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.

China ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli O157 ; classification ; genetics ; Genes, Bacterial ; Humans ; Molecular Typing ; Virulence

This paper introduced a new method of "ingredient difference phonetypical cloning" for functional gene clone of medicinal plants, which might solve the difficulties in isolating genes encoding enzymes for the biosynthesis of secondary metabolites by usual ways. Concepts, mechanisms and methods were systematically introduced and possibility was proved by experiments. The method showed the extra superiority of for the isolation of the genes belonged to unknown metabolic pathway and little information about its sequences. The method provides a new way to isolate functional gene cloning from Chinese herbs and a fundament for the further study on medicinal plant genetic engineering.
Cloning, Molecular ; Drugs, Chinese Herbal ; metabolism ; Genes, Plant ; genetics ; Genetic Engineering ; methods ; Phenotype ; Plants, Medicinal ; genetics ; metabolism ; Reproducibility of Results ; Time Factors

OBJECTIVETo study the relationship between functional gene expression in Salvia miltiorrhiza from different producing areas and active principles, which might provide scientific basis for the gene regulation of tanshinones.

METHODThe quantitative determination of cryptotanshinone and tanshinone II A was carried out by using HPLC method, expression level of 3 functional genes of SmAACT, SmCMK and SmIPPI were investigated by real-time PCR method.

RESULTThe content of active principles together with expression level of SmAACT and SmCMK were higher in S. miltiorrhiza from genuine producing areas including Henan and Shanxi, but lower in samples from Beijing which was non-genuine producing area.

CONCLUSIONExpression level of SmAACT and SmCMK had close relationships involving tanshinones' accumulation, but the SmIPPI gene had not.

Diterpenes, Abietane ; metabolism ; Gene Expression ; Real-Time Polymerase Chain Reaction ; Salvia miltiorrhiza ; genetics ; metabolism

OBJECTIVETo establish the new EST-SSR markers for analyzing the genetic variation of different population of Salvia miltiorrhiza.

METHODIt was dealt with ESTs newly downloaded from Genbank and that of acquired from HMPL lab EGassembler software, and then carried out SSR loci search and SSR type analysis by SSRIT software. After that, it was designed the EST-SSR primer pairs for PCR amplification condition optimization.

RESULTAbundant and high coverage of SSR loci distribution were found in S. miltiorrhiza with having one SSR per 5.8 kb ESTs. Among them, the occurrences of different repeat units were mainly the di- (63.0%) and tri- (35.5%). The CT/AG was the most frequent motif in dinucleotide motif type and the GAA/TCC was the most frequent motif in trinucleotide repeats. Out off 36 primer pairs, 29 primer pairs (80.5%) were successfully amplified in all samples of S. miltiorrhiza while the rest failed to give PCR products at various annealing temperature and Mg2+ concentrations. The selected primer pairs also showed the polymorphism in samples from different S. miltiorrhiza populations.

CONCLUSIONThe newly establishment of EST-SSR markers showed high SSR loci coverage and genetic polymorphisms in S. miltiorrhiza population. It could be used for genetic variation analysis.

Alleles ; Expressed Sequence Tags ; Gene Frequency ; Genetic Markers ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Salvia miltiorrhiza ; genetics

OBJECTIVETo establish a convenient, quick and accurate molecular method for the identification of crude drugs of antlers due to the difficult discrimination between the genuine antler and its adulterants.

METHODAccording to the alignment analysis of full length sequences of Cyth gene from closely relate species of Cervus, one pair of allele-specific diagnostic PCR primers was designed. Factors such as annealing temperature, dosage of polymerase, times of cycles and dosage of template DNA that influence the PCR results were also investigated.

RESULTBased on the study mentioned above, about 323 bp positive band was amplified under the annealing temperature of 65 degrees C in the total volume of 25 microL PCR reaction using the genuine antler DNA as the template. Sequencing results proved that the positive band was the fragment of Cytb gene from both C. elaphus Linnaeus and C. nippon Temminck.

CONCLUSIONThe established method, with higher specificity and reproducibility, could accurately differentiate genuine antler from its adulterants and would be widely used in Cervus related Chinese crude drugs' identification.

Alleles ; Animals ; Antlers ; chemistry ; China ; DNA Primers ; genetics ; Deer ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Species Specificity

This paper introduced a new identification method, the 2-dimensional molecular marking method (2-DM), for Chinese materia medica identification. It can be used in genuine/false discriminating and quality evaluating for the Chinese materia medica. Concept, principle and process of 2-DM method were introduced in this paper. The technical advantages and contributions of 2-DM method in the study of Chinese materia medica were also discussed. Generally speaking, the occurring of 2-DM method would not only expand connotation of identification of Chinese materia medica but provide another effective way for quality evaluating.
Drugs, Chinese Herbal ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control

In this experiment the data of the infrared radiation spectrum of acupoints in human body were obtained through an ingenious device. In thirty-one healthy volunteer subjects and thirty-one patients suffering from coronary heart disease, we detected the infrared radiation spectra from some acupoints and compared them with those of the healthy subjects. The curves of patients showed more differences. After analyzing the data, we noted statistically significant differences in some fields between the healthy subjects and the patients. And after reducing the data, we observed significant differences in larger fields. So utilization of reduced data from patients for analysis is our choice.
Acupuncture Points ; Adult ; Aged ; Aged, 80 and over ; Coronary Disease ; physiopathology ; Female ; Humans ; Infrared Rays ; Male ; Middle Aged ; Skin ; physiopathology ; Spectrum Analysis

OBJECTIVETo clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODSVP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTSVP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONSVP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Enterovirus A, Human ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Immunoglobulin M ; blood ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo investigate the antimicrobial resistance status and pulsed field gel electrophoresis (PFGE) patterns of Salmonella Enteritidis (S.Enteritidis) strains in Henan province.

METHODSS. Enteritidis strains were isolated from seven sentinel hospitals from March 2011 to December 2013. According to molecular typing and Salmonella (Kirby-Bauer, K-B) drug susceptibility testing method published by the international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI), we analyzed drug sensitivity of 8 kinds antibiotics and PFGE molecule characteristics of 120 S. Enteritidis isolates from seven sentinel hospitals.

RESULTSAmong 120 strains of S. Enteritidis, 77 were isolated from male patients, 43 from female patients. A total of 78 strains S. Enteritidis were isolated from young children ranged from 0 to 5 years old (65.0%), including 57 strains isolated from 6 months to 2 years old (47.5%). The isolated time mainly centralized on May to October of the year, 11 strains isolated in March-April (9.2%), 48 were in May-July (40.0%),54 in August-October (45.0%), 7 in other months, with a typical summer seasonal characteristics. The resistance rate of 120 strains S. Enteritidis to ampicillin was 50.0% (n=60); to ceftazidime was 14.2% (n=17), to cefotaxime was 18.3% (n=22); to cefepime was 5.8% (n=7); to nalidixic acid was 61.7% (n=74); to ciprofloxacin was 8.3% (n=10), to norfloxacin was 5.8% (n=7); to gentamicin was 42.5% (n=51); to streptomycin was 21.7% (n=26); to chloramphenicol was 30.0% (n=36); resistance to methicillin benzyl ammonium was 11.7% (n=14), compound sulfamethoxazole resistance rate was 71.7% (n=86); the tetracycline resistant rate was 47.5% (n=57). All 120 strains of S. Enteritidis had different levels of resistance to 8 kinds of antibiotics, all strains were multidrug resistant strains, 28 isolates were resistant to 3-4 kinds of antibiotics (23.3%), 38 isolates were resistant to 5-6 kinds of antibiotics (31.7%), 39 isolates were resistant to 7-8 kinds of antibiotics (32.5%). All 120 strains of S. Enteritidis were divided into 44 molecular patterns by digestion with XbaI and pulsed field gel electrophoresis. each pattern contained 1-35 strains with similarity ranged from 54.3%-100%. EN14 and EN19 were the main PFGE types, including 35 and 29 strains respectively.

CONCLUSIONThe status of drug resistance of clinical isolates of Salmonella in Henan province was rather serious, PFGE patterns showed advantages and partial strain's corresponding resistant spectrum have certain relevance and the same aggregation relationship.

Anti-Bacterial Agents ; pharmacology ; Child, Preschool ; China ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Microbial Sensitivity Tests ; Molecular Typing ; Salmonella Infections ; microbiology ; Salmonella enteritidis ; classification ; drug effects