Multicellular magnetotactic prokaryotes （MMPs） are a group of aggregates composed of 7-45 gram-negative cells synthesizing intracellular magnetic crystals. Although they are thought to be globally distributed, MMPs ...Multicellular magnetotactic prokaryotes （MMPs） are a group of aggregates composed of 7-45 gram-negative cells synthesizing intracellular magnetic crystals. Although they are thought to be globally distributed, MMPs have been observed only in marine environments in America and Europe. Most MMPs share a rosette-like morphology and biomineralize iron sulfide crystals. In the present study, abundant MMPs were observed, with a density of 26 ind./cm^3, in the sediments of a coastal lagoon, Lake Yuehu, in the Yellow Sea. Optical microscopy showed that all of them were rosette shaped with a diameter of 5.5±0.8 μm. Transmission electron microscopy revealed that these MMPs were composed of 10- 16 ovoid cells and flagellated peritrichously. High-resolution transmission electron microscopy and energy dispersive X-ray analysis indicated that they biomineralized bullet-shaped magnetite crystals in highly organized parallel chains within which the magnetosomes were oriented in the same direction. This is the first report of MMPs from Asia and demonstrates the ubiquitous distribution of MMPs.展开更多
Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenopr...Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli,Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the Nterminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion展开更多
Prokaryotes play a fundamental role in global ocean biogeochemical cycles.However,how the abundance and metabolic activity of ecologically distinct subgroups(i.e.,high nucleic acid(HNA)and low nucleic acid(LNA)cells),...Prokaryotes play a fundamental role in global ocean biogeochemical cycles.However,how the abundance and metabolic activity of ecologically distinct subgroups(i.e.,high nucleic acid(HNA)and low nucleic acid(LNA)cells),and their regulating factors,change in response to changing marine environmental conditions remains poorly understood.Here,we delved into the time-evolving dynamic responses of the HNA and LNA prokaryotic subgroups to declining resource availability and selective grazing by protozoa by conducting a 73-day incubation experiment in a large-volume(117,000 L)macrocosm that facilitates community-level exploration.We found that the metabolic activity of the HNA subgroup was higher than that of the LNA subgroup when the macrocosm was resource replete but that the HNA subgroup declined more rapidly than the LNA subgroup as the resources became increasingly scarce,leading to a steadily increasing contribution of LNA cells to prokaryotic activity.Meanwhile,as resources in the macrocosm became limited,protozoan grazing preference shifted from the HNA to the LNA subgroup and the contributions of the LNA subgroup to the carbon flow within the macrocosm increased.The findings highlight the resilience of LNA cells in resource-limited environments,illuminate the critical role of selective grazing by protozoa in balancing distinct prokaryotic subgroups under changing resource conditions,and demonstrate the complex and adaptive interactions between protozoa and prokaryotes across diverse environmental contexts.展开更多
Many investigations suggest that dissimilatory arsenate-respiring prokaryotes (DARPs) play a key role in stimulating reductive mobilization of As from solid phase into groundwater,but it is not clear how environmental...Many investigations suggest that dissimilatory arsenate-respiring prokaryotes (DARPs) play a key role in stimulating reductive mobilization of As from solid phase into groundwater,but it is not clear how environmental Mn(Ⅱ) affects the DARPs-mediated reductive mobilization of arsenic.To resolve this issue,we collected soil samples from a realgar tailingsaffected area.We found that there were diverse arsenate-respiratory reductase (arr) genes in the soils.The microbial communities had high arsenate-respiring activity,and were able to efficiently stimulate the reductive mobilization of As.Compared to the microcosms without Mn(Ⅱ),addition of 10 mmol/L Mn(Ⅱ) to the microcosms led to 23.99%-251.79% increases in the microbial mobilization of As,and led to 133.3%-239.2% increases in the abundances of arr genes.We further isolated a new cultivable DARP,Bacillus sp.F11,from the arseniccontaminated soils.It completely reduced 1 mmol/L As(V) in 5 days under the optimal reaction conditions.We further found that it was able to efficiently catalyze the reductive mobilization and release of As from the solid phase;the addition of 2 mmol/L Mn(Ⅱ) led to 98.49%-248.78% increases in the F11 cells-mediated reductive mobilization of As,and70.6%-104.4% increases in the arr gene abundances.These data suggest that environmental Mn(Ⅱ) markedly increased the DARPs-mediated reductive mobilization of As in arseniccontaminated soils.This work provided a new insight into the close association between the biogeochemical cycles of arsenic and manganese.展开更多
We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxo...We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxonomy-primarily the latest online Outline of the Bergey's Manual of Systematic Bacteri-ology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.展开更多
In order to show that the newly developed K-string composition distance method, based on counting oligopeptide frequencies, for inferring phylogenetic relations of prokaryotes works equally well without requiring the ...In order to show that the newly developed K-string composition distance method, based on counting oligopeptide frequencies, for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data, we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species. The latter group has been known to yield inconsistent trees if used individually. Our trees are obtained without making any sequence alignment. Altogether 16 Archaea, 105 Bacteria and 2 Eucarya are represented on the tree. Most of the lower branchings agree well with the latest, 2003, Outline of the second edition of the Bergeys Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.展开更多
Globally,various types of pollution affect coastal waters as a result of human activities.Bioaugmentation and biostimulation are effective methods for treating water pollution.However,few studies have explored the res...Globally,various types of pollution affect coastal waters as a result of human activities.Bioaugmentation and biostimulation are effective methods for treating water pollution.However,few studies have explored the response of coastal prokaryotic and eukaryotic communities to bioaugmentation and biostimulation.Here,a 28-day outdoor mesocosm experiment with two treatments(bioaugmentation-A and combined treatment of bioaugmentation and biostimulation-AS)and a control(untreated-C)were carried out.The experiment was conducted in Meishan Bay to explore the composition,dynamics,and co-occurrence patterns of prokaryotic and eukaryotic communities in response to the A and AS using 16S rRNA and 18S rRNA gene amplicon sequencing.After treatment,Gammaproteobacteria and Epsilonproteobacteria were significantly increased in group AS compared to group C,while Flavobacteriia and Saprospirae were significantly reduced.Dinoflagellata was significantly reduced in AS compared to C,while Chrysophyta was significantly reduced in both AS and A.Compared to C,the principal response curve analyses of the prokaryotic and eukaryotic communities both showed an increasing trend followed by a decreasing trend for AS.Furthermore,the trends of prokaryotic and eukaryotic communities in group A were similar to those in group AS compared with group C,but AS changed them more than A did.According to the species weight table on principal response curves,a significant increase was observed in beneficial bacteria in prokaryotic communities,such as Rhodobacterales and Oceanospirillales,along with a decrease in autotrophs in eukaryotic communities,such as Chrysophyta and Diatom.Topological properties of network analysis reveal that A and AS complicate the interactions between the prokaryotic and eukaryotic communities.Overall,these findings expand our understanding of the response pattern of the bioaugmentation and biostimulation on coastal prokaryotic and eukaryotic communities.展开更多
Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts...Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.展开更多
High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlappin...High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlapping architecture when compared to the existing genome annotation. Therefore, there is a need for customized tiling array data analysis tools. Since most of the initial tiling arrays were conducted in eukaryotes, data analysis methods are well suited for eukaryofic genomes. For using whole-genome tiling arrays to identify previously unknown transcriptional elements like small RNA and antisense RNA in prokaryotes, existing data analysis tools need to be tailored for prokaryotic genome architecture. Furthermore, automation of such custom data analysis workflow is necessary for biologists to apply this powerful platform for knowledge discovery. Here we describe TAAPP, a web-based package that consists of two modules for prokaryotic tiling array data analysis. The transcript generation module works on normalized data to generate transcriptionally active regions （TARs）. The feature extraction and annotation module then maps TARs to existing genome annotation. This module further categorizes the transcription profile into potential novel non-coding RNA, antisense RNA, gene expression and operon structures. The implemented workflow is microarray platform independent and is presented as a web-based service. The web interface is freely available for acedemic use at http：//lims.lshi.mafes.msstate.edu/TAAPP-HTML/.展开更多
Prokaryotic organisms have evolved sophisticated immune systems for self-protection to resist bacteriophage invasion.With further research on prokaryotic immune systems,several important molecular biology tools have b...Prokaryotic organisms have evolved sophisticated immune systems for self-protection to resist bacteriophage invasion.With further research on prokaryotic immune systems,several important molecular biology tools have been developed,including the widely used restriction endonuclease in molecular cloning experiments and the CRISPR-Cas system in gene editing.展开更多
The short term(hourly scale)variability of heterotrophic prokaryote(HP)vertical distribution and respiratory activity,was investigated in the north-western(NW)Mediterranean Sea.HP vertical distribution was determined ...The short term(hourly scale)variability of heterotrophic prokaryote(HP)vertical distribution and respiratory activity,was investigated in the north-western(NW)Mediterranean Sea.HP vertical distribution was determined on board by flow cytometry analysis of seawater samples collected by series of CTD casts.Cell counts and viability were determined for all samples.HP respiratory rates were determined later in the laboratory from filtered seawater samples(23 dm^(3))from 300-1150-m depth.The average cell viability was 94.8%±2.2%(n=240).There was no accumulation of dead cells,due to quick decay of damaged cells.In the epipelagic layer,three HP groups were distinguished,two(HNA1,HNA2)who se cells exhibited a high nucleic acid content and one(LNA)with low nucleic acid content cells.HNA2 was most populated at 50 m but not detected at 90 m and below,presumably aerobic anoxygenic photoheterotrophic bacteria(AAPs).The variability in HP abundance was mainly confined in the upper 80 m.A few secondary peaks of HP abundance were observed(80-150 m)in connection with abundance troughs in the surface layer.HP cells were continuously present in a wide layer around 500 m(mean 191×10^(3)cells/cm^(3)).Below this layer,HP abundance randomly exhibited peaks,coupled to respiratory rate peaks.The HP abundance and variability in the water column was suppressed during a strong wind event.The observed sporadic variability was tentatively interpreted through a pulsed carbon-export mechanism induced by the microorganism production of dissolved poly saccharide s,followed by flocculation and rapid sinking.This mechanism would thus contribute to(ⅰ)preventing organic matter accumulation in the epipelagic layer,(ⅱ)seeding the water column with live HP cells,and(ⅲ)supplying the aphotic water column with fre sh and labile organic matter.This important vertical flux mechanism needs further observations and modelling.展开更多
Prokaryotic diversity and community composition in the water column of eight stations(63 samples) around the Antarctic Peninsula of the Southern Ocean were investigated. Through pyrosequencing of the V3–V4hypervariab...Prokaryotic diversity and community composition in the water column of eight stations(63 samples) around the Antarctic Peninsula of the Southern Ocean were investigated. Through pyrosequencing of the V3–V4hypervariable regions of the 16S ribosomal RNA gene, we characterized 4 720 089 valid reads representing 48 188operational taxonomic units(OTUs, 97% similarity). The community was dominated by the phyla Pseudomonadota(original name: Proteobacteria, 47%), Oxyphotobacteria(26%), and Bacteroidota(original name: Bacteroidetes, 18%), which comprised an average of 91% of the total OTUs in all samples. The prokaryotic community composition varied vertically within the water column. Water column prokaryotic communities exhibited a clear depth profile, with higher microbial richness and higher diversity observed with increasing water depth. Cluster analysis of the community composition of water column samples exhibited a similar trend with depth. Correlation with environmental factors suggested distinct variation in prokaryotic community composition with changes in depth, salinity, temperature and dissolved oxygen levels. Functional prediction showed presence of active nitrogen, sulphur and methane metabolic cycles along the vertical transect of the studied region. These results will improve our knowledge of prokaryotic diversity and community composition at different depth of water column for better understanding of the microbial ecology and nutrient cycles in Antarctic Peninsula region of the Southern Ocean.展开更多
Microorganisms are fundamental for the functioning of marine ecosystems and are involved in the decomposition of organic matter, transformation of nutrients and circulation of biologically-important chemicals. Based o...Microorganisms are fundamental for the functioning of marine ecosystems and are involved in the decomposition of organic matter, transformation of nutrients and circulation of biologically-important chemicals. Based on the complexity of the natural geographic characteristics of the Changjiang River Estuary, the geographic distribution of sedimentary microorganisms and the causes of this distribution are largely unexplored. In this work, the surface sediment samples from the adjacent sea area of the Changjiang River Estuary were collected. Their prokaryotic diversity was examined by high-throughput sequencing technology, and the environmental factors of the bacterial community were investigated. The results indicated that the distribution of prokaryotic communities in the sediments of the study areas showed obvious spatial heterogeneity. The sampling sequences divided the sample regions into three distinct clusters. Each geographic region had a unique community structure, although Proteobacteria, Bacteroidota, Desulfobacterota, Acidobacteriota, and Actinobacteriota all existed in these three branches. Canonical correspondence analysis demonstrated that prokaryotic diversity and community distribution were significantly correlated with the geographic location of sediment, seawater depth, and in particular, nutrient content(e.g., total phosphorus, total organic carbon and dissolved oxygen). Moreover, it was found for the first time that the metal ions obviously affected the composition and distribution of the prokaryotic community in this area. In general, this work provides new insights into the structural characteristics and driving factors of prokaryotic communities under the background of the ever-changing Changjiang River Estuary.展开更多
The potential involvement of calcium in signalling in cyanobacteria has been investigated in recent years. Enough evidences showed that the cyanobacteria were capable of sensing and distinguishing different environmen...The potential involvement of calcium in signalling in cyanobacteria has been investigated in recent years. Enough evidences showed that the cyanobacteria were capable of sensing and distinguishing different environmental stimuli, and making responses in ways of Ca^2＋ transients, which were the results of influx or efflux of Ca^2＋ aroused by different environmental stimuli. The calcium signal elicited by nitrogen starvation was crucial to heterocyst differentiation in filamentous cyanobacteria Anabaena species. Identification of a calcium-binding protein （CcbP） from Anabaena sp. PCC 7120 provided further evidence, and the degradation and down-regulation of CcbP accounted for the generation of calcium signal when nitrogen starvation exits. However, the encoding and decoding mechanisms of the calcium signals in cyanobacteria still remain unclear. In order to reveal the exact role of it, a detailed, systematic investigation will be needed, especially for the calcium dynamics at the single cell level.展开更多
[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucl...[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.展开更多
Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doubl...Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.展开更多
[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding...[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.展开更多
A full-length sequence coding for △^12 fatty acid desaturase gene from peanut（Arachis hypogaea L.）was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently tr...A full-length sequence coding for △^12 fatty acid desaturase gene from peanut（Arachis hypogaea L.）was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21（DE3）pLysS. The △^12 fatty acid desaturase was highly expressed in E. coli BL21（DE3）pLysS in the presence of isopropyl-D-thiogalactopyranoside （IPTG）. The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20℃ for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS （gas chromatogram and gas chromatogram-mass spectrometry） analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited △^12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.展开更多
Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombi...Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.展开更多
[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification...[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.展开更多
基金Supported by the National Natural Science Foundation of China(Nos. 40906069,40776094)Shangdong 908 Project (No. SD-908-02-08)+1 种基金the Haiwaijiechuxuezhe-Fund of Chinese Academy of Sciences (No.2006-1-15)K. C. WONG Education Foundation
文摘Multicellular magnetotactic prokaryotes （MMPs） are a group of aggregates composed of 7-45 gram-negative cells synthesizing intracellular magnetic crystals. Although they are thought to be globally distributed, MMPs have been observed only in marine environments in America and Europe. Most MMPs share a rosette-like morphology and biomineralize iron sulfide crystals. In the present study, abundant MMPs were observed, with a density of 26 ind./cm^3, in the sediments of a coastal lagoon, Lake Yuehu, in the Yellow Sea. Optical microscopy showed that all of them were rosette shaped with a diameter of 5.5±0.8 μm. Transmission electron microscopy revealed that these MMPs were composed of 10- 16 ovoid cells and flagellated peritrichously. High-resolution transmission electron microscopy and energy dispersive X-ray analysis indicated that they biomineralized bullet-shaped magnetite crystals in highly organized parallel chains within which the magnetosomes were oriented in the same direction. This is the first report of MMPs from Asia and demonstrates the ubiquitous distribution of MMPs.
文摘Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli,Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the Nterminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion
基金supported by the National Natural Science Foundation of China(Grant Nos.42188102,41861144018)the Natural Science Foundation of Fujian Province of China(Grant No.2023J05017)+3 种基金the Marine Economic Development Special Fund Project of Fujian Province of China(Grant No.FJHJF-L-2022-11)supported by the China Postdoctoral Science Foundation(Grant No.2021M691863)supported by the Innovation Team Project of Universities in Guangdong Province(Grant No.2023KCXTD028)supported by the Ph.D.Fellowship of the State Key Laboratory of Marine Environmental Science at Xiamen University。
文摘Prokaryotes play a fundamental role in global ocean biogeochemical cycles.However,how the abundance and metabolic activity of ecologically distinct subgroups(i.e.,high nucleic acid(HNA)and low nucleic acid(LNA)cells),and their regulating factors,change in response to changing marine environmental conditions remains poorly understood.Here,we delved into the time-evolving dynamic responses of the HNA and LNA prokaryotic subgroups to declining resource availability and selective grazing by protozoa by conducting a 73-day incubation experiment in a large-volume(117,000 L)macrocosm that facilitates community-level exploration.We found that the metabolic activity of the HNA subgroup was higher than that of the LNA subgroup when the macrocosm was resource replete but that the HNA subgroup declined more rapidly than the LNA subgroup as the resources became increasingly scarce,leading to a steadily increasing contribution of LNA cells to prokaryotic activity.Meanwhile,as resources in the macrocosm became limited,protozoan grazing preference shifted from the HNA to the LNA subgroup and the contributions of the LNA subgroup to the carbon flow within the macrocosm increased.The findings highlight the resilience of LNA cells in resource-limited environments,illuminate the critical role of selective grazing by protozoa in balancing distinct prokaryotic subgroups under changing resource conditions,and demonstrate the complex and adaptive interactions between protozoa and prokaryotes across diverse environmental contexts.
基金supported by the General Programs (No. 41472219)the Foundations for Innovative Research Groups (No. 41521001) from the National Natural Science Foundation of China。
文摘Many investigations suggest that dissimilatory arsenate-respiring prokaryotes (DARPs) play a key role in stimulating reductive mobilization of As from solid phase into groundwater,but it is not clear how environmental Mn(Ⅱ) affects the DARPs-mediated reductive mobilization of arsenic.To resolve this issue,we collected soil samples from a realgar tailingsaffected area.We found that there were diverse arsenate-respiratory reductase (arr) genes in the soils.The microbial communities had high arsenate-respiring activity,and were able to efficiently stimulate the reductive mobilization of As.Compared to the microcosms without Mn(Ⅱ),addition of 10 mmol/L Mn(Ⅱ) to the microcosms led to 23.99%-251.79% increases in the microbial mobilization of As,and led to 133.3%-239.2% increases in the abundances of arr genes.We further isolated a new cultivable DARP,Bacillus sp.F11,from the arseniccontaminated soils.It completely reduced 1 mmol/L As(V) in 5 days under the optimal reaction conditions.We further found that it was able to efficiently catalyze the reductive mobilization and release of As from the solid phase;the addition of 2 mmol/L Mn(Ⅱ) led to 98.49%-248.78% increases in the F11 cells-mediated reductive mobilization of As,and70.6%-104.4% increases in the arr gene abundances.These data suggest that environmental Mn(Ⅱ) markedly increased the DARPs-mediated reductive mobilization of As in arseniccontaminated soils.This work provided a new insight into the close association between the biogeochemical cycles of arsenic and manganese.
文摘We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxonomy-primarily the latest online Outline of the Bergey's Manual of Systematic Bacteri-ology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.
基金This work was partly supported by the Special Funds for Major State Basic Research Projects(Grant No.G2000077308)National Natural Science Foundation of China(Grant No.30170232)+1 种基金the Innovation Project of Chinese Academy of Sciencesby a grant from Shaghai Municipality via Fudan University.
文摘In order to show that the newly developed K-string composition distance method, based on counting oligopeptide frequencies, for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data, we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species. The latter group has been known to yield inconsistent trees if used individually. Our trees are obtained without making any sequence alignment. Altogether 16 Archaea, 105 Bacteria and 2 Eucarya are represented on the tree. Most of the lower branchings agree well with the latest, 2003, Outline of the second edition of the Bergeys Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.
基金supported by the National Natural Science Foundation of China(No.42077219)the Ningbo Municipal Natural Science Foundation(No.2019A610443)+1 种基金the Hangzhou Municipal Agriculture and Social Development Project(No.2020ZDSJ0697)the Fundamental Research Funds for the Provincial Universities of Zhejiang(No.SJLY2020011)
文摘Globally,various types of pollution affect coastal waters as a result of human activities.Bioaugmentation and biostimulation are effective methods for treating water pollution.However,few studies have explored the response of coastal prokaryotic and eukaryotic communities to bioaugmentation and biostimulation.Here,a 28-day outdoor mesocosm experiment with two treatments(bioaugmentation-A and combined treatment of bioaugmentation and biostimulation-AS)and a control(untreated-C)were carried out.The experiment was conducted in Meishan Bay to explore the composition,dynamics,and co-occurrence patterns of prokaryotic and eukaryotic communities in response to the A and AS using 16S rRNA and 18S rRNA gene amplicon sequencing.After treatment,Gammaproteobacteria and Epsilonproteobacteria were significantly increased in group AS compared to group C,while Flavobacteriia and Saprospirae were significantly reduced.Dinoflagellata was significantly reduced in AS compared to C,while Chrysophyta was significantly reduced in both AS and A.Compared to C,the principal response curve analyses of the prokaryotic and eukaryotic communities both showed an increasing trend followed by a decreasing trend for AS.Furthermore,the trends of prokaryotic and eukaryotic communities in group A were similar to those in group AS compared with group C,but AS changed them more than A did.According to the species weight table on principal response curves,a significant increase was observed in beneficial bacteria in prokaryotic communities,such as Rhodobacterales and Oceanospirillales,along with a decrease in autotrophs in eukaryotic communities,such as Chrysophyta and Diatom.Topological properties of network analysis reveal that A and AS complicate the interactions between the prokaryotic and eukaryotic communities.Overall,these findings expand our understanding of the response pattern of the bioaugmentation and biostimulation on coastal prokaryotic and eukaryotic communities.
基金Major Project of Science and Technology of Shandong Province(Grant No.2022CXGC010605)Fruit Industrial Technology System of Shandong Province(Grant No.SDAIT-06-03)+1 种基金Key Research and Development Program of Shandong Province(Grant No.2022LZGCQY019)Agriculture Improved Variety Project of Shandong Province(Grant No.2020 LZGC008).
文摘Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.
基金supported by a grant from the National Science foundation of USA (Mississippi EPSCoR-0903787)
文摘High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlapping architecture when compared to the existing genome annotation. Therefore, there is a need for customized tiling array data analysis tools. Since most of the initial tiling arrays were conducted in eukaryotes, data analysis methods are well suited for eukaryofic genomes. For using whole-genome tiling arrays to identify previously unknown transcriptional elements like small RNA and antisense RNA in prokaryotes, existing data analysis tools need to be tailored for prokaryotic genome architecture. Furthermore, automation of such custom data analysis workflow is necessary for biologists to apply this powerful platform for knowledge discovery. Here we describe TAAPP, a web-based package that consists of two modules for prokaryotic tiling array data analysis. The transcript generation module works on normalized data to generate transcriptionally active regions （TARs）. The feature extraction and annotation module then maps TARs to existing genome annotation. This module further categorizes the transcription profile into potential novel non-coding RNA, antisense RNA, gene expression and operon structures. The implemented workflow is microarray platform independent and is presented as a web-based service. The web interface is freely available for acedemic use at http：//lims.lshi.mafes.msstate.edu/TAAPP-HTML/.
文摘Prokaryotic organisms have evolved sophisticated immune systems for self-protection to resist bacteriophage invasion.With further research on prokaryotic immune systems,several important molecular biology tools have been developed,including the widely used restriction endonuclease in molecular cloning experiments and the CRISPR-Cas system in gene editing.
基金Supported by the PROOF Grant provided by the INSU-CNRS and was part of the PECHE project“Production and exportation of carbon:control by heterotrophic organisms at small time scales”。
文摘The short term(hourly scale)variability of heterotrophic prokaryote(HP)vertical distribution and respiratory activity,was investigated in the north-western(NW)Mediterranean Sea.HP vertical distribution was determined on board by flow cytometry analysis of seawater samples collected by series of CTD casts.Cell counts and viability were determined for all samples.HP respiratory rates were determined later in the laboratory from filtered seawater samples(23 dm^(3))from 300-1150-m depth.The average cell viability was 94.8%±2.2%(n=240).There was no accumulation of dead cells,due to quick decay of damaged cells.In the epipelagic layer,three HP groups were distinguished,two(HNA1,HNA2)who se cells exhibited a high nucleic acid content and one(LNA)with low nucleic acid content cells.HNA2 was most populated at 50 m but not detected at 90 m and below,presumably aerobic anoxygenic photoheterotrophic bacteria(AAPs).The variability in HP abundance was mainly confined in the upper 80 m.A few secondary peaks of HP abundance were observed(80-150 m)in connection with abundance troughs in the surface layer.HP cells were continuously present in a wide layer around 500 m(mean 191×10^(3)cells/cm^(3)).Below this layer,HP abundance randomly exhibited peaks,coupled to respiratory rate peaks.The HP abundance and variability in the water column was suppressed during a strong wind event.The observed sporadic variability was tentatively interpreted through a pulsed carbon-export mechanism induced by the microorganism production of dissolved poly saccharide s,followed by flocculation and rapid sinking.This mechanism would thus contribute to(ⅰ)preventing organic matter accumulation in the epipelagic layer,(ⅱ)seeding the water column with live HP cells,and(ⅲ)supplying the aphotic water column with fre sh and labile organic matter.This important vertical flux mechanism needs further observations and modelling.
基金The Impact and Response of Antarctic Seas to Climate Change under contract No. IRFSOCC2020-2022。
文摘Prokaryotic diversity and community composition in the water column of eight stations(63 samples) around the Antarctic Peninsula of the Southern Ocean were investigated. Through pyrosequencing of the V3–V4hypervariable regions of the 16S ribosomal RNA gene, we characterized 4 720 089 valid reads representing 48 188operational taxonomic units(OTUs, 97% similarity). The community was dominated by the phyla Pseudomonadota(original name: Proteobacteria, 47%), Oxyphotobacteria(26%), and Bacteroidota(original name: Bacteroidetes, 18%), which comprised an average of 91% of the total OTUs in all samples. The prokaryotic community composition varied vertically within the water column. Water column prokaryotic communities exhibited a clear depth profile, with higher microbial richness and higher diversity observed with increasing water depth. Cluster analysis of the community composition of water column samples exhibited a similar trend with depth. Correlation with environmental factors suggested distinct variation in prokaryotic community composition with changes in depth, salinity, temperature and dissolved oxygen levels. Functional prediction showed presence of active nitrogen, sulphur and methane metabolic cycles along the vertical transect of the studied region. These results will improve our knowledge of prokaryotic diversity and community composition at different depth of water column for better understanding of the microbial ecology and nutrient cycles in Antarctic Peninsula region of the Southern Ocean.
基金The National Natural Science Foundation of China under contract Nos 32000074 and 42176130the Science and Technology Planning Project of Guangxi under contract No. AA21196002+4 种基金the Natural Science Foundation of Shandong Province under contract No. ZR2021MD044the Tai Mountain Industry Leading Talent of Shandong under contract No. 2019TSCYCX-06the Key Research and Development Program of Shandong Province under contract No. 2021TZXD008the Biosafety Research Program under contract No.20SWAQX04the Shandong Program of Pilot National Laboratory for Marine Science and Technology (Qingdao)under contract No. 2022QNLM030003-1。
文摘Microorganisms are fundamental for the functioning of marine ecosystems and are involved in the decomposition of organic matter, transformation of nutrients and circulation of biologically-important chemicals. Based on the complexity of the natural geographic characteristics of the Changjiang River Estuary, the geographic distribution of sedimentary microorganisms and the causes of this distribution are largely unexplored. In this work, the surface sediment samples from the adjacent sea area of the Changjiang River Estuary were collected. Their prokaryotic diversity was examined by high-throughput sequencing technology, and the environmental factors of the bacterial community were investigated. The results indicated that the distribution of prokaryotic communities in the sediments of the study areas showed obvious spatial heterogeneity. The sampling sequences divided the sample regions into three distinct clusters. Each geographic region had a unique community structure, although Proteobacteria, Bacteroidota, Desulfobacterota, Acidobacteriota, and Actinobacteriota all existed in these three branches. Canonical correspondence analysis demonstrated that prokaryotic diversity and community distribution were significantly correlated with the geographic location of sediment, seawater depth, and in particular, nutrient content(e.g., total phosphorus, total organic carbon and dissolved oxygen). Moreover, it was found for the first time that the metal ions obviously affected the composition and distribution of the prokaryotic community in this area. In general, this work provides new insights into the structural characteristics and driving factors of prokaryotic communities under the background of the ever-changing Changjiang River Estuary.
基金supported by the Start-up Foundation for Introduced Talents in Qingdao University of Science and Technology
文摘The potential involvement of calcium in signalling in cyanobacteria has been investigated in recent years. Enough evidences showed that the cyanobacteria were capable of sensing and distinguishing different environmental stimuli, and making responses in ways of Ca^2＋ transients, which were the results of influx or efflux of Ca^2＋ aroused by different environmental stimuli. The calcium signal elicited by nitrogen starvation was crucial to heterocyst differentiation in filamentous cyanobacteria Anabaena species. Identification of a calcium-binding protein （CcbP） from Anabaena sp. PCC 7120 provided further evidence, and the degradation and down-regulation of CcbP accounted for the generation of calcium signal when nitrogen starvation exits. However, the encoding and decoding mechanisms of the calcium signals in cyanobacteria still remain unclear. In order to reveal the exact role of it, a detailed, systematic investigation will be needed, especially for the calcium dynamics at the single cell level.
基金Supported by Subproject of"Development and Utilization of Plant Resources under Special Environment"from the National Project"863"(2007AA021401)Corps Doctoral Foundation of"Study on Transgenic Breeding Technology"(2006JC07)~~
文摘[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.
基金This work was supported by the National Basic Research Program of China(No.2004CB117401)Chinese National Programsfor High Technology Research and Development(No.2004AA243060).
文摘Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.
基金Supported by National Natural Science Foundation of China(30771596)Ph.D.Programs Foundation of Ministry of Education of China(20060183010)~~
文摘[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.
基金This work was supported by Chinese National Programs for High Technology Research and Development (No. 2002AA207004).
文摘A full-length sequence coding for △^12 fatty acid desaturase gene from peanut（Arachis hypogaea L.）was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21（DE3）pLysS. The △^12 fatty acid desaturase was highly expressed in E. coli BL21（DE3）pLysS in the presence of isopropyl-D-thiogalactopyranoside （IPTG）. The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20℃ for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS （gas chromatogram and gas chromatogram-mass spectrometry） analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited △^12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.
文摘Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.
基金Supported by the National Natural Science Fundation Item of China(30970578,31070651)"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~
文摘[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.
