Pengguo Xia

High-performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

Molecular cloning and characterization of five SmGRAS genes associated with tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

The gibberellin-responsive element binding factor (GRAS) family of proteins plays an important role in the transcriptional regulation of plant development and hormone signaling. To date, there are no reports on GRAS family proteins expressed in Salvia miltiorrhiza. In this study, 28 ESTs that contained the GRAS domain were identified from a S. miltiorrhiza cDNA library. Of these, full-length sequences of five genes were cloned and sequence analysis indicated that all five proteins contain only one GRAS domain and therefore, belong to the GRAS family. The five genes were designated S. miltiorrhiza GRAS1-5 (SmGRAS1-5), which belong to groups I (SmGRAS2 and SmGRAS4), II (SmGRAS3), III (SmGRAS1), and VIII (SmGRAS5) respectively. Additionally, SmGRAS1-5 have different expression patterns in the reed head, stems, leaves, flowers, and roots of S. miltiorrhiza. In this study, the expression of SmGRAS1-5 was sensitive to Gibberellin (GA) stress and that of SmGRAS1, SmGRAS4 and SmGRAS5 was sensitive to Ethephon (Eth) stress respectively. Moreover, S. miltiorrhiza copalyl diphosphate synthases 1 (SmCPS1) and S. miltiorrhiza kaurene synthase like 1 (SmKSL1), which are two key enzymes gene in the diterpenoid biosynthesis pathway, were also response to GA and Eth stress. In addition, Dihydrotanshinone (DT-I) and Tanshinone I (T-I) content were enhanced by GA and Eth stress, Tanshinone IIA (T-IIA) content was increased by GA stress, and the accumulation of Cryptotanshinone (CT) was insensitive to both GA and Eth stress. Together, these results provide insights into functional conservation and diversification of SmGRASs and are useful information for further elucidating SmGRAS functions.

Identification of seeds based on molecular markers and secondary metabolites in Senna obtusifolia and Senna occidentalis

BackgroundSenna obtusifolia and Senna occidentalis (Leguminosae), whose seeds have similar appearance and chemical constituents, are easily confused in using their seeds. To elucidate the similarities and differences between S. obtusifolia seeds and S. occidentalis seeds, three molecular markers and high performance liquid chromatography (HPLC) were employed to evaluate the seeds characteristics of these two medicinal herbs.ResultsThe results showed that selected 3 ISSR and 7 SCoT primers could distinguish S. obtusifolia seeds from S. occidentalis seeds based on the specific band and UPGMA dendrogram. ITS2 sequence indicated that the intra-specific similarity of 20 S. obtusifolia and 16 S. occidentalis was 99.79 and 100.0%, respectively, while the inter-specific similarity between S. obtusifolia and S. occidentalis was 89.58%. Although phylogenetic analysis revealed that these two species had a close relationship, they were assigned to different branches. HPLC fingerprint results showed that seeds of S. obtusifolia and S. occidentalis shared some secondary metabolites, but aurantio-obtusin was not detected in S. occidentalis seeds which could differentiate S. obtusifolia seeds from S. occidentalis seeds.ConclusionsThe present study not only compared the seeds characters of S. obtusifolia and S. occidentalis from molecular and secondary metabolites levels, but also provided a convenient method to identify S. obtusifolia seeds and S. occidentalis seeds effectively.

Genetic diversity and population structure assessment of Chinese Senna obtusifolia L. by molecular markers and morphological traits of seed

Senna obtusifolia L. is an important medicinal plant in Asia. This study was the first report on the genetic diversity and population structure of S. obtusifolia which were collected from 47 geographic populations widespread in China. Inter-Simple Sequence Repeat (ISSR) and Start Codon Target Polymorphism (SCoT) combined with seeds morphological traits were used to investigate the relationship of 47 populations. 11 ISSR primers yielded 98 polymorphic bands with 81.67% polymorphism. 24 SCoT primers yielded 267 polymorphic bands with 89.59% polymorphism. The number of allele (Na), the number of effective allele (Ne), Nei’s diversity index (H), and Shannon’s information index (I) reflected a high level of genetic diversity of S. obtusifolia species. The greatest genetic distance (GD) existed between Southwest and Northwest (0.4022ISSR/0.5019SCoT), while the Eastern and Northern showed the least genetic distance (0.1751ISSR/0.2186SCoT). The genetic differentiation (Gst) was 0.4875ISSR/0.4434SCoT, and the gene flow (Nm) was 0.5256ISSR/0.6275SCoT, which indicated that gene exchange among four regions was limited. 47 samples were divided into four clusters mainly according to their geographic distribution through clustering and structure analysis. The analysis on the combined data of ISSR and SCoT showed more reliable and superior results than single analysis of ISSR and SCoT. This study explored the effectiveness of ISSR and SCoT markers to evaluate the genetic diversity and population structure of S. obtusifolia and provided useful information for S. obtusifolia germplasm research and breeding program.

The ethylene response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Main ConclusionThe SmERF6, which recognizes the GCC-box of SmCPS1 and SmKSL1 promoter in nucleus, regulates the tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.Tanshinone, an important medicinal ingredient in Salvia miltiorrhiza, is best known for its use in medicine. However, the transcription factor regulation of tanshinone biosynthesis is unclear. Here, we isolated and identified a transcription factor in the ERF family of S. miltiorrhiza, SmERF6, which was screened from an S. miltiorrhiza cDNA library by the promoters of two key tanshinone synthesis genes (SmKSL1 and SmCPS1); this factor regulated tanshinone biosynthesis. The gene was highly expressed in the root and responded to ethylene treatment. SmERF6 modulated tanshinone biosynthesis by directly binding to an ethylene-responsive element (GCC-box) of the SmKSL1 and SmCPS1 promoters and activating their transcription. Overexpression of SmERF6 in the hairy roots increased their tanshinone accumulation, and SmERF6 silencing by RNAi led to a lower tanshinone content. Furthermore, tanshinone accumulation maintained homeostasis with the total phenolic acid and flavonoid contents in S. miltiorrhiza. These findings elucidated how SmERF6 directly co-regulates the transcription of SmCPS1 and SmKSL1 and modulates tanshinone synthesis to accelerate the metabolic flux of tanshinone accumulation in S. miltiorrhiza.

Diverse responses of tanshinone biosynthesis to biotic and abiotic elicitors in hairy root cultures of Salvia miltiorrhiza and Salvia castanea Diels f . tomentosa

Salvia miltiorrhiza (S. miltiorrhiza) and Salvia castanea Diels f. tomentosa (S. castanea) are both used for treatment of cardiovascular diseases. They have the same bioactive compound tanshinones, but whose contents are hugely different. This study illustrated diverse responses of tanshinone biosynthesis to yeast extract (YE) and Ag+ in hairy roots of the two species. YE enhanced both the growth and tanshinone biosynthesis of two hairy roots, and contributed more to tanshinone accumulation in S. castanea than that in S. miltiorrhiza. Genes encoding 1-deoxy-d-xylulose 5-phosphate synthase (DXS2), geranylgeranyl diphosphatesynthase (GGPPS1), copalyl diphosphate synthase (CPS1), and two cytochromes P450 (CYP76AH1 and CYP76AH3) were also more responsive to YE in S. castanea than those in S. miltiorrhiza. Accumulations of dihydrotanshinone I and tanshinone I, and most biosynthetic genes in S. miltiorrhiza were more responsive to Ag+ than those in S. castanea. Accumulations of dihydrotanshinone I and cryptotanshinone were more responsive to YE, while tanshinone IIA accumulation was more responsive to Ag+ in S. miltiorrhiza. However, accumulations of other four tanshinones and related genes in S. castanea were more responsive to YE than Ag+. This study provides foundations for studying diverse specialized metabolism between the related species.