Novel SNP markers on ginsenosides biosynthesis functional gene for authentication of ginseng herbs and commercial products / 中国天然药物

Panax ginseng and Panax quinquefolius have similar bioactive components and morphological characteristics, but they are known to have different medicinal values, high-sensitive and accurate method is expected to identify the sources of ginseng products and evaluate the quality, but with a huge challenge. Our established UHPLC-TOF/MS method coupled with orthogonal partial least squares discriminant analysis (OPLS-DA) model based on 18 ginsenosides was applied to discriminate the sources of raw medicinal materials in ginseng products, and nested PCR strategy was used to discover 6 novel single nucleotide polymorphism (SNP) sites in functional dammarenediol synthase (DS) gene for genetic authentication of P. ginseng and P. quinquefolius for the first time. OPLS-DA model could identify the sources of raw ginseng materials are real or not. SNP markers were applied to identify ginseng fresh samples as well as commercial products, and proved to be successful. This established molecular method can tell exact source information of adulterants, and it was highly sensitive and specific even when total DNA amount was only 0.1 ng and the adulteration was as low as 1%. Therefore, this study made an attempt at the exploration of new type SNP marker for variety authentication and function regulation at the same time, and the combination of chemical and molecular discrimination methods provided the comprehensive evaluation and authentication for the sources of ginseng herbs and products.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Discovery of differential sequences for improving breeding and yield of cultivated Ophiocordyceps sinensis through ITS sequencing and phylogenetic analysis / 中国天然药物

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.

Comparison of identification and medicinal progress of Hedyotis diffusa and H. corymbosa / 中草药

Hedyotis diffusa and H. corymbosa belong to the genus Hedyotis Linn. in family Rubiaceae. They shared similar heat-clearing and toxin-resolving effects and pharmacological activities, such as anti-cancer activity, and H. diffusa is frequently adulterated or substituted by H. corymbosa in clinical application. But whether they can completely replace with each other or whether the therapeutic effect could be changed after the substitution, is still a problem needs to be resolved. Therefore, the two herbs were compared in terms of the textual research, medical history, mixed use, commercial medicine status, macro-characters, micro-characteristic, chemical compositions and pharmacologic activities, and found that they shared similar macro-characters and micro-characteristic but were obviously different in chemical composition and pharmacologic activities, and now they could be identified quickly and accurately by molecular identification method. Therefore, we suggest that they should be used as two different medicinal materials at this stage.

Cloning and bioinformatics analysis of ent-kaurene oxidase synthase gene in Salvia miltiorrhiza / 中国中药杂志

Based on the transcriptome database of Salvia miltiorrhiza, specific primers were designed to clone a full-length cDNA of ent-kaurene oxidase synthase (SmKOL) using the RACE strategy. ORF Finder was used to find the open reading frame of SmKOL cDNA, and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5.1. The transcription level of SmKOL from the hairy roots induced by elicitor methyl jasmonate (MeJA) was qualifiedby real-time quantitative PCR. The full length of SmKOL cDNA was of 1 884 bp nucleotides encoding 519 amino acids. The molecular weight of the SmKOL protein was about 58.88 kDa with isoelectric point (pI) of 7.62. Results of real-time quantitative PCR analyses indicated that the level of SmKOL mRNA expression in hairy roots was increased by elicitor oMeJA, and reached maximum in 36 h. The full-length cDNA of SmKOL was cloned from S. miltiorrhiza hairy root, which provides a target gene for further studies of its function, gibberellin biosynthesis and regulation of secondary metabolites.

Cloning and induced expression analysis of 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase gene (smHDR) of Salvia miltiorrhiza / 药学学报

This study reported the obtainment of the full-length cDNA of Salvia miltiorrhiza hairy roots (Abbr: SmHDR, GenBank number: JX233817), via extracting Salvia miltiorrhiza hairy roots total RNA, designing specific primers according to the transcriptome data and using the RACE strategy, and then analyzed it with bioinformatics approaches. On this basis, using the real-time PCR to detect SmHDR gene expression after Ag+ induction, and testing tanshinones contents of corresponding samples by UPLC. SmHDR has 1 647 nucleotides, and an open reading frame (ORF) encoding a protein of 463 amino acid residues. The deduced protein has isoelectric point (pI) of 5.72 and a calculated molecular weight about 51.88 kD. In the secondary structure, the percentage of alpha helix, beta turn and random coil were 35.64%, 20.30% and 44.06%, respectively. Sequence alignment and phylogenetic analysis demonstrated that SmHDR had relative close relationship to the HDR of Picrorhiza kurrooa, similar to HDR from other species of plants. Real time PCR results indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmHDR. At the same time, results of ultra performance liquid chromatography (UPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy roots of Salvia miltiorrhiza were increased dramatically at 12 h after treated with Ag+, and then decreased significantly. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by Ag+. The content of tanshinones was gradually raised, and it had an obvious increase at 120 h. The bioinformatics analysis and gene expression indicated that SmHDR might be involved in tanshinones biosynthesis, which laid the foundation for further study of secondary metabolic regulation mechanism of tanshinones.

Advances in studies on 3-hydroxy-3-metllylglutaryl coenzyme A reductase in terpenoids biosynthesis of medicinal plants / 中国中药杂志

There exists many kinds and a huge number of terpenoid in medicinal plants, which show a wide range of pharmacological activities. 3-Hydroxy-3-metllylglutaryl coenzyme A reductase(HMGR) is a key rate-limiting enzyme in terpenoid biosynthetic pathway . HMGR plays an important role in the regulation of secondary metabolism of the terpenoid. The paper summarized the biological function and the catalytic mechanism of HMGR, the cloning and the structure of the gene as well as its research progress in some medicinal plants.

Cloning and bioinformatics analysis of 2-c-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene in Salvia miltiorrhiza / 中国中药杂志

<p><b>OBJECTIVE</b>To clone and analysis a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (SmMCS) full-length eDNA from Salvia miltiorrhiza hairy roots.</p><p><b>METHOD</b>A full-length eDNA of SmMCS has been cloned by designing specific primers according to the transcriptome database and using the RACE strategy. ORF Finder was used to find the open reading frame of SmMCS cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5. 1. Real-time quantitative PCR have been applied to detect the transcription level of SmMCS from hairy roots after elicitor Ag+ supplied.</p><p><b>RESULT</b>The SmMCS cDNA sequence was obtained. The full length of SmMCS (DNA was 988 bp encoding 234 amino acids. The deduced protein had isoelectric point (pI) of 8.53 and a calculated molecular weight about 24. 6 kDa. Results of real time PCR indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmMCS in hairy roots, and were increased dramatically at 12 h.</p><p><b>CONCLUSION</b>The full-length cDNA of SmMCS was cloned from S. miltiorrhiza hairy root,which can provide a gene target for further studies of tanshinones biosynthesis and terpenoid secondary metabolites.</p>