Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora / 中国中药杂志

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza / 中国中药杂志

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

Research progress of natural borneol resources / 中国中药杂志

Natural borneol is an important traditional Chinese medicine herb with resuscitation-inducing, antipyretic and analgesic effects, and has been widely used in the fields of medicine, perfume and chemical industry. At present, natural borneol is short supply, with promising market development prospects. This paper summarized the distribution of borneol plant resources, cultivation status and molecular biological research progress, in the expectation of providing basis and ideas for the research and application of natural borneol.

Biopathway construction of plant natural products / 药学学报

Plant natural products (PNPs) are important sources of innovative drugs. They are mainly obtained by isolation or extraction from plants. Low content and with structural analogues in plants result in high production cost, which restricts the research and application of PNPs. While biopathway construction by synthetic biology provides an alternative for production of PNPs. By biosynthetic pathway analysis of PNPs and reconstructing the biopathway in microorganisms, we can produce PNPs in cell factories efficiently. Recently, several predominantly international reports about biosynthesis of PNPs and its synthetic biology production, triggered the researches of PNPs. Abundant traditional Chinese medicine resources and profound cultural heritage of Chinese medicine make biosynthesis pathway analysis of PNPs to be a research hotspot. And some of the studies have achieved significant progress. Here, recent progress in the biosynthesis of plant natural products and its synthetic biology was reviewed. In particular, the application of new methods and technologies in recent years were summarized and discussed. This will provide reference for the biopathway construction of plant natural products.

Transcriptome analysis to identify genes involved in the biosynthesis of aconitines in Aconitum pendulum / 药学学报

italic>Aconitum pendulum is a Tibetan medicine that is rich in bioactive compounds such as aconitine-type C19-diterpenoid alkaloids. To investigate the key enzymes in the aconitine biosynthesis pathway, roots, leaves and flowers of Aconitum pendulum were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM2000. Trinity de novo assembly yielded 47 264 unigenes with an average length of 1 140 bp and N50 of 1 678 bp, of which 30 231 unigenes (63.96%) were annotated. In the KEGG database, 542 unigenes were implicated in 17 secondary metabolic pathways; the analysis showed that 44 genes encoded 20 key enzymes in the diterpene skeleton of aconitine biosynthesis and 12 BAHD acyltransferase genes were related to the acetylation modification, with differential expression among three organs. For example, ApTPS8 was the only committed enzyme in the upstream aconitine biosynthetic pathway. The high expression level of ApTPS8 in root indicated that it is the main tissue for the production of precursors of diterpene alkaloids. Consistent with the accumulation of aconitine, we propose that ApBAHD1/2/8 is involved in the biosynthesis of 2-hydroxyaconitine, dehydrated 14-benzoylaconitine, 8-O-methyl-14-benzoylaconine, benzoyldeoxyaconitine and benzoylaconitine, and ApBAHD10 is involved in the biosynthesis of acontine, lucidusculine, 14-O-acetylneoline and 14-O-acetylvirescenin. Comparative transcriptome analysis of A. pendulum and A. carmichaeli indicates significant gene loss in the family of diterpene synthases and acyltransferases in A. pendulum, which is in accordance with the significantly fewer type and quantity of aconitine compounds in this species. Therefore, A. pendulum has proved to be an ideal material for the study of the aconitine biosynthesis pathway. This work provides basic scientific data for further study of aconitine biosynthesis, the discussion of molecular mechanisms of toxicity, and the synthesis of genuine medicinal materials.

Research progress of tissue culture of Aconitum / 中国中药杂志

Aconitum is a kind of important medicinal plant, which has been used in China for more than 2 000 years, with both a good medicinal and ornamental value. However, due to the lack of effective breeding methods and low seed and root propagation coefficients, the comprehensive development and utilization of Aconitum were greatly restricted. Tissue culture is an important basis for seed selection, germplasm conservation and genetic engineering. Therefore, this paper summarized the research on tissue culture of Aconitum, put forward the main problems and corresponding countermeasures, and provided important references for accelerating the seedling breeding of Aconitum and conducting the basic research of molecular biology.

Research progress of P450 in the biosynthesis of bioactive compound of medicinal plants / 药学学报

Most of the active ingredients of herbs are secondary metabolites of plants. Cytochrome P450s (P450s) are hemoglobin-containing monooxygenases encoded by a super-gene family, which play important roles in the metabolic network of plants. This review focuses on the role of P450s on biosynthesis of secondary metabolites such as terpenoids, alkaloids, flavonoids and phenylpropanoids. This will provide references for biosynthesis and regulation of secondary metabolites in medicinal plants.

Limitation standard of toxic aconitines in proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 proprietary Chinese medicines (APCMs). Multi-type dosage forms (, tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

Accumulation and biosynthetic of curcuminoids and terpenoids in turmeric rhizome in different development periods / 中国中药杂志

The dynamic accumulation rule of active substances in medicinal plants is of great value not only for medicinal material production and application,but also for the genetic mechanism study on the formation of medicinal ingredients,especially vital to guide medicinal material collection as well as experiment material selection and candidate gene screening in the analysis of biosynthesis pathway. This study investigated the accumulation of curcumins and terpenoids,and the biosynthesis of these metabolites,which are the active metabolites in Curcuma longa,a commonly used traditional Chinese medicine. Rhizoma of C. longa from leaf growing period,rhizome swelling period and dry matter accumulating period were used as experimental materials,to analyze the changes of metabolites and biosynthesis in the three periods by comparative transcriptome and metabolomes analysis.The results indicated that terpenoids accumulation and biosynthesis mainly occurred in leaf growing period,while curcumin accumulation and biosynthesis mainly occurred in dry matter accumulating period. Therefore,we suggested that turmeric rhizomes in leaf growth period were suitable for terpenoids biosynthetic pathway characterization,and rhizome in accumulation of dry matter period was suitable for curcuminoid biosynthesis pathway characterization. This study provides references for medicinal materialproduction and application,as well as biopathway analysis of active compounds for C. longa.

Molecular cloning and SNP analysis of a acetyl-CoA C-acetyltransferase gene (SmAACT) from Salvia miltiorrhiza / 药学学报

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.

Cloning and polymorphism analysis of SmERF in Salvia miltiorrhiza / 药学学报

The transcription factor of ethylene responsive factor binding protein (ERF) is belonged to AP2/ERF superfamily, which is known to be unique in plants. AP2/ERF proteins have important functions in the transcriptional regulation of a variety of biological processes related to growth and development, as well as various responses to environmental stimuli. An ERF gene from Salvia miltiorrhiza is cloned and divided into ERF gene family group VII of Arabidopsis and Rice. It contains a MCGGAI (I/L) motif referred to as CMVII-1 and a single intron in the 5'-flanking region of the AP2/ERF domain. Sequence analysis reveals that the region of second extron has abundant polymorphism sites. There are 21 single nucleotide polymorphism sites (SNPs) in the 264 bp region, among them, 14 SNPs are synonymous substitutions and 7 SNPs are non-synonymous substitutions. Though analysis of 181 samples from Shandong, Shaanxi and Sichuan Provinces, it reveals that each production area has its own special genotypes, 5 SNPs show significant difference. Cluster based on UPGMA method reveals that different populations from specific province have clustered together. It shows that SmERF gene will be a candidate molecular marker for the identification of Salvia miltiorrhiza from different areas.

The establishment of PCR system to identify Bungarus multicinctus rapidly / 药学学报

The purpose of the present study is to establish a rapid and effective PCR method for the identification of B. multicinctus. Based on sequence alignment of B. multicinctus and its adulterants, we found that Cyt b gene is a good molecular genetic marker for the authentication of B. multicinctus. On the basis of the sequence data, a pair of highly specialized primers was designed. The templates were extracted by the DNA purification system. Key factors such as annealing temperature, concentration of Taq enzyme and cycle numbers were analyzed and optimized. The modified PCR program consisted of an initial denaturation step at 95 degrees C for 5 min, followed by 30 cycles of 95 degrees C for 30 s and 55 degrees C for 45 s and a final extension at 72 degrees C for 5 min. Thirteen samples of B. multicinctus were identified accurately from their 20 adulterants in 4 hours. The results indicated it is a highly accurate, rapid and applicable method for the authentication of B. multicinctus.

A taxonomic study on the original plant of radix astragali / 药学学报

There are dispute about the status of taxonomy among Astragalus membranaceus (Fisch.) Bge, A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. and A. pallidipurpureus stat. nov. The varieties and taxa of the complex are still in need of revision. With molecular biology study used trnH-psbA intergenic region, the taxonomic revision of Radix Astragali has been made. A. pallidipurpureus stat. nov is suggested as a new species.

Genetic relationships of Atractylodes plants / 药学学报

The phylogenetic relationships of the genus Atractylodes DC. was analyzed using the nuclear ribosomal ITS and three chloroplast fragments, including atpB-rbcL, psbB-psbF and trnL-trnF intergenic spacer (IGS) sequences. Phylogenetic analyses revealed that A. lancea subsp. luotianensis and A. lancea, A. chinensis var. liaotungensis and A. coreana form monophyletic terminal clade, separately. The trees, within each the pairwise genetic distances, did not support Hu's classification. Authors suggested that A. lancea Subsp. luotianensis should be included in A. lancea rather than be treated as a separate subspecies. A. carlinoides was placed in the basal position of Atractylodes, which had a distant relationship with the others of the genus. The results lead us to suggest that A. chinensis var. liaotungensis be put into A. coreana, A. chinensis as a subspecies of A. lancea.

A full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase cloning and analysis of introduced gene expression in Salvia miltiorrhiza / 药学学报

This paper firstly introduced the acquired full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from hairy roots of Salvia miltiorrhiza (Abbr: SmCMK, GenBank number: EF534309). Results of KEGG analysis showed that SmCMK was belong to the upstream of nonemevalonate pathway, the only one kinase of the pathway. The full-length cDNA was deduced as encoding 4-(cytidine 5'-diphospho)-2-C-methylerythritol kinase (designated as SmCMK), and the sequence had a 1493 bp including 5' UTR 71 bp and 3' UTR 232 bp, an open reading frame (ORF) encoding a protein of 396 amino acid residues. The deduced protein had isoelectric point (pI) of 6.78 and a calculated molecular weight about 43 kDa, similar to cloned diterpene of CMK from other species of plants such as Mentha piperita and Lycopersicon esculentum reported previously. Real time PCR results indicated that elicitors of MJ stimulated the increase of mRNA expression of SmCMK. At the same time, results of high performance liquid chromatography (HPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy root of Salvia miltiorrhiza were increased dramatically after treated with methyl jasmonate (MJ). This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by MJ. It proved primarily that the increased expression level of mRNA of SmCMK helps to enhance tanshinones' accumulation, which will be the basis for further study on the mechanism of gene regulation of secondary metabolism of tanshinones.

Preliminary study on variation pattern of Cistanche deserticola / 中国中药杂志

<p><b>OBJECTIVE</b>Through analyze of shape, structure and content of effective components of Cistanche deserticola, the variation of "you cong rong" is discussed.</p><p><b>METHOD</b>The color surface, texture, the size of pith and the arrange of vascular bundle was observed with freshed, dried drugs and the transverse section, the effective component of echinacoside and acteoside was analyzed with hplc and the amylose was analyzed with ultraviolet spectrophotometry.</p><p><b>RESULT</b>The variation of "you cong rong" is obvious different with the common drug in shape and structure, the content of echinacoside is 2.5 times, acteoside is 3.8 times, amylose yield is 1.6 times and the content is 2 times of the common drugs.</p><p><b>CONCLUSION</b>"you cong rong" is an independent variation pattern of C. deserticola, it has no relationship with the ecological and geographical conditions.</p>

Optimizing expression and purification of recombinant Salvia miltiorrhiza copalyl diphosphate synthase protein in E. coli and preparation of rabbit antiserum against SmCPS / 药学学报

The expression plasmid pET32CPS harboring SmCPS gene was transformed into E. coli BL21 trxB (DE3) resulting in recombinant strain E. coli [pET32CPS]. The induction of E. coli [pET32CPS] in different temperatures, induction time, IPTG concentrations and A600 values of E. coli were performed. The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis, and the ratio of the interest protein to total proteins reached to 35.6%. The recombinant SmCPS protein purified by Ni2+ affinity chromatography column was identified by SDS-PAGE and Western blotting, and then used for rabbit immunization. The titer of the rabbit antiserum against SmCPS was about 1:24 300 after the third immunization, and could specifically recognize the antigen of SmCPS protein by Western blotting analysis. The successful preparation of polyclonal antibody against SmCPS laid a foundation for further correlative study between expression of SmCPS and the production of tanshinones in protein level.

Authentication of Pinellia pedatisecta and their adulterants by AS-PCR / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a molecular method for the authentication of Pinellia pedatisecta and its adulterants.</p><p><b>METHOD</b>DNA sequences of some species from P. tenore, Typhonium and Arisaema were downloaded from GenBank, the sequences were aligned using DNAMAN. Allele-specific primers for P. pedatisecta and P. tenore were designed according to their SNPs in rpl 20 sequence. The designed primers were used to amplify 10 samples of P. pedatisecta, P. ternata and T. flagelliforme.</p><p><b>RESULT</b>A 351 bp band was amplified from P. pedatisecta but not form P. ternata and T. flagelliforme by primer Pprpl149F and Pprpl484R. A 630 bp band was amplified from P. ternate and P. pedatisecta but not from T. flagelliforme by primer Ptrpl94F and Ptrpl699R.</p><p><b>CONCLUSION</b>AS-PCR has the advantages of highly specific and good reproducibility, by which P. pedatisecta can be identified from part of its adulterants quickly. It is a potential method to be used in the molecular identification of other materia medica.</p>

Functional genomics studies of Salvia miltiorrhiza III. analyze of metallothionein (MT-2) genes / 中国中药杂志

<p><b>OBJECTIVE</b>To study the metallothionein genes of Salvia miltiorrhiza through bioinformatics and characterization of tissue expression in regenerated shoots.</p><p><b>METHOD</b>Metallothionein genes were obtained by cDNA microarray analyze. BLAST was used for align, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT-PCR method was used to detect the gene expression level.</p><p><b>RESULT</b>Two metallothionein genes were obtained which were contained a deduced amino acid sequence of 80 and 79 residues, named as SmMT-2a and SmMT-2b, they had a homology of 71.25%. Semiquantitative RT-PCR indicated that metallothionein genes were expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in leaf than in root and stem.</p><p><b>CONCLUSION</b>It was the first time that metallothionein genes were obtained from S. miltiorrhiza. It provides a good basis for further functional study of S. miltiorrhiza.</p>

A new molecular identification method: anchored primer amplification polymorphism DNA / 药学学报

To build up a stable and easy doing method for molecular identification in traditional Chinese medicine, on basis of RAPD, the new method mainly changed the primer length and PCR annealing temperature. Panax ginseng, Panax quinquefolius and its nine adulterants were used to establish the method and test it using MARMS primers published in 2004. The new method also used to authenticate Chinese Materia Medica of Tian-hua-fen (Radix Trichosanthes) and Bai-zhi (Radix Angelica). Primer Pg-q36F obtained polymorphic bands of P. Ginseng, P. quinquefolius and its adulterants. The identification result is identical to that published before and more stable. Primer TkS1-64F obtained polymorphic bands of Tian-hua-fen and its nine adulterants. Primer AfS1-100F obtained polymorphic bands of Bai-zhi and its three adulterants. The method has good stability and reproducibility and can easily identify authertic medicines from their adulterants. It was a potential molecular method to identify other Chinese Materia Medica. The method was named as anchored primer amplification polymorphism DNA (APAPD).