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1.
Acta Pharmaceutica Sinica ; (12): 3285-3299, 2021.
Article in Chinese | WPRIM | ID: wpr-906851

ABSTRACT

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.

2.
Acta Pharmaceutica Sinica ; (12): 3353-3361, 2021.
Article in Chinese | WPRIM | ID: wpr-906835

ABSTRACT

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.

3.
Article in Chinese | WPRIM | ID: wpr-887953

ABSTRACT

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.


Subject(s)
Abietanes , Microbiota , Plant Roots , Salvia miltiorrhiza
4.
Article in Chinese | WPRIM | ID: wpr-878911

ABSTRACT

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.


Subject(s)
Camphanes , Drugs, Chinese Herbal , Medicine, Chinese Traditional
5.
Acta Pharmaceutica Sinica ; (12): 1573-1589, 2020.
Article in Chinese | WPRIM | ID: wpr-823295

ABSTRACT

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.

6.
Article in Chinese | WPRIM | ID: wpr-878824

ABSTRACT

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.


Subject(s)
Aconitum , China , Plant Breeding , Plants, Medicinal , Seedlings
7.
Acta Pharmaceutica Sinica B ; (6): 1511-1520, 2020.
Article in English | WPRIM | ID: wpr-828793

ABSTRACT

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.

8.
Article in Chinese | WPRIM | ID: wpr-777536

ABSTRACT

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.


Subject(s)
Curcuma , Chemistry , Curcumin , Phytochemicals , Plants, Medicinal , Chemistry , Rhizome , Chemistry , Terpenes
9.
Acta Pharmaceutica Sinica ; (12): 1327-1332, 2010.
Article in Chinese | WPRIM | ID: wpr-250661

ABSTRACT

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.


Subject(s)
Animals , Bungarus , Classification , Genetics , Cytochromes b , Genetics , DNA Primers , Genetics , Drug Contamination , Molecular Sequence Data , Polymerase Chain Reaction , Methods , Sequence Alignment , Sequence Analysis, DNA
10.
Acta Pharmaceutica Sinica ; (12): 1188-1193, 2010.
Article in Chinese | WPRIM | ID: wpr-353402

ABSTRACT

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.


Subject(s)
Cloning, Molecular , DNA, Complementary , Genetics , DNA, Plant , Genetics , DNA-Binding Proteins , Genetics , Gene Expression Regulation, Plant , Gene Frequency , Genotype , Phylogeny , Plant Proteins , Genetics , Plants, Medicinal , Genetics , Polymorphism, Single Nucleotide , Salvia miltiorrhiza , Genetics
11.
Acta Pharmaceutica Sinica ; (12): 785-790, 2010.
Article in Chinese | WPRIM | ID: wpr-354532

ABSTRACT

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.


Subject(s)
Acetyl-CoA C-Acetyltransferase , Genetics , Metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Gene Expression Regulation, Plant , Genotype , Introns , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Phylogeny , Plant Leaves , Genetics , Plant Roots , Genetics , Plant Stems , Genetics , Plants, Medicinal , Classification , Genetics , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain Reaction , Salvia miltiorrhiza , Classification , Genetics
12.
Acta Pharmaceutica Sinica ; (12): 680-686, 2009.
Article in Chinese | WPRIM | ID: wpr-278199

ABSTRACT

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.


Subject(s)
Atractylodes , Classification , Genetics , DNA, Intergenic , Genetics , DNA, Plant , Genetics , Genes, Plant , Phylogeny
13.
Acta Pharmaceutica Sinica ; (12): 1429-1433, 2009.
Article in Chinese | WPRIM | ID: wpr-344059

ABSTRACT

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.


Subject(s)
Astragalus Plant , Classification , Genetics , Astragalus propinquus , Classification , Genetics , Chloroplasts , Genetics , DNA, Plant , Genetics , Haplotypes , Phylogeny , Plant Roots , Genetics , Plants, Medicinal , Classification , Genetics , Species Specificity
14.
Acta Pharmaceutica Sinica ; (12): 1251-1257, 2008.
Article in Chinese | WPRIM | ID: wpr-232608

ABSTRACT

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.


Subject(s)
Acetates , Pharmacology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Conserved Sequence , Cyclopentanes , Pharmacology , DNA, Complementary , Genetics , Abietanes , Gene Expression Regulation, Plant , Genes, Plant , Molecular Sequence Data , Open Reading Frames , Oxylipins , Pharmacology , Phenanthrenes , Metabolism , Phosphotransferases (Alcohol Group Acceptor) , Genetics , Metabolism , Plant Proteins , Genetics , Metabolism , Plant Roots , Plants, Medicinal , Genetics , Metabolism , RNA, Messenger , Metabolism , Salvia miltiorrhiza , Genetics , Metabolism , Sequence Homology, Amino Acid
15.
Article in Chinese | WPRIM | ID: wpr-295402

ABSTRACT

<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>


Subject(s)
Alleles , China , Consumer Product Safety , DNA Primers , Genetics , Pinellia , Genetics , Plants, Medicinal , Genetics , Polymerase Chain Reaction , Methods , Polymorphism, Single Nucleotide , Quality Control
16.
Acta Pharmaceutica Sinica ; (12): 766-772, 2008.
Article in Chinese | WPRIM | ID: wpr-277798

ABSTRACT

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.


Subject(s)
Alkyl and Aryl Transferases , Genetics , Metabolism , Animals , Antibody Formation , Escherichia coli , Metabolism , Gene Expression , Immune Sera , Allergy and Immunology , Isopropyl Thiogalactoside , Chemistry , Male , Plant Proteins , Genetics , Metabolism , Plant Roots , Chemistry , Plants, Medicinal , Chemistry , Plasmids , Rabbits , Recombinant Proteins , Genetics , Metabolism , Salvia miltiorrhiza , Chemistry , Temperature , Time Factors , Transformation, Genetic
17.
Article in Chinese | WPRIM | ID: wpr-283771

ABSTRACT

<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>


Subject(s)
Amylose , Chemistry , Chromatography, High Pressure Liquid , Cistanche , Chemistry , Drugs, Chinese Herbal , Chemistry , Glucosides , Chemistry , Glycosides , Chemistry , Phenols , Chemistry
18.
Article in Chinese | WPRIM | ID: wpr-235276

ABSTRACT

<p><b>OBJECTIVE</b>Searching a new molecular method to authenticate Panax ginseng and P. quenquefolium.</p><p><b>METHOD</b>Single primers based on rDNA sequences of Panax species were designed to obtain polymorphic bands of P. ginseng and P. quinquefolius and then sequenced. Four PCR primers (two forword and two reverse primers) specific to P. ginseng and P. quinquefolius were designed.</p><p><b>RESULT</b>Primer Pg-6F, Pg-479R only amplified 474 bp band for P. ginseng and primer Pq-442F, Pq-658R only amplified 217 bp band for P. quinquefolius. It is indicated that the four primers could serve as specific STS primers for Panax species.</p><p><b>CONCLUSION</b>A new way to obtain STS primers of Panax species was established. This method is more quick and efficient than SCAR-PCR method and can serve as a model to obtain molecular markers for other Chinese material medica.</p>


Subject(s)
Base Sequence , DNA Primers , DNA, Plant , Genetics , DNA, Ribosomal , Genetics , Genetic Markers , Genetics , Molecular Sequence Data , Panax , Classification , Genetics , Plant Roots , Genetics , Plants, Medicinal , Genetics , Polymorphism, Genetic , Random Amplified Polymorphic DNA Technique , Methods , Sequence Analysis, DNA , Sequence Tagged Sites , Species Specificity
19.
Article in Chinese | WPRIM | ID: wpr-235251

ABSTRACT

<p><b>OBJECTIVE</b>Establishing cDNA microarray, in order to study functional genomics of Salvia miltiorrhiza.</p><p><b>METHOD</b>Total RNA samples were prepared from S. miltiorrhiza roots using a modified CTAB method. mRNA was isolated by Quichprep Micro mRNA Purification Kit from Pharmacia. Then cDNA was synthesized and cloned into the EcoRI-XhoI sites of the ZAP Express vector using a cDNA synthesis kit, and the ligation mixture was packaged using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene). The single phage was isolated for PCR amplification, Aliquots of the PCR reactions were analyzed in a 1.5% agarose gel to verify the quality of PCR. The remaining cDNA was purified by Multiscreen filter plates (Millipore) and aliquots were analyzed by agarose gel again to verify the quality of purification. Clones passed verification was resuspended in 15 microL 50% DMSO for arraying. An actin gene from S. miltiorrhiza was used for positive control. PloyA and 50% DMSO was used for negative controls.</p><p><b>RESULT</b>Bacterial colonies containing cNDAs of S. miltiorrhiza were inserted with average insert size of 0. 5 kb to 2. 5 kb. Total 4 354 genes were singled out from the first 8 736 PCR product and used for cDNA microarray manufacture. Single color fluorescence hybridization showed that all positive controls had signals while negative controls had no signals.</p><p><b>CONCLUSION</b>It was the first cDNA microarray about traditional Chinese herbs especially for geoherbs. It could be a powerful tool for studying functional genomics of S. miltiorrhiza.</p>


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , Genomics , Methods , Oligonucleotide Array Sequence Analysis , Methods , Plant Roots , Genetics , Plants, Medicinal , Genetics , RNA, Messenger , Genetics , Metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Salvia miltiorrhiza , Genetics
20.
Article in Chinese | WPRIM | ID: wpr-235226

ABSTRACT

<p><b>OBJECTIVE</b>Studying the gene expression profiling of different stage hairy root of Salvia miltiorrhiza, in order to find functional genes.</p><p><b>METHOD</b>The contents of second metabolites were determined by HPLC and gene expression profiling was detected by cDNA microarray. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. The microarrays were scanned with a ScanArray Express scanner using ScanArray 2.0 software and quantified by signal intensities of individual spots from the 16-bit TIFF images using GenePix Pro 4.0. The linear normalization method was used for data analyze. Northern blot was used to test the gene expression results obtained by microarray. Different expressed genes were sequenced and analyzed by gap4 software, and then they were analyzed with BLASTX, BLASTN, GO and KEGG.</p><p><b>RESULT</b>Growth rate and second metabolites analysis indicated that the stage from 30 d to 45 d was the growth stage, while the stage from 45 d to 60 d was the second metabolites accumulation stage. Accordingly 30 d hairy root was chosen as a reference, which was hybridized with 45 d and 60 d hairy root separately. Total 203 different expressed genes were obtained. Northern blot showed that the result was identical with the microarray result. After sequenced, there were 172 genes clustered into 114 clusters (Unigenes). Among them, 62 unigenes had known functions, 34 unigenes were hypothetical protein, 9 unigenes were homologues with no similarity and 9 unigenes were unidentified protein with low similarity. Total 67 genes were classified into cellular component ontology, molecular function ontology and biological process ontology based on GO analysis. Total 26 genes, which represented 29 metabolic-related enzymes, were located in metabolic maps based on KEGG pathway classification.</p><p><b>CONCLUSION</b>Several important functional genes related to second metabolite synthesis were cloned such as P450 and copalyl diphosphate synthase genes. cDNA microarray was a useful tool for functional genomics of traditional Chinese medicine.</p>


Subject(s)
Alkyl and Aryl Transferases , Genetics , Metabolism , Cytochrome P-450 Enzyme System , Genetics , Metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genomics , Methods , Oligonucleotide Array Sequence Analysis , Plant Proteins , Genetics , Metabolism , Plant Roots , Genetics , Metabolism , Plants, Medicinal , Genetics , Metabolism , Salvia miltiorrhiza , Genetics , Metabolism
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