ABSTRACT
Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.
Subject(s)
Genes, Essential , Gelsemium/genetics , Peptide Elongation Factor 1/genetics , Transcriptome , Gene Expression Profiling/methods , Alkaloids , Real-Time Polymerase Chain Reaction/methods , Reference StandardsABSTRACT
Objective: To screen reference genes for real time quantitative PCR (qRT-PCR) research in Ampelopsis grossedentata. Methods: On the basis of the conserved sequences among plant species, six candidate reference genes (including Actin, 18 S-rRNA, GAPDH, α-Tubulin, β-Tubulin, and UBQ) were cloned from A. grossedentata by RT-PCR in this study. The expression stability of each reference gene in different tissues (shoot tip, young leaf, mature leaf, old leaf, stem, and root) were analyzed by three softwares (GeNorm, NormFinder, and BestKeeper), followed by validation of the expression pattern of AgPAL by qRT-PCR. Results: Actin, 18 S-rRNA, and GAPDH expressed most stably in all samples and were suitable for reference genes, which were further confirmed by the transcript level analysis result of AgPAL in different tissues. Conclusion: This is the first report on the screening and validation of reference genes for qRT-PCR in A. grossedentata, which benefits future studies on gene expression in this species.
ABSTRACT
Objective To obtain knowledge of microbial colonial structures and epidemic strains of clinically isolated multidrug-resistant Acinetobacter baumannii in Anhui in recent years.To analyze gene homologies and phylogenetic relationships of multidrug-resistant Acinetobacter baumannii.To provide some laboratory data for clinical antimicrobial application.Methods Eighty-seven strains of clinically isolate dmultidrug-resistant Acinetobacter baumannii were collected in this study,and the minimal inhibitory concentrations of these strains to 11 common antimicrobials were measured by agar dilution.Evolutionary relationships between the strains were revealed with employment of multilocus sequence typing,and clustering figures were constructed with the aid of the BioNumerics software,thus enabling genotyping.Results The drug-resistance rates of 87 strains of Acinetobacter baumannii to imipenem and meropenem were 74.7％ and 66.7％,respectively,while the drug-resistance rates to other antimicrobials were considerably high.All the 87 strains were sub-divided into 42 ST-types,6 of which were already involved in the database while 36 (temporarily named STnew01 ～STnew36) were newly established.Thirty-seven strains were proved dominant types and sub-listed in the ST2 category.The ST2 Acinetobacter baumannii belongs to the clone complex CC1.Conclusion All multidrug-resistant Acinetobacter baumannii in research show high drug-resistance rates when treated with 11 common antimicrobials.The ST2 category is the principal epidemic clones of multidrug-resistant Acinetobacter baumannii in Anhui province,and a highly recogonizable homology is observed between the principal epidemic strains(ST2) in Anhui and those in the world.
ABSTRACT
Objective To explore the postmortem stability of nine Housekeeping gene mRNA—β-actin, GAPDH, RPL32, PKG1, SDHA, rRPL13, HPRT, TBP, YWHAZ and the correlation between its relative expression and postmortem interval (PMI) in rat brain tissue and skeletal muscle. Methods 33 healty adult SD rats were randomly divided into 11 groups at 11 postmortem interval(0h, 1h, 3h, 6h, 12h, 24h, 2d, 4d, 8d, 16d, 24d), sacrificed by cervic al dislocation and it's anterior tibial muscle and brain tissue (about 50mg) were extracted, respectively. The total RNA was extracted from organizations and the nine mRNA were detected by Real-time RT-PCR. Proper internal reference was selected by geNorm software. To evaluate the stability of genes by GeNorm, NormFinder and BestKeeper software. Regression analysis by spss software. Results The expression of HPRT is the most stable in both brain and skeletal muscle, suitable for reference gene. The relative expression levels of SDHA, RPL32 and TBP in brain tissue had a certain linear relationship with PMI. While there was no significant correlation between the relative expression of RNA and PMI in skeletal muscle. Conclusion Brain tissue and skeletal muscle could be suitable materials for extracting RNA in advanced stage PMI. The relative expression levels of SDHA, RPL32 and TBP had a certain linear relationship with PMI, which could be auxiliary indices for the estimation of PMI.
ABSTRACT
Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection.
ABSTRACT
Objective: To screen the reference genes of Siraitia grosvenorii for gene expression analysis, and to study the spatio-temporal expression characteristics of 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) which was the key enzyme of mogroside V biosynthesis. Methods: In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), microtubule associated protein (α-tubulin), actin (β-actin), and ubiquitin (UBQ5) fragments of S. grosvenorii were cloned, and the stabilities of the four housekeeping genes were evaluated in different positions (leaves, stems, and fruits) and different periods of fruit development. In addition, the spatio-temporal expression of HMGR gene was analyzed. Results: UBQ5 was the most suitable reference gene for spatio-temporal expression analysis in S. grosvenorii; The relative expression of HMGR was low in leaves, and that in stems and fruits was higher; The relative expression quantity of HMGR in fruits showed the fluctuation changes that increased firstly and then decreased, then increased and decreased again; The highest expression of HMGR was at 70 d of fruit development period, followed by 5, 30, 10, and 50 d. Conclusion: UBQ5 is the most suitable reference gene in S. grosvenorii. The relative expression of HMGR changes significantly in leaves, stems, and fruits. The relative expression quantity of HMGR in fruits shows the fluctuation changes, which is similar to synthetic accumulation pattern of mogroside V.
ABSTRACT
Objective Reverse transcription-polymerase chain reaction (RT-PCR) is widely used in gene expression analysis.Selection of proper housekeeping gene is very important.Rat model of myocardial infarction is a major animal model of myocardial infarction.Few reports are found about the selection of rat housekeeping gene after myocardial infarction.This study was aimed to explore the stability of the housekeeping genes expression in the rat model of myocardial infarction.Methods Myocardial infarction models were made using the anterior descending coronary artery ligation method.Combining with practical work,analysis and selection were made of four widely used standard housekeeping genes:glyceraldehydes-3-phosphate dehydrogenase (GAPDH),ribosomal protein L13A (RPL13A),beta-actin (ACTB) and acidic ribosomal phosphoprotein P0 (ARBP).The expressions in the rat heart were compared using real-time fluorescent RT-PCR and analysis was carried out to figure out which was the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm program.Results The M values of RPL13A,GAPDH,ARBP and ACTB gene were 0.812,0.721,0.812 and 1.2 respectively.Conclusion GAPDH and ARBP are the most stable genes for the rat myocardial infarction model.
ABSTRACT
Purpose: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. Materials and Methods: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. Results: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. Conclusion: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.
Subject(s)
Bacterial Typing Techniques/methods , Environmental Microbiology , Humans , Polymerase Chain Reaction/methods , Rec A Recombinases/genetics , Sensitivity and Specificity , Vibrio cholerae/classification , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicityABSTRACT
Multilocus sequence typing (MLST) is a molecular genotyping method based on nucleotide sequencing. The procedure of this method characterizes isolates of bacterial species using the DNA sequencing of multiple housekeeping genes(usually seven). For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles.For each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST). MLST has the advantages of being robust (based on genetic data) and electronically portable to generate data that allow rapid and global comparisons between different laboratories. In this paper, the principle, method, data analysis, application, advantages and flaws of MLST are introduced.
ABSTRACT
Background: Evaluation of genetic relationship of pathogenic Vibrio cholerae clones isolated from specimens and different areas throughout analysis of various genes\u2019 sequence, in particularly, housekeeping genes provides the most accurate molecular database to molecular epidemiological surveillance. Objective: To evaluate the genetic relationship of pathogenic Vibrio cholerae clones in Vietnam to some other pathogenic clones throughout analysis of 2 housekeeping genes\u2019 sequence mdh and hlyA. Subject and methods: 2 housekeeping genes, mdh (malate dehydrogenase) and hlyA (hemolysin) were sequenced and submitted to the GeneBank with accession numbers AJ575356 and AJ576090. These sequences were compared with mdh and hlyA sequences from pathogenic strains of sixth and seventh cholera pandemics and from environmental strains. Results and Conclusion: The analysis results by MEGA3.0 software showed that the mdh and hlyA sequences from the pathogenic clones of Vietnam, sixth pandemic and seventh pandemic were rather similar, although having 11-bp deletion in hlyA gene of sixth pandemic clone. The 11-pb deletion in hlyA of the sixth pandemic clone was a characterization that distinguished the classical and El Tor types. Phylogenetic tree were constructed by the neighbor-joining method based on the mdh and hlyA sequences indicated that the Vietnam strain was very closely related to strains of sixth and seventh pandemics (the genetic distance: 0.2%). This evidenct suggested that the pathogenic clone in Vietnam diverged from a common ancestor with the sixth and seventh pandemic clones which had the intact properties of the pathogenic agent. \r\n', u'\r\n', u'