The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monit...The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.展开更多
Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,...Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.展开更多
[Objectives]To investigate the structure and function of the lipoxygenase(LOX)gene family in Physcomitrella patens.[Methods]This study employed bioinformatics methods to identify and predict LOX gene family members.Qu...[Objectives]To investigate the structure and function of the lipoxygenase(LOX)gene family in Physcomitrella patens.[Methods]This study employed bioinformatics methods to identify and predict LOX gene family members.Quantitative real-time PCR(qRT-PCR)was utilized to analyze the expression patterns of LOX genes at different stages of Botrytis cinerea infection.[Results]The P.patens LOX gene family comprises eight putative proteins,including two 12-LOX-type members and six 13-LOX-type members.Among the eight LOX proteins,PpLOX7 exhibited the lowest molecular weight and shortest amino acid sequence.PpLOX7 was identified as a basic protein with an isoelectric point(pI)of 8.54,while all other members were acidic.Subcellular localization analysis indicated that PpLOX7 was localized to the chloroplast,whereas the remaining members were distributed in the cytoplasm.Secondary structure prediction showed that all eight proteins were predominantly composed of random coils andα-helixes.Chromosomal mapping revealed that the LOX genes were distributed across 7 of the 27 chromosomes in P.patens,with PpLOX1 and PpLOX2 tandemly arranged on chromosome 15.The qRT-PCR analysis demonstrated distinct expression patterns among the eight PpLOX genes following B.cinerea infection.PpLOX1-3 and PpLOX7 were upregulated to varying degrees,suggesting their potential involvement in the early defense response of P.patens against B.cinerea.Notably,PpLOX2 exhibited highly significant differential expression,making it a key candidate for further investigation.[Conclusions]This study provides foundational insights into the functional roles of the LOX gene family in P.patens during biotic stress responses.展开更多
The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examinin...The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examining their phylogenetic relationships,conserved motifs,gene structures,and syntenic relationships.The analysis identified 54 Ah PR10 genes,which were classified into eight groups based on phylogenetic relationships,supported by gene structure and conserved motif characterization.Analysis of chromosomal distribution and synteny demonstrated that segmental duplications played a crucial role in the expansion of the Ah PR10 gene family.The identified Ah PR10 genes exhibited both constitutive and inducible expression patterns.Significantly,Ah PR10-7,Ah PR10-33,and Ah PR10-41 demonstrated potential importance in peanut resistance to Aspergillus flavus.In vitro fungistatic experiments demonstrated that recombinant Ah PR10-33 effectively inhibited A.flavus mycelial growth.These findings provide valuable insights for future investigations into Ah PR10 functions in protecting peanut from A.flavus infection.展开更多
Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)a...Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)and linearly polarized light(LPL),but also are the only animals capable of recognizing circularly polarized light(CPL).Here,we integrated single-cell RNA sequencing,previously published Illumina data,and in-situ hybridization(ISH)to quantify and localize functional opsin genes in Oratosquilla oratoria,a common stomatopoda species in the China Sea.A total of high-quality 31777 cells were captured for the first time in the O.oratoria compound eye,which were classified into 25 cell subpopulations,and hypothesized that cluster 22 is a critical cell subpopulation responsible for light(whether NL,LPL,or CPL)response in O.oratoria.Furthermore,we propose that the long-wavelengthsensitive opsin gene(lws)gene family,retinol dehydrogenase(rdh),voltage-gated ion channel(vgic),arrestin(arr),and myosin(myo)collectively mediate the light response in O.oratoria.Considering that very few vision-related opsin genes show differential expression in right-handed CPL(RCPL)-vs.-dark(DL),which provides additional evidence that stomatopoda cannot recognize RCPL.Meanwhile,we believe that UV-stimulated scaffold protein A(uvssa)and red pigment concentrating hormone(rpch)play special contributions in the left-handed CPL(LCPL)environment response.ISH revealing that 16 lws,6 middle-wavelength-sensitive(mws),and 2 ultraviolet(uv)opsin genes were expressed in the photoreceptors of the O.oratoria compound eye.Although the inability to determine the functional types of cell subpopulations limits the resolution of opsin genes,these findings systematically elucidate the specific expression patterns of opsin genes in O.oratoria and represent a significant step toward refining the visual ecological theory of O.oratoria and other stomatopod species.展开更多
While methodology for determining the mode of evolution in coding sequences has been well established,evaluation of adaptation events in emerging types of phenotype data needs further development.Here,we propose an an...While methodology for determining the mode of evolution in coding sequences has been well established,evaluation of adaptation events in emerging types of phenotype data needs further development.Here,we propose an analysis framework(expression variance decomposition,EVaDe)for comparative single-cell expression data based on phenotypic evolution theory.After decomposing the gene expression variance into separate components,we use two strategies to identify genes exhibiting large between-taxon expression divergence and small within-cell-type expression noise in certain cell types,attributing this pattern to putative adaptive evolution.In a dataset of primate prefrontal cortex,we find that such humanspecific key genes enrich with neurodevelopment-related functions,while most other genes exhibit neutral evolution patterns.Specific neuron types are found to harbor more of these key genes than other cell types,thus likely to have experienced more extensive adaptation.Reassuringly,at the molecular sequence level,the key genes are significantly associated with the rapidly evolving conserved non-coding elements.An additional case analysis comparing the naked mole-rat(NMR)with the mouse suggests that innateimmunity-related genes and cell types have undergone putative expression adaptation in NMR.Overall,the EVaDe framework may effectively probe adaptive evolution mode in single-cell expression data.展开更多
Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challengin...Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.展开更多
Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily...Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.展开更多
Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribo...Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.展开更多
Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fol...Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fold higher biological activities,compared with the monomer.Unfortunately,the yield of di-AFN A_(1) is very low(0.09±0.03 mg·L^(−1))in the engineered strain Streptomyces alboflavus 313_hmtS(S.albo/313_hmtS),which is not friendly to be genetically engineered for titer improvement of di-AFN A_(1) production.In this study,we constructed a biosynthetic gene cluster for di-AFN A_(1) and increased its production through heterologous expression.During the collection of di-AFN A_(1) biosynthetic genes,the afn genes were located at three sites of S.alboflavus 313 genome.The di-AFN A_(1) biosynthetic gene cluster(BGC)was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24,which produced di-AFN A_(1) at a titer of 0.43±0.01 mg·L^(−1).To further increase the yield of di-AFN A_(1),the di-AFN A_(1) BGC was multiplied and split to mimic the natural afn biosynthetic genes,and the production of di-AFN A_(1) increased to 0.62±0.11 mg·L^(−1) in S.lividans TK24 by the later strategy.Finally,different Streptomyces hosts were tested and the titer of di-AFN A_(1) increased to 0.81±0.17 mg·L^(−1),about 8.0-fold higher than that in S.albo/313_hmtS.Successful heterologous expression of di-AFN A_(1) with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.展开更多
The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their ...The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.展开更多
Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and t...Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.展开更多
The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genom...The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.展开更多
Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterolog...Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.展开更多
Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different envir...Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different environmental conditions and bacterial pathogenesis. The outer membrane protein R (OmpR) is a member of the two-component regulatory system of Aeromonas hydrophila which differentially regulates the expression of OmpF or OmpC depending on the osmolarity conditions. Role of OmpR has been demonstrated in its virulence in other infectious bacteria and it is found to be a potential drug target/vaccine candidate. However, the OmpR of A. hydrophila has not been characterized. In the present study, we report recombinant expression, purification of the OmpR of A. hydrophila strain Ah17 in salt inducible E. coli GJ1158 cells. Leaky expression of rOmpR was confirmed by Western blot analysis using anti-6 × His antibody. The histidine tagged recombinant OmpR (rOmpR) (~29 kDa) was purified using Ni-NTA affinity chromatography from the soluble fraction of induced E. coli cells. The rOmpR was found to be highly immunogenic with end point titres of greater than 1:80,000. The anti-rOmpR antisera were capable of agglutinating live A. hydrophila cells, thus showing vaccine potential of the rOmpR.展开更多
Objective:To clone,express and purify a putative parasitic nematode specific protein of Setaria digitata(S.digitata),filarial nematode that infects livestock and cause significant economic losses in Far East and Asia ...Objective:To clone,express and purify a putative parasitic nematode specific protein of Setaria digitata(S.digitata),filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to he used for structural and functional analyses.Methods:To characterize uneharacterized gene of,S.digitata(SDUG),the herterologous expression of SDUG was carried out in the pET[cloned into pET45b(+)]expression system initially and co-expression of SDUC using chaperoiie plasmids pG-KJE8,pGro 7,pKJE7,pG-Tf2 and pTf16 containing chapcrone proteins of dnaK-dnaJ-grpE-groES-gro-E,groES-groEL,dnaK-dnaJ-grpE,groES-groEL-tig,and tig respectively,was carried out subsequently.Results:Expression of SDUG was seen when Escherichia coli strain BI.21(DE3)is used,while concentrating protein largely into the insoluble fraction.The co-expression of SDUG using chaperoiie plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasniid sets,the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of2 mg/mL L-arabinosc and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28℃.Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of^24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.展开更多
The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A,under the control of the AOX1 promoter.The recombina...The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A,under the control of the AOX1 promoter.The recombinant plasmid pHBM905A-85B linearized by Sal I was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method.After phenotype screening and PCR identification,the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol.The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×10^3 approximately detected by SDS-PAGE and Western blot.EI,ISA experiment proved that the protein had good antigen specificity.Secretory expression of recombinant M.tuberculosis Ag85B in P.pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.展开更多
Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHS...Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHSPs in Lenzites gibbosa,a common polypore in northern temperate forests that causes spongy white rot of broadleaf trees,under temperature stress,L.gibbosa mycelia were grown at 25℃ for 9 d,treated at 33℃ for 15,30,60,and 120 min before sequencing the transcriptomes.From among 32 heat shock protein(HSP)genes found in the screen of the transcriptome data,a highly expressed gene was cloned and named Lghsp17.4.RT-qPCR was used to analyze the expression of the gene Lghsp17.4 under heat shock and dye stress.Both treatments induced higher expression of Lghsp17.4 at the transcriptional level,indicating that Lghsp17.4 might function in the response to heat stress and dye degradation.We previously found that L.gibbosa generally had a heat shock reaction(HSR)during degradation of aromatic compounds,and HSPs were always produced with manganese peroxidases(MnPs)and other lignin-degrading enzymes.Therefore,we measured the activity of MnPs in L.gibbosa after 33℃ heat shock to analyze the relationship between MnPs expression and Lghsp17.4 expression.Heat shocks of 0–30 min increased MnPs activity,and the change in MnPs activity were closely positively correlated with the expression levels of Lghsp17.4 over time,indicating a potential connection and interaction between LgHSP17.4 and MnPs during the HSR in L.gibbosa.Thus,LgHSP17.4 might have a positive regulatory effect on the HSR in L.gibbosa and be a critical component of a stress resistance mechanism.展开更多
Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situ...Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.展开更多
Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confi...Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confirmed that OsADCS and OsGTPCHI,encoding the initial enzymes necessary for folate synthesis,positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds.The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter.We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes;the folate accumulation in brown rice and polished rice reached 5.65μg/g and 2.95μg/g,respectively,representing 37.9-fold and 26.5-fold increases compared with the wild type.Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances,while the expression of other folate synthesis genes was weakly regulated.In addition,we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation.This study provides an alternative and effective complementary strategy for rice biofortification,promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.展开更多
基金湖南省教育厅基金优秀青年项目(No.22B0482)湖南科技大学博士启动基金(No.E51992 and E51993)资助。
文摘The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.
基金supported by the National Natural Science Foundation of China(32271580,42020104009)the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes(JX6311101923)。
文摘Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.
基金Supported by the Science and Technology Program of Guizhou Provence(Qiankehejichu-ZK[2023]Yiban 271).
文摘[Objectives]To investigate the structure and function of the lipoxygenase(LOX)gene family in Physcomitrella patens.[Methods]This study employed bioinformatics methods to identify and predict LOX gene family members.Quantitative real-time PCR(qRT-PCR)was utilized to analyze the expression patterns of LOX genes at different stages of Botrytis cinerea infection.[Results]The P.patens LOX gene family comprises eight putative proteins,including two 12-LOX-type members and six 13-LOX-type members.Among the eight LOX proteins,PpLOX7 exhibited the lowest molecular weight and shortest amino acid sequence.PpLOX7 was identified as a basic protein with an isoelectric point(pI)of 8.54,while all other members were acidic.Subcellular localization analysis indicated that PpLOX7 was localized to the chloroplast,whereas the remaining members were distributed in the cytoplasm.Secondary structure prediction showed that all eight proteins were predominantly composed of random coils andα-helixes.Chromosomal mapping revealed that the LOX genes were distributed across 7 of the 27 chromosomes in P.patens,with PpLOX1 and PpLOX2 tandemly arranged on chromosome 15.The qRT-PCR analysis demonstrated distinct expression patterns among the eight PpLOX genes following B.cinerea infection.PpLOX1-3 and PpLOX7 were upregulated to varying degrees,suggesting their potential involvement in the early defense response of P.patens against B.cinerea.Notably,PpLOX2 exhibited highly significant differential expression,making it a key candidate for further investigation.[Conclusions]This study provides foundational insights into the functional roles of the LOX gene family in P.patens during biotic stress responses.
基金supported by the National Key R&D Program of China(2022YFD1200400)the National Natural Science Foundation of China(32301851)。
文摘The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examining their phylogenetic relationships,conserved motifs,gene structures,and syntenic relationships.The analysis identified 54 Ah PR10 genes,which were classified into eight groups based on phylogenetic relationships,supported by gene structure and conserved motif characterization.Analysis of chromosomal distribution and synteny demonstrated that segmental duplications played a crucial role in the expansion of the Ah PR10 gene family.The identified Ah PR10 genes exhibited both constitutive and inducible expression patterns.Significantly,Ah PR10-7,Ah PR10-33,and Ah PR10-41 demonstrated potential importance in peanut resistance to Aspergillus flavus.In vitro fungistatic experiments demonstrated that recombinant Ah PR10-33 effectively inhibited A.flavus mycelial growth.These findings provide valuable insights for future investigations into Ah PR10 functions in protecting peanut from A.flavus infection.
基金Supported by the Natural Science Foundation of Shandong Province(No.ZR2021QD110)the National Natural Science Foundation of China(No.42106128)。
文摘Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)and linearly polarized light(LPL),but also are the only animals capable of recognizing circularly polarized light(CPL).Here,we integrated single-cell RNA sequencing,previously published Illumina data,and in-situ hybridization(ISH)to quantify and localize functional opsin genes in Oratosquilla oratoria,a common stomatopoda species in the China Sea.A total of high-quality 31777 cells were captured for the first time in the O.oratoria compound eye,which were classified into 25 cell subpopulations,and hypothesized that cluster 22 is a critical cell subpopulation responsible for light(whether NL,LPL,or CPL)response in O.oratoria.Furthermore,we propose that the long-wavelengthsensitive opsin gene(lws)gene family,retinol dehydrogenase(rdh),voltage-gated ion channel(vgic),arrestin(arr),and myosin(myo)collectively mediate the light response in O.oratoria.Considering that very few vision-related opsin genes show differential expression in right-handed CPL(RCPL)-vs.-dark(DL),which provides additional evidence that stomatopoda cannot recognize RCPL.Meanwhile,we believe that UV-stimulated scaffold protein A(uvssa)and red pigment concentrating hormone(rpch)play special contributions in the left-handed CPL(LCPL)environment response.ISH revealing that 16 lws,6 middle-wavelength-sensitive(mws),and 2 ultraviolet(uv)opsin genes were expressed in the photoreceptors of the O.oratoria compound eye.Although the inability to determine the functional types of cell subpopulations limits the resolution of opsin genes,these findings systematically elucidate the specific expression patterns of opsin genes in O.oratoria and represent a significant step toward refining the visual ecological theory of O.oratoria and other stomatopod species.
文摘While methodology for determining the mode of evolution in coding sequences has been well established,evaluation of adaptation events in emerging types of phenotype data needs further development.Here,we propose an analysis framework(expression variance decomposition,EVaDe)for comparative single-cell expression data based on phenotypic evolution theory.After decomposing the gene expression variance into separate components,we use two strategies to identify genes exhibiting large between-taxon expression divergence and small within-cell-type expression noise in certain cell types,attributing this pattern to putative adaptive evolution.In a dataset of primate prefrontal cortex,we find that such humanspecific key genes enrich with neurodevelopment-related functions,while most other genes exhibit neutral evolution patterns.Specific neuron types are found to harbor more of these key genes than other cell types,thus likely to have experienced more extensive adaptation.Reassuringly,at the molecular sequence level,the key genes are significantly associated with the rapidly evolving conserved non-coding elements.An additional case analysis comparing the naked mole-rat(NMR)with the mouse suggests that innateimmunity-related genes and cell types have undergone putative expression adaptation in NMR.Overall,the EVaDe framework may effectively probe adaptive evolution mode in single-cell expression data.
基金supported by Central Public-interest Scientific Institution Basal Research Fund(CATAS-Nos.1630152023007,1630152023011,1630152023012,1630152023013)the National Natural Science Foundation of China(Grant No.32071805).
文摘Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.
基金This work was supported by the China Postdoctoral Science Foundation(Grant No.2016M602873)the Fundamental Research Funds for the Central Universities(Grant No.2452016182,2452017074)+1 种基金the earmarked fund for Modern Agro-industry Technology Research System(Grant No.CARS-19)the special fund for University-Supported Extension Model(Grant No.TGZX2018-39).
文摘Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.
基金supported by the National Natural Science Foundation of China(Nos.81573311 and 82173702).
文摘Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.
基金supported by the Ministry of Science and Technology of the People’s Republic of China(No.2020YFA0907703)the National Natural Science Foundation of China(Nos.32070044,32025002)the Biological Resources Programme,Chinese Academy of Sciences(No.KFJ-BRP-009)。
文摘Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fold higher biological activities,compared with the monomer.Unfortunately,the yield of di-AFN A_(1) is very low(0.09±0.03 mg·L^(−1))in the engineered strain Streptomyces alboflavus 313_hmtS(S.albo/313_hmtS),which is not friendly to be genetically engineered for titer improvement of di-AFN A_(1) production.In this study,we constructed a biosynthetic gene cluster for di-AFN A_(1) and increased its production through heterologous expression.During the collection of di-AFN A_(1) biosynthetic genes,the afn genes were located at three sites of S.alboflavus 313 genome.The di-AFN A_(1) biosynthetic gene cluster(BGC)was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24,which produced di-AFN A_(1) at a titer of 0.43±0.01 mg·L^(−1).To further increase the yield of di-AFN A_(1),the di-AFN A_(1) BGC was multiplied and split to mimic the natural afn biosynthetic genes,and the production of di-AFN A_(1) increased to 0.62±0.11 mg·L^(−1) in S.lividans TK24 by the later strategy.Finally,different Streptomyces hosts were tested and the titer of di-AFN A_(1) increased to 0.81±0.17 mg·L^(−1),about 8.0-fold higher than that in S.albo/313_hmtS.Successful heterologous expression of di-AFN A_(1) with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.
文摘The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.
基金The study was supported by National Basic Science Key Infrastructure Development Program (973 program), National Ministry of Science and Technology (Grant No: G19990559) and in part by E-institutes fund of Shanghai Municipal Education Commission. (Grant No:E03003)
文摘Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.
基金supported by the National High Technology Research and Development Program of China(Grant no.2007AA091905)the Basic Scientific Research Operation Fund of China(Grant no.GY02-2007-T11)
文摘The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.
基金funded by the National Key Research and Development Program of China(2021YFC2102700)was Supported by the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)as well as the Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.
文摘Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different environmental conditions and bacterial pathogenesis. The outer membrane protein R (OmpR) is a member of the two-component regulatory system of Aeromonas hydrophila which differentially regulates the expression of OmpF or OmpC depending on the osmolarity conditions. Role of OmpR has been demonstrated in its virulence in other infectious bacteria and it is found to be a potential drug target/vaccine candidate. However, the OmpR of A. hydrophila has not been characterized. In the present study, we report recombinant expression, purification of the OmpR of A. hydrophila strain Ah17 in salt inducible E. coli GJ1158 cells. Leaky expression of rOmpR was confirmed by Western blot analysis using anti-6 × His antibody. The histidine tagged recombinant OmpR (rOmpR) (~29 kDa) was purified using Ni-NTA affinity chromatography from the soluble fraction of induced E. coli cells. The rOmpR was found to be highly immunogenic with end point titres of greater than 1:80,000. The anti-rOmpR antisera were capable of agglutinating live A. hydrophila cells, thus showing vaccine potential of the rOmpR.
基金supported by a grant(SIDA/2006/BT/04) awarded by National Science Foundation of Sri Lanka
文摘Objective:To clone,express and purify a putative parasitic nematode specific protein of Setaria digitata(S.digitata),filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to he used for structural and functional analyses.Methods:To characterize uneharacterized gene of,S.digitata(SDUG),the herterologous expression of SDUG was carried out in the pET[cloned into pET45b(+)]expression system initially and co-expression of SDUC using chaperoiie plasmids pG-KJE8,pGro 7,pKJE7,pG-Tf2 and pTf16 containing chapcrone proteins of dnaK-dnaJ-grpE-groES-gro-E,groES-groEL,dnaK-dnaJ-grpE,groES-groEL-tig,and tig respectively,was carried out subsequently.Results:Expression of SDUG was seen when Escherichia coli strain BI.21(DE3)is used,while concentrating protein largely into the insoluble fraction.The co-expression of SDUG using chaperoiie plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasniid sets,the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of2 mg/mL L-arabinosc and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28℃.Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of^24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.
基金Supported by the Key Scientific and Technologica1 Project of Wuhan(30112l028)
文摘The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A,under the control of the AOX1 promoter.The recombinant plasmid pHBM905A-85B linearized by Sal I was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method.After phenotype screening and PCR identification,the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol.The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×10^3 approximately detected by SDS-PAGE and Western blot.EI,ISA experiment proved that the protein had good antigen specificity.Secretory expression of recombinant M.tuberculosis Ag85B in P.pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.
基金supported by the Fundamental Research Funds for the Central Universities(Grant No.:2572016AA04)Northeast Asia Biodiversity Research Center Double First class Funds(Grant No.:411146030416 and No.:411147021003).
文摘Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHSPs in Lenzites gibbosa,a common polypore in northern temperate forests that causes spongy white rot of broadleaf trees,under temperature stress,L.gibbosa mycelia were grown at 25℃ for 9 d,treated at 33℃ for 15,30,60,and 120 min before sequencing the transcriptomes.From among 32 heat shock protein(HSP)genes found in the screen of the transcriptome data,a highly expressed gene was cloned and named Lghsp17.4.RT-qPCR was used to analyze the expression of the gene Lghsp17.4 under heat shock and dye stress.Both treatments induced higher expression of Lghsp17.4 at the transcriptional level,indicating that Lghsp17.4 might function in the response to heat stress and dye degradation.We previously found that L.gibbosa generally had a heat shock reaction(HSR)during degradation of aromatic compounds,and HSPs were always produced with manganese peroxidases(MnPs)and other lignin-degrading enzymes.Therefore,we measured the activity of MnPs in L.gibbosa after 33℃ heat shock to analyze the relationship between MnPs expression and Lghsp17.4 expression.Heat shocks of 0–30 min increased MnPs activity,and the change in MnPs activity were closely positively correlated with the expression levels of Lghsp17.4 over time,indicating a potential connection and interaction between LgHSP17.4 and MnPs during the HSR in L.gibbosa.Thus,LgHSP17.4 might have a positive regulatory effect on the HSR in L.gibbosa and be a critical component of a stress resistance mechanism.
基金supported by China Academy of Railway Sciences Corporation Limited(No.2021YJ127).
文摘Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.
基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,China(Grant No.CPSIBRF-CNRRI-202403)。
文摘Rice is a poor source of folate,an essential micronutrient for the body.Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans.In this study,we confirmed that OsADCS and OsGTPCHI,encoding the initial enzymes necessary for folate synthesis,positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds.The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter.We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes;the folate accumulation in brown rice and polished rice reached 5.65μg/g and 2.95μg/g,respectively,representing 37.9-fold and 26.5-fold increases compared with the wild type.Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances,while the expression of other folate synthesis genes was weakly regulated.In addition,we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation.This study provides an alternative and effective complementary strategy for rice biofortification,promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.
