Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient co...Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.展开更多
Syringa species are important ornamentals with strong floral scent,of which monoterpenes are the main component.In this study,a new monoterpene synthase gene,named SoLIM,was collected from the flowers of Syringa oblat...Syringa species are important ornamentals with strong floral scent,of which monoterpenes are the main component.In this study,a new monoterpene synthase gene,named SoLIM,was collected from the flowers of Syringa oblata and S.oblata var.alba using a homologous cloning method.The full-length cDNA of SoLIM was1746 bp and encoded 581 amino acids.Sequence analysis showed that SoLIM contained the DDxxD and RRx8 W motifs,which are two typical conserved monoterpene synthase motifs,and was thus classified as belonging to the Tpsb subfamily.Using quantitative reverse-transcription PCR,SoLIM was significantly expressed in the petals and pistils of S.oblata and S.oblata var.alba,respectively.SoLIM expression peaked earlier than the D-limonene emissions in the diurnal experiments,but occurred later when D-limonene had peaked during the flowering phase,indicating that differences in SoLIM gene expression and D-limonene emissions existed.The synthesis of floral scent is thus associated with diverse regulatory mechanisms that require further investigation.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.展开更多
We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maxi...We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.展开更多
The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guenée) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis sh...The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guenée) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis showed that the full- length of HassGαq open reading frame (ORF) is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights (MW) and isoelectric point (PI) are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coli BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.展开更多
Pseudoalteromonas sp.SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment.The genes encoding chaperones DnaJ and DnaK of P.sp.SM9913 were cloned by normal PCR and TAIL-PCR(GenBank accession Nos DQ6...Pseudoalteromonas sp.SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment.The genes encoding chaperones DnaJ and DnaK of P.sp.SM9913 were cloned by normal PCR and TAIL-PCR(GenBank accession Nos DQ640312,DQ504163).The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria,and show some cold-adapted characteristics in their structures when compared with those from psychro-,meso-and themophilic bacteria.It is indicated that chaperones DnaJ and DnaK of P.sp.SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding,assembling and translocation of proteins at low temperature.This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.展开更多
Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(...Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.展开更多
Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Thermus sp. IM6501, B. stearothermophilus ET-1, and B. acidopullulytics. Primers were designed a...Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Thermus sp. IM6501, B. stearothermophilus ET-1, and B. acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.展开更多
To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was ampl...To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.展开更多
White-brown complex(WBC) transporters, also called half-size ATP binding cassette G(ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevine...White-brown complex(WBC) transporters, also called half-size ATP binding cassette G(ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic(seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs(cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR(qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.展开更多
The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains we...The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains were connected by a flexible linker to form asingle chain variable fragment(ScFv)gene with 720bp,which was in turn fused topCANTAB 5 phage.The single chain Fv was expressed as fusion protein displayed on thephage surface.The phagemid is used to transform compepent E.Coli TG1 cells,then infect-ed with M13K07 helper phage to rescue the phagemid and antibody ScFv gene.All rand-mized 12 clones were shown reacting with colon cancer cell line Ls174t,which expresses S-Tnantigen.The recombinant phage has been infected E.Coli HB2151 cells to produe soluble an-tibody,which can be used for immunodetection and immunotherapy for cancer.展开更多
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer m...Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.展开更多
Objective:To amplify antigen genes from patients with human immunodeficiency virus type 1 (HIV-1) in Guangdong Province for candidate AIDS vaccine design. Methods:Viral nucleic acid was isolated from 10 HIV-1 infected...Objective:To amplify antigen genes from patients with human immunodeficiency virus type 1 (HIV-1) in Guangdong Province for candidate AIDS vaccine design. Methods:Viral nucleic acid was isolated from 10 HIV-1 infected individuals' peripheral blood collected during 1995-2000 in Guangdong Province. The viral gag p24 gene and env gp120 gene were amplified by nested-PCR and sequenced. The homologies among HIV-1 isolates were compared with HIV-BLAST. Results: Among 10 HIV-1 isolates, nine are homologous to viruses of subtype B, and one is homologous to viruses of subtype E. Conclusion: Subtype B viruses of HIV-1 are predominantly present in Guangdong Province.展开更多
Objective: To evaluate the humoral immune induction in rats of a candidate AIDS vaccine expressing the gag p24 gene from a subtype B HIV-1 isolate. Methods: The amplified p24 gene was inserted into an eukaryotic expre...Objective: To evaluate the humoral immune induction in rats of a candidate AIDS vaccine expressing the gag p24 gene from a subtype B HIV-1 isolate. Methods: The amplified p24 gene was inserted into an eukaryotic expression vector to form the supercoiled DNA vaccine. The linearized expressed DNA vaccine was preparedfrom the expression plasmid by polymerase chain reaction(PCR). The antigen gene expression in rats of the linearized and supercoiled DNA vaccines were in vitro and in vivodetected. Results: In vitro transcription and Northern hybridizationshowed that the linearized DNA vaccine could synthesizeamounts of p24 mRNA similar to the supercoiled DNA vaccine.Antibody assays of inoculated rats confirmed that thelinearized expression DNA could induce a slightly higherantibody titer than the expression plasmid, while the highestantibody titer had been induced by plasmid plus adjuvantinoculation. Conclusion: The construction of a candidate AIDS vaccinebased on the p24 gene could shed light on a potential HIVvaccine, meriting evaluation in a rhesus macaque SHIV-AIDSmodel.展开更多
RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little succe...RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.展开更多
[Objectives]To explore the function of hcp gene in Aeromonas hydrophila.[Methods]A pair of specific primers was designed referring to the hcp gene sequence of A.hydrophila.The hcp gene was amplified by PCR,and perform...[Objectives]To explore the function of hcp gene in Aeromonas hydrophila.[Methods]A pair of specific primers was designed referring to the hcp gene sequence of A.hydrophila.The hcp gene was amplified by PCR,and performed bioinformatics analysis.[Results]The hcp gene had a total length of 1650 bp and encoded 549 amino acids.The theoretical molecular weight of the protein predicted was about 59476.44 kDa.After predicting the N-terminal signal peptide structure of the amino acid sequence,neither obvious signal peptide cleavage site nor signal peptide was found,and the protein had no transmembrane region.The amino acid sequence had a N-glycosylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,9 N-myristoylation sites,4 isoprene binding sites,10 microbody C-terminal target signal sites,and an ATP/GTP binding site motif A(P-ring).The amino acid sequence of hcp gene of A.hydrophila was performed homology analysis with other Aeromonas strains,and it showed higher homology with A.veronii.In the secondary structure,theα-helix,β-sheet,random coil and extended strand accounted for 45.36%,6.01%,37.52%and 11.11%,respectively.The tertiary structure model consisted of 18α-helix and 22β-sheet.Analysis of protein-protein network interaction demonstrated that the proteins interacting with Hcp protein were AHA_3407,nrfA,nirB-1,nirB-2 and AHA_1112.[Conclusions]Through the bioinformatics prediction results,the basic information of hcp gene of A.hydrophila is preliminarily understood,and the possible function of this protein is predicted,in order to provide guidance for subsequent vaccine research.展开更多
For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids w...For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.展开更多
[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The ...[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.展开更多
Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput wa...Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.展开更多
基金Supported by National Natural Science Foundation of China(81060028)Key Project of Science and Technology of Guangxi Zhuang Autonomous Region(Gui Ke Zhong 1598005-9)Key Science and Technology Program of Guangxi Zhuang Autonomous Region(Gui Ke Gong 1355005-3-7)
文摘Dendrobium officinale is one of the most precious medicinal plants in China. Its main medicinal ingredients are its secondary metabolites. However,it has the characteristics of limited sources,low active ingredient content and high cost,limiting the use of D. officinale.Studying the network structure and rate-limiting steps of secondary metabolites of medicinal components of D. officinale,analyzing the secondary metabolic synthesis process,mastering the production rules of its medicinal components and carrying out gene cloning or biosynthesis,etc.are of great significance for the rational development and utilization of D. officinale resources. This paper briefly reviews the progress of the research on the secondary metabolites of D. officinale,including the detection and identification of metabolites and the identification and cloning of key metabolic enzymes.
基金supported by the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality(IDHT20180509)the National Natural Science foundation of China(31201645)+1 种基金the Beijing Municipal Natural Science Foundation(6172006)key project of Beijing Municipal Education Commission(KZ201510020021)
文摘Syringa species are important ornamentals with strong floral scent,of which monoterpenes are the main component.In this study,a new monoterpene synthase gene,named SoLIM,was collected from the flowers of Syringa oblata and S.oblata var.alba using a homologous cloning method.The full-length cDNA of SoLIM was1746 bp and encoded 581 amino acids.Sequence analysis showed that SoLIM contained the DDxxD and RRx8 W motifs,which are two typical conserved monoterpene synthase motifs,and was thus classified as belonging to the Tpsb subfamily.Using quantitative reverse-transcription PCR,SoLIM was significantly expressed in the petals and pistils of S.oblata and S.oblata var.alba,respectively.SoLIM expression peaked earlier than the D-limonene emissions in the diurnal experiments,but occurred later when D-limonene had peaked during the flowering phase,indicating that differences in SoLIM gene expression and D-limonene emissions existed.The synthesis of floral scent is thus associated with diverse regulatory mechanisms that require further investigation.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.
基金Qingdao Municipal Science and Technology Commission,Qingdao,China for providing financial support to this work(06-2-2-22-jch)
文摘We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.
文摘The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta (Guenée) by reverse transcription polymerase chain reaction (RT-PCR) and named as HassGαq. Sequencing analysis showed that the full- length of HassGαq open reading frame (ORF) is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights (MW) and isoelectric point (PI) are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coli BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.
基金The work was supported by the Hi-Tech Research and Development Program of China under contract Nos 2006AA09Z414 and 2007AA091903；the China Ocean Mineral Resources R ＆ D Association under contract No. DYXM - 115 - 02 - 2 - 6；the National Natural Science Foundation of China under contract No. Z2004D02；the Natural Science Foundation of Shandong Province of China under contract No. Z2004D02；the Foundation for Young Excellent Scientists in Shandong Province of China under contract No. 2006BS02002；the Program for New Century Excellent Talents in University under contract No. NCET - 06 - 0578.
文摘Pseudoalteromonas sp.SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment.The genes encoding chaperones DnaJ and DnaK of P.sp.SM9913 were cloned by normal PCR and TAIL-PCR(GenBank accession Nos DQ640312,DQ504163).The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria,and show some cold-adapted characteristics in their structures when compared with those from psychro-,meso-and themophilic bacteria.It is indicated that chaperones DnaJ and DnaK of P.sp.SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding,assembling and translocation of proteins at low temperature.This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.
文摘Objective To clone the ACP(acyl carrier protein)gene in Jatropha curcas L.,a potential anti-tumour and anti-fungal plant.And to determinate the expression of ACP in Jatropha curcas L.Methods A cDNA clone encoding ACP(acyl carrier protein)was isolated from Jatropha curcas L.endosperm cDNA library by random sequencing.The expression of ACP gene was investigated by semi-quantitative RT-PCR in leaves,stems and seeds of J.curca.The expression of ACP was also investigated in germinating seeds.The fragment encoding ACP protein in J.curca.was inserted into a prokaryotic expression vector pET28a(+).The gene was overexpressed in E.coli BL21 to produce abundant protein.Immunohistochemical analysis was used to detect the expression of ACP in different tissues of J.curca.Results The cDNA sequence was 806 bp in length and the ORF was 393 bp.The predicted molecular weight of the putative protein was 14.4 kD,pI=5.2.It contained a 4'-phosphopantetheine-binding motif.This prosthetic group can be combined with Serine of ACP protein.Semi-quantitative RT-PCR analysis showed that ACP gene was expressed in leaves,stems and seeds of J.curcas.The expression level of ACP was the highest in seeds and it was not detected in roots.After seeds germinated,the expression level of ACP in seeds increased progressively and reached a peak at 96 h.After induced by IPTG,SDS-PAGE analysis showed that the ACP protein of 20 kD was expressed.Immunohistochemical analysis showed that ACP specifical expressed abundantly in embyo of the seeds,and it was not detected in roots and the emdosperm while expressed in leaves and stems.Conclusions A cDNA clone encoding ACP which had all the typical characteristics of ACPs was isolated.It was expressed successfully in E.coli.The results of semi-quantitative RT-PCR analysis and immunohistochemical analysis were very similar,which showed that the expression of ACP in J.curcas.was abundant in seeds.The results indicated the expression related to the high metabolism.
基金Natural Science Foundation of Heilongjiang Province (C9912)
文摘Primers and probes were established according to the sequences of the alpha-amylase genes of Bacillus. halodurans C-125, Thermus sp. IM6501, B. stearothermophilus ET-1, and B. acidopullulytics. Primers were designed and a 0.2 kb DNA fragment was amplified, the fragment was successfully used for the detection of the amylase Ⅱ gene in a 2 842 bp region from Bacillus halodurans strain 38C1-1.
文摘To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
基金supported by the National Natural Science Foundation of China (31372023)the earmarked fund for China Agricultural Research System (CARS-30-yz-7)
文摘White-brown complex(WBC) transporters, also called half-size ATP binding cassette G(ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic(seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs(cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR(qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.
基金Supported by Grant of Medical Science from the Ministry of Health of PLA.
文摘The gene encoding the heavy-and light-chain Fv regions of monoclonal antibody PS-9,which recognizes a cancer-associated antigen S-Tn on the most adenocarcinoma,was clonedby PCR techniques.The light and heavy chains were connected by a flexible linker to form asingle chain variable fragment(ScFv)gene with 720bp,which was in turn fused topCANTAB 5 phage.The single chain Fv was expressed as fusion protein displayed on thephage surface.The phagemid is used to transform compepent E.Coli TG1 cells,then infect-ed with M13K07 helper phage to rescue the phagemid and antibody ScFv gene.All rand-mized 12 clones were shown reacting with colon cancer cell line Ls174t,which expresses S-Tnantigen.The recombinant phage has been infected E.Coli HB2151 cells to produe soluble an-tibody,which can be used for immunodetection and immunotherapy for cancer.
基金Supported by the Key Laboratory Foundation of the Educational Department of Liaoning Province (No. 2009S024)the Dalian Municipal Government of China (No. 2007B11NC069)the Grant of Dalian Ocean University (No. SY2007005)
文摘Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.
基金Financially supported by Natural Science Foundation of China(No 39870725)Natural Science Foundation of Guangdong(No 980642)Research Foundation of Guangdong Education Beaureu(No.20032).
文摘Objective:To amplify antigen genes from patients with human immunodeficiency virus type 1 (HIV-1) in Guangdong Province for candidate AIDS vaccine design. Methods:Viral nucleic acid was isolated from 10 HIV-1 infected individuals' peripheral blood collected during 1995-2000 in Guangdong Province. The viral gag p24 gene and env gp120 gene were amplified by nested-PCR and sequenced. The homologies among HIV-1 isolates were compared with HIV-BLAST. Results: Among 10 HIV-1 isolates, nine are homologous to viruses of subtype B, and one is homologous to viruses of subtype E. Conclusion: Subtype B viruses of HIV-1 are predominantly present in Guangdong Province.
基金Financially supported by Natural Science Foundation of China(No 39870725)Natural Science Foundation of Guangdong(No 980642)Research Foundation of Guangdong Education Buresu(No.20032).
文摘Objective: To evaluate the humoral immune induction in rats of a candidate AIDS vaccine expressing the gag p24 gene from a subtype B HIV-1 isolate. Methods: The amplified p24 gene was inserted into an eukaryotic expression vector to form the supercoiled DNA vaccine. The linearized expressed DNA vaccine was preparedfrom the expression plasmid by polymerase chain reaction(PCR). The antigen gene expression in rats of the linearized and supercoiled DNA vaccines were in vitro and in vivodetected. Results: In vitro transcription and Northern hybridizationshowed that the linearized DNA vaccine could synthesizeamounts of p24 mRNA similar to the supercoiled DNA vaccine.Antibody assays of inoculated rats confirmed that thelinearized expression DNA could induce a slightly higherantibody titer than the expression plasmid, while the highestantibody titer had been induced by plasmid plus adjuvantinoculation. Conclusion: The construction of a candidate AIDS vaccinebased on the p24 gene could shed light on a potential HIVvaccine, meriting evaluation in a rhesus macaque SHIV-AIDSmodel.
基金This study was supported by Jiangsu Agricultural Science and Technology Innovation Fund[CX(21)3088,CX(22)2038]Jiangsu Province Key R&D Program(Modern Agriculture)Project:Surface Project(BE2021303).
文摘RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2023008)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To explore the function of hcp gene in Aeromonas hydrophila.[Methods]A pair of specific primers was designed referring to the hcp gene sequence of A.hydrophila.The hcp gene was amplified by PCR,and performed bioinformatics analysis.[Results]The hcp gene had a total length of 1650 bp and encoded 549 amino acids.The theoretical molecular weight of the protein predicted was about 59476.44 kDa.After predicting the N-terminal signal peptide structure of the amino acid sequence,neither obvious signal peptide cleavage site nor signal peptide was found,and the protein had no transmembrane region.The amino acid sequence had a N-glycosylation site,4 protein kinase C phosphorylation sites,7 casein kinase II phosphorylation sites,9 N-myristoylation sites,4 isoprene binding sites,10 microbody C-terminal target signal sites,and an ATP/GTP binding site motif A(P-ring).The amino acid sequence of hcp gene of A.hydrophila was performed homology analysis with other Aeromonas strains,and it showed higher homology with A.veronii.In the secondary structure,theα-helix,β-sheet,random coil and extended strand accounted for 45.36%,6.01%,37.52%and 11.11%,respectively.The tertiary structure model consisted of 18α-helix and 22β-sheet.Analysis of protein-protein network interaction demonstrated that the proteins interacting with Hcp protein were AHA_3407,nrfA,nirB-1,nirB-2 and AHA_1112.[Conclusions]Through the bioinformatics prediction results,the basic information of hcp gene of A.hydrophila is preliminarily understood,and the possible function of this protein is predicted,in order to provide guidance for subsequent vaccine research.
基金This research was funded by National Natural Science Foundation of China,Grant Number(31871576)National Keypoint Research and Invention Program of the Thirteenth,Grant Number(2019YFD1002205)The APC was funded by National Keypoint Research and Invention Program of the Thirteenth.
文摘For the purpose of functional validation,the mung bean(Vigna radiata)VrPR gene was cloned and overexpressed in Arabidopsis thaliana.Thefindings revealed that the ORF of VrPR contained 1200 bp,in which 399 amino acids were encoded.Bioinformatics analysis showed that the VrPR protein belonged to the NADB Rossmann superfamily,which was one of the non-transmembrane hydrophilic proteins.VrPR was assumed to have 44 amino acid phosphorylation sites and be contained in chloroplasts.The VrPR secondary structure comprised of random coil,αhelix,βangle,and extended chain,all of which were quite compatible with the anticipated tertiary structure.Moreover,analysis of the phylogenetic tree indicated that the soybean PR(Glyma.12G222200)and VrPR were closely related.Furthermore,chlorophyll content in leaves is markedly increased in Arabidopsis when VrPR is overexpressed.Ourfindings will serve as a reference for more functional studies on the PR genes in mung bean.
基金Supported by Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityGrants from Shenzhen Science and Technology Project(JCYJ20190813104207152)+3 种基金National Natural Science Foundation of China(32073015)Natural Science Foundation of Guangdong Province(2021A1515011078)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2022005)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘[Objectives]To clone and analyze the vscB gene of Vibrio alginolyticus HY9901 by bioinformatics.[Methods]A pair of specific primers were designed according to the vscB gene sequence of Vibrio alginolyticus HY9901.The full length of the primers was cloned by PCR and analyzed by bioinformatics.[Results]The vscB gene was 429 bp long,encoding 142 amino acids,with a theoretical molecular weight of 16.4 kDa and a pI value of 5.48.Amino acid sequence analysis of VscB showed that VscB was not a secretory protein,without signal peptide and transmembrane region,and there were protein kinase C phosphorylation site and casein kinase II phosphorylation site in the sequence.Homologous comparison of amino acid sequences showed that VscB of V.alginolyticus had the highest protein similarity with Vibrio Parahaemolyticus,reaching 91%.Phylogenetic tree analysis showed that the corresponding proteins of V.alginolyticus VscB,Vibrio Parahaemolyticus and Vibrio diabolicus were clustered in the same subfamily.Functional domain analysis showed that it had CesT family domain.Tertiary structure prediction showed that there were 3α-helices and 5β-turns in VscB protein.[Conclusions]This study provided a theoretical basis for further study on the function of chaperone of V.alginolyticus.
基金funded by the National Natural Science Foundation of China(21904139)。
文摘Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.
