3-KH2PO4, the germination percentage of treating with 300 mg x L(-1) GA3 increased from 52.67% to 80.67%, while the activity of SOD increased from 138.01 to 444.61.

[Wild resources survey of Marsdenia tenacissima in Honghe, Yunnan].

To ascertain current situation of wild Marsdenia tenacissima resources in Honghe, Yunnan province, the distribution, habitat characteristic and resources reserves of M. tenacissima were surveyed based on interviews and investigation. The results showed that M. tenacissima was found in 7 counties such as Jinping, Mengzi etc, and distributed mainly on the mountainsides from 800 m to 1 200 m. And distribution was affected by many factors, such as light, heat, topography, soil, and vegetation. M. tenacissima grew well in distribution areas. M. tenacissima had averagely a weight of 2.8 kg per plant. Resources reserve of M. tenacissima in Honghe was estimated to 1 300 tons by now but it reduced rapidly in resent years, the wild resources reserve may not meet demand of market. Resources protection and wildlife tending would be conducted to deal with increasing medication requirements.

[Contents of total anthocyanins and total saponins as well as composition of saponin monomers of Purple and Green Notoginseng Radix et Rhizoma].

OBJECTIVE: The contents of total anthocyanins and total saponins as well as the composition of saponin monomers of Purple and Green Notoginseng Radix et Rhizoma were studied to compare the medicinal quality and commercial values. METHODS: Three-year-old Notoginseng Radix et Rhizoma was selected as the research materials. The contents of total anthocyanins and total saponins were determined by spectrophotometry. The compositions of saponin monomers were monitored by HPLC. The significance of content differences was determined by variance analysis. RESULTS: The contents of total anthocyanins and total saponins of Purple Notoginseng Radix et Rhizomawere about 204.85% and 33.86% higher than those of Green Notoginseng Radix et Rhizoma respectively. The Purple and Green Notoginseng Radix et Rhizoma both contained five saponin monomers whose contents were as follows: ginsenoside Rg1 > ginsenoside Rb1 > notoginsenoside R1 > ginsenoside Rd > ginsenoside Re. The contents of notoginsenoside R1, ginsenoside Rd and ginsenoside Re of Purple Notoginseng Radix et Rhizoma were about 16.03%, 10.83% and 5.39% higher than those of Green Notoginseng Radix et Rhizoma respectively. However, the contents of ginsenoside Rg1 and ginsenoside Rb1 of Green Notoginseng Radix et Rhizoma were about 0.93% and 3.33% higher than those of Purple Notoginseng Radix et Rhizoma respectively. With respect to Green Notoginseng Radix et Rhizoma, the increase of the total anthocyanins in Purple Notoginseng Radix et Rhizoma reached a significant level, but the increases of total saponins, notoginsenoside R1, ginsenoside Rd and ginsenoside Re and the decreases of ginsenoside Rg1 and ginsenoside Rb1 did not. CONCLUSION: The total anthocyanins accumulation in Notoginseng Radix et Rhizoma implies the content increases of the total saponins, notoginsenoside R1, ginsenoside Rd and ginsenoside Re, and the slight decreases of ginsenoside Rg1 and ginsenoside Rb1 contents; but the type and relative contents of saponin monomers remain unchanged. The medicinal quality and commercial value of Purple Notoginseng Radix et Rhizoma are higher than those of Green Notoginseng Radix et Rhizoma.

[FTIR fingerprint spectrograms of traditional Chinese medicine Marsdenia tenacissima].

In this paper, Fourier transform infrared spectroscopy fingerprint analysis of Marsdenia tenacissima samples was used to develop a reliable method of tracing the geographical origins. Forty-eight samples from four provinces of China were analyzed by FTIR. We analyzed and characterized the fingerprints in both the full spectrum peaks and characteristic peaks, then the principal component analysis and the cluster analysis were carried out. The results of fingerprint analysis, correlation analysis, principal component analysis and cluster analysis can identify the geographic origins correctly, which verified and supplemented each other; the identification results and the actual location showed a high degree of consistency, namely the lower the space distance, the greater the similarity of different samples. These results revealed the obvious superiority and practical value in comparison to the more tedious and time-consuming wet chemistry method normally used. Using appropriate metrology methods can trace the geographical source correctly. The M. tenacissima materials from the region of Maguan should be considered as genuine medicinal materials taking into account the good quality.

[SSR information in Erigeron breviscapus transcriptome and polymorphism analysis].

OBJECTIVE: The SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation. METHOD: SSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places. RESULT: A total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups. CONCLUSION: There are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.

[SSR information in transcriptome of Pinellia ternata].

OBJECTIVE: In order to establish new molecular markers and to design SSR primers,the SSR information was analyzed in the transcriptome of Pinellia ternata. METHODS: The unigenes in transcriptome were filtered and then redundant sequences were removed by the tool of EST-trimmer and cross-match, respectively. Meanwhile, the non-redundant unigenes were examined by the tool of MISA. RESULTS: 14,468 SSRs were found, and these SSRs were distributed in 12 000 unigenes in the examined sequences. Frequency of occurrences for SSRs was 16.24%, and density of distribution was on average 1/4.33 kb in the present study. Di-nucleotide and tri-nucleotide were the major repeated types,accounting for 51.15% and 41.50%, respectively. AG/CT and CCG/CGG were most frequent motifs in di-nucleotide and tri-nucleotide, accounting for 41.54% and 11. 84%, respectively. 87.14% motifs in the length ranged from 12 bp to 20 bp. CONCLUSION: The SSR site in the transcriptome of Pinellia ternata have the characteristics of high frequency of occurrences, type richness, high density and high potential of polymorphism, and these characteristics might be applied in the study on genetic diversity, marker-assisted breeding,and genetic map of Pinellia ternata.

Optimization of saponin extraction from the leaves of Panax notoginseng and Panax quinquefolium and evaluation of their antioxidant, antihypertensive, hypoglycemic and anti-inflammatory activities.

Panax notoginseng and Panax quinquefolium are important economic plants that utilize dried roots for medicinal and food dual purposes; there is still insufficient research of their stems and leaves, which also contain triterpenoid saponins. The extraction process was developed with a total saponin content of 12.30 ± 0.34% and 12.19 ± 0.64% for P. notoginseng leaves (PNL) and P. quinquefolium leaves (PQL) extracts, respectively. PNL and PQL saponin extracts showed good antioxidant, antihypertensive, hypoglycemic, and anti-inflammatory properties in vitro and RAW264.7 cells. A total of 699 metabolites were identified in PNL and PQL saponin extracts, with the majority being triterpenoid saponins, flavonoids and amino acids. Fourteen ginsenosides, 18 flavonoids or alkaloids, and 16 amino acids were enriched in both saponin extracts. Overall, the utilization of saponins from medicinal plants PNL and PQL has been developed to facilitate systematic research in the functional food and natural product industries.

Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Three unusual sesquineolignans from Alpinia conchigera.

Three unusual sesquineolignans conchignans A, B, and C, together with two known compounds vanillin and phloroglucinol, were isolated from the whole plants of Alpinia conchigera. Their structures were established by spectroscopic analysis, including 2D NMR spectroscopic techniques.