[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the ...[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...展开更多
The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Bas...The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.展开更多
A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15...A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.展开更多
A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single...A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.展开更多
[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine ...[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine populations, which is considered to be associated with some diseases such as post-weaning multi- systemic wasting syndrome （PMWS）. This study aimed to provided data for epidemiology of Tl＇suV in Guangdong Province. [Method] PCR primers were synthe- sized based on untranslated region （UTR） segment of TYsuV genome, to conduct PCR detection of 14 swine serum samples from two swine farms in Guangdong Province. A total of four PCR products of TrsuV1 and TrsuV2 from two swine farms were selected for cloning, sequencing and analysis. [ Result] Ten TrsuVl pos- itive samples （71% ） and eight TrsuV2 positive samples （57%） were obtained by PCR, including five double-positive samples （36%）. Sequence analysis of PCR products and reference strains showed that the UTR segments of samples GDTI-1 and GDT1-2 were both 305 bp, sharing 90.2% -95.1% similarity with Tl＇suVl reference strain, and the UTR segments of samples GDT2-1 and GDT2-2 were respectively 259 bp and 248 bp, sharing 67.3% - 100% similarity with TrsuV2 refer- ence strain. [ Conclusion] These results conformed that there are at least two types of Tl＇suV in Guangdong Province, with relatively high detection rates in some swine farms. Despite the putative harmlessness of Tl＇suV, the public health significance of TTsuV is noticeable due to its potential pathogenicity.展开更多
Objective： Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to th...Objective： Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Prl gene. Methods： The genomic DNA was firstly digested with BamH I and then treated with calf intestinal alkaline phosphatase（CIAP）. Next, a 5＇ phosphorylated oligonucleotide was ligated to the 5＇ ends of BamH I -digested DNA. After denaturation, intmstrand annealing and polymemse extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results： A 864 bp product was amplified,which was adjacent to the known sequence of Prl gene.The gene has been accessed by GenBank （Accession：JQ975036）. Conclusion： Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Prl gene展开更多
Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establ...Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.展开更多
Aging is linked to the deterioration of many physical and cognitive abilities and is the leading risk factor for Alzheimer’s disease. The growing aging population is a significant healthcare problem globally that res...Aging is linked to the deterioration of many physical and cognitive abilities and is the leading risk factor for Alzheimer’s disease. The growing aging population is a significant healthcare problem globally that researchers must investigate to better understand the underlying aging processes. Advances in microarrays and sequencing techniques have resulted in deeper analyses of diverse essential genomes(e.g., mouse, human, and rat) and their corresponding cell types, their organ-specific transcriptomes, and the tissue involved in aging. Traditional gene controllers such as DNA-and RNA-binding proteins significantly influence such programs, causing the need to sort out long non-coding RNAs, a new class of powerful gene regulatory elements. However, their functional significance in the aging process and senescence has yet to be investigated and identified. Several recent researchers have associated the initiation and development of senescence and aging in mammals with several well-reported and novel long non-coding RNAs. In this review article, we identified and analyzed the evolving functions of long non-coding RNAs in cellular processes, including cellular senescence, aging, and age-related pathogenesis, which are the major hallmarks of long non-coding RNAs in aging.展开更多
A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was clon...A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.展开更多
Hepatocellular carcinoma(HCC) is one of the most common malignancies leading to high mortality rates in the general population and the sixth most common cancer worldwide. HCC is characterized by deregulation of multip...Hepatocellular carcinoma(HCC) is one of the most common malignancies leading to high mortality rates in the general population and the sixth most common cancer worldwide. HCC is characterized by deregulation of multiple genes and signalling pathways. These genetic effects can involve both protein coding genes as well as non-coding RNA genes. Long noncoding RNAs(lnc RNAs) are transcripts longer than 200 nt, constituting a subpopulation of nc RNAs. Their biological effects are not well understood comparedto small non-coding RNA(micro RNAs), but they have been recently recognized to exert a crucial role in the regulation of gene expression and modulation of signalling pathways. Notably, several studies indicated that lnc RNAs contribute to the pathogenesis and progression of HCC. Investigating the molecular mechanisms underlying lnc RNAs expression opens potential applications in diagnosis and treatment of liver disease. This editorial provides three examples(MALAT-1 metastasis associated lung adenocarcinoma transcript, HULC highly upregulated in liver cancer and HOTAIR HOX transcript antisense intergenic RNA) of well-known lnc RNAs upregulated in HCC, whose mechanisms of action are known, and for which therapeutic applications are delineated. Targeting of lnc RNAs using several approaches(siR NA-mediated silencing or changing their secondary structure) offers new possibility to treat HCC.展开更多
AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of p...AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivu- dine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, re- spectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Com- pletely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was de- tected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-ef- fective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.展开更多
Long non-coding RNAs(lncRNAs)are abundantly expressed in the central nervous system and exert a critical role in gene regulation via multiple biological processes.To uncover the functional significance and molecular m...Long non-coding RNAs(lncRNAs)are abundantly expressed in the central nervous system and exert a critical role in gene regulation via multiple biological processes.To uncover the functional significance and molecular mechanisms of lncRNAs in spinal cord injury(SCI),the expression signatures of lncRNAs were profiled using RNA sequencing(RNA-seq)technology in a Sprague-Dawley rat model of the 10th thoracic vertebra complete transection SCI.Results showed that 116 of 14,802 detected lncRNAs were differentially expressed,among which 16—including eight up-regulated(H19,Vof16,Hmox2-ps1,LOC100910973,Ybx1-ps3,Nnat,Gcgr,LOC680254)and eight down-regulated(Rmrp,Terc,Ngrn,Ppp2r2b,Cox6a2,Rpl37a-ps1,LOC360231,Rpph1)—demonstrated fold changes>2 in response to transection SCI.A subset of these RNA-seq results was validated by quantitative real-time PCR.The levels of 821 mRNAs were also significantly altered post-SCI;592 mRNAs were up-regulated and 229 mRNAs were down-regulated by more than 2-fold.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that differentially expressed mRNAs were related to GO biological processes and molecular functions such as injury and inflammation response,wound repair,and apoptosis,and were significantly enriched in 15 KEGG pathways,including cell phagocytosis,tumor necrosis factor alpha pathway,and leukocyte migration.Our results reveal the expression profiles of lncRNAs and mRNAs in the rat spinal cord of a complete transection model,and these differentially expressed lncRNAs and mRNAs represent potential novel targets for SCI treatment.We suggest that lncRNAs may play an important role in the early immuno-inflammatory response after spinal cord injury.This study was approved by the Administration Committee of Experimental Animals,Guangdong Province,China.展开更多
To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical se...To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.展开更多
[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum ...[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum leaves with the modified CTAB method. CHS gene sequences were downloaded from the NCBI and aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer- premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR and ligated to pMD18-T vector. The identified positive colonies were sequenced. [Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the obtained fragment encoded 365 amino acids and shared above 90% homology to nucleotide sequence of CHS gene from A. palmatum and A. [Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for the first time, which laid the foundation for efficient utilization of CHS gene.展开更多
A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemip...A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.展开更多
Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) hu...Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) human osteosarcoma cell. The amplified products were cloned into pGEM T Easy vector and sequenced. Then the expressed vector pBV220 hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE Sephadex A 50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.展开更多
基金Supported by National 863 Program of China(2006AA10A113)Natural Science foundation of Zhejiang Province(Y306097)~~
文摘[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...
基金Shandong Provincial Excellent YoungScientist Fund (2004BS06002)Innovation Fund ofShandong Agricultural University
文摘The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.
文摘【目的】早发性肌无力综合征(early onset muscle weakness syndrome,MW)是新近发现的一种荷斯坦牛遗传缺陷,患病犊牛表现为出生后趴卧不起、后肢肌肉萎缩等,其遗传机制与L型钙通道蛋白α1S亚基编码基因CACNA1S的单碱基突变相关。本研究旨在建立该遗传缺陷的分子检测方法,并探究其在国内荷斯坦牛群体中的扩散情况。【方法】基于MW致病位点特异性DNA序列,设计扩增阻滞突变系统-聚合酶链式反应(ARMS-PCR)检测引物,对317份荷斯坦牛冻精和毛囊样本进行MW遗传缺陷基因筛查,通过Sanger测序对ARMS-PCR检测结果进行验证,并利用系谱数据追溯MW突变源头。【结果】ARMS-PCR检出MW携带者21头,携带率为6.62%(21/317),未发现缺陷基因纯合子,提示该突变为隐性纯合致死。Sanger测序与ARMS-PCR所得基因型完全一致,证明该技术具有高度准确性。系谱追溯表明,MW携带者的遗传来源可追溯至1984年出生的荷斯坦公牛Southwind Bell of Bar-Lee。【结论】MW遗传缺陷已在中国荷斯坦牛群体中扩散且携带率较高,建议牧场应尽早开展MW遗传缺陷基因检测和风险评估,采取科学的选种选配措施以减少遗传缺陷导致的经济损失。
文摘A technique of direct sequence analysis of β-globin gene with the products of amplifi-cation by polymerase chain reaction (PCR) was reported and a case of β-thalassemia with therare mutation in Chinese,‘codon 14/15 (+G)’ was detected by this method.After the se-quence of the mutation site was determined,an analysis of the restriction map of the gene anddot blot hybridization with radioactive allele specific oligonucleotide probe was designed to con-firm the result of DNA sequencing.
基金partially supported by the Program 30470120,30671419,30700541 from the National Natural Science Foundation of Chinaby the 863 Programs 2006AA10Z108,2006AA100108 from the Ministry of Science and Technology of China+2 种基金by the Ph.D Funding 20050307009 from the Ministry of Education of Chinaby the Program BK2006139 from the Natural Science Foundation of Jiangsu Provinceby Research Fund KJ05006 from Nanjing Agricultural University,China.
文摘A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.
基金Supported by Funding Project of Guangdong Science and Technology Department(No.2011B010500023)Funding Project of Guangzhou Science and Information Technology Department(No.12A64071507)
文摘[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine populations, which is considered to be associated with some diseases such as post-weaning multi- systemic wasting syndrome （PMWS）. This study aimed to provided data for epidemiology of Tl＇suV in Guangdong Province. [Method] PCR primers were synthe- sized based on untranslated region （UTR） segment of TYsuV genome, to conduct PCR detection of 14 swine serum samples from two swine farms in Guangdong Province. A total of four PCR products of TrsuV1 and TrsuV2 from two swine farms were selected for cloning, sequencing and analysis. [ Result] Ten TrsuVl pos- itive samples （71% ） and eight TrsuV2 positive samples （57%） were obtained by PCR, including five double-positive samples （36%）. Sequence analysis of PCR products and reference strains showed that the UTR segments of samples GDTI-1 and GDT1-2 were both 305 bp, sharing 90.2% -95.1% similarity with Tl＇suVl reference strain, and the UTR segments of samples GDT2-1 and GDT2-2 were respectively 259 bp and 248 bp, sharing 67.3% - 100% similarity with TrsuV2 refer- ence strain. [ Conclusion] These results conformed that there are at least two types of Tl＇suV in Guangdong Province, with relatively high detection rates in some swine farms. Despite the putative harmlessness of Tl＇suV, the public health significance of TTsuV is noticeable due to its potential pathogenicity.
基金Supported by the Guangxi Department of education scientific research funds,China(No.200103YB154)
文摘Objective： Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Prl gene. Methods： The genomic DNA was firstly digested with BamH I and then treated with calf intestinal alkaline phosphatase（CIAP）. Next, a 5＇ phosphorylated oligonucleotide was ligated to the 5＇ ends of BamH I -digested DNA. After denaturation, intmstrand annealing and polymemse extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results： A 864 bp product was amplified,which was adjacent to the known sequence of Prl gene.The gene has been accessed by GenBank （Accession：JQ975036）. Conclusion： Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Prl gene
基金National natural science foundation of China,No.81860367,31460017,81672072Key Research and Development Program of Hainan Province,No.ZDYF2017091+1 种基金Special Project for Scientific Research of Institutions of Higher Learning in Hainan Province,No.Hnky2017ZD-16,Hnkyzx2014-08Open Fund Project of State key Laboratory of Virology in 2018.Project,No.2018IOV002.
文摘Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.
文摘Aging is linked to the deterioration of many physical and cognitive abilities and is the leading risk factor for Alzheimer’s disease. The growing aging population is a significant healthcare problem globally that researchers must investigate to better understand the underlying aging processes. Advances in microarrays and sequencing techniques have resulted in deeper analyses of diverse essential genomes(e.g., mouse, human, and rat) and their corresponding cell types, their organ-specific transcriptomes, and the tissue involved in aging. Traditional gene controllers such as DNA-and RNA-binding proteins significantly influence such programs, causing the need to sort out long non-coding RNAs, a new class of powerful gene regulatory elements. However, their functional significance in the aging process and senescence has yet to be investigated and identified. Several recent researchers have associated the initiation and development of senescence and aging in mammals with several well-reported and novel long non-coding RNAs. In this review article, we identified and analyzed the evolving functions of long non-coding RNAs in cellular processes, including cellular senescence, aging, and age-related pathogenesis, which are the major hallmarks of long non-coding RNAs in aging.
基金The article was a part of the research program financed by the Science and Technology Bureau of Hebei Province, China (06220106D)
文摘A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.
文摘Hepatocellular carcinoma(HCC) is one of the most common malignancies leading to high mortality rates in the general population and the sixth most common cancer worldwide. HCC is characterized by deregulation of multiple genes and signalling pathways. These genetic effects can involve both protein coding genes as well as non-coding RNA genes. Long noncoding RNAs(lnc RNAs) are transcripts longer than 200 nt, constituting a subpopulation of nc RNAs. Their biological effects are not well understood comparedto small non-coding RNA(micro RNAs), but they have been recently recognized to exert a crucial role in the regulation of gene expression and modulation of signalling pathways. Notably, several studies indicated that lnc RNAs contribute to the pathogenesis and progression of HCC. Investigating the molecular mechanisms underlying lnc RNAs expression opens potential applications in diagnosis and treatment of liver disease. This editorial provides three examples(MALAT-1 metastasis associated lung adenocarcinoma transcript, HULC highly upregulated in liver cancer and HOTAIR HOX transcript antisense intergenic RNA) of well-known lnc RNAs upregulated in HCC, whose mechanisms of action are known, and for which therapeutic applications are delineated. Targeting of lnc RNAs using several approaches(siR NA-mediated silencing or changing their secondary structure) offers new possibility to treat HCC.
文摘AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivu- dine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, re- spectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Com- pletely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was de- tected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-ef- fective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.
基金financially supported by the National Natural Science Foundation of China,No.81371366(to HFW)Characteristic Innovation Project of Colleges and Universities in Guangdong Province of China,No.2018KTSCX075(to HFW)+3 种基金the Key Project of Social Development of Dongguan of China,No.20185071521640(to HFW)College Students’ Science and Technology Innovation Training Project,China,Nos.201810571058,GDMU2018024,GDMU2018056,GDMU2018061(to HFW)College Students’ Innovative Experimental Project in Guangdong Medical University,China,No.ZZDS001(to HFW)College Students’ Science and Technology Innovation Cultivation Project in Guangdong of China,No.pdjh2019b0217(to HFW)
文摘Long non-coding RNAs(lncRNAs)are abundantly expressed in the central nervous system and exert a critical role in gene regulation via multiple biological processes.To uncover the functional significance and molecular mechanisms of lncRNAs in spinal cord injury(SCI),the expression signatures of lncRNAs were profiled using RNA sequencing(RNA-seq)technology in a Sprague-Dawley rat model of the 10th thoracic vertebra complete transection SCI.Results showed that 116 of 14,802 detected lncRNAs were differentially expressed,among which 16—including eight up-regulated(H19,Vof16,Hmox2-ps1,LOC100910973,Ybx1-ps3,Nnat,Gcgr,LOC680254)and eight down-regulated(Rmrp,Terc,Ngrn,Ppp2r2b,Cox6a2,Rpl37a-ps1,LOC360231,Rpph1)—demonstrated fold changes>2 in response to transection SCI.A subset of these RNA-seq results was validated by quantitative real-time PCR.The levels of 821 mRNAs were also significantly altered post-SCI;592 mRNAs were up-regulated and 229 mRNAs were down-regulated by more than 2-fold.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that differentially expressed mRNAs were related to GO biological processes and molecular functions such as injury and inflammation response,wound repair,and apoptosis,and were significantly enriched in 15 KEGG pathways,including cell phagocytosis,tumor necrosis factor alpha pathway,and leukocyte migration.Our results reveal the expression profiles of lncRNAs and mRNAs in the rat spinal cord of a complete transection model,and these differentially expressed lncRNAs and mRNAs represent potential novel targets for SCI treatment.We suggest that lncRNAs may play an important role in the early immuno-inflammatory response after spinal cord injury.This study was approved by the Administration Committee of Experimental Animals,Guangdong Province,China.
文摘To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.
基金Supported by Agricultural Improved Variety Project of Shandong Province(LKZ[2014]No.96)
文摘[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum leaves with the modified CTAB method. CHS gene sequences were downloaded from the NCBI and aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer- premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR and ligated to pMD18-T vector. The identified positive colonies were sequenced. [Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the obtained fragment encoded 365 amino acids and shared above 90% homology to nucleotide sequence of CHS gene from A. palmatum and A. [Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for the first time, which laid the foundation for efficient utilization of CHS gene.
基金Funding for the studies described was provided by the University of Greenwich Proof of Concept and Research Funds,UK(E0162/RAE-NRI-009/09and K0070)
文摘A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.
文摘Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) human osteosarcoma cell. The amplified products were cloned into pGEM T Easy vector and sequenced. Then the expressed vector pBV220 hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE Sephadex A 50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.
