Effects of Biostimulation-Bioaugmentation on Coastal Microbial Community in an in situ Mesocosm System

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.

Structural Basis of Gabija Bacterial Defense System

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.

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine. METHODS： The TGF-β1 encoding epitope gene （the mature TGF-β1 from 78-109 amino acid residues, TGF-β1^32） was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β1 ^32 vector. The HBcAg gene fragments （encoding HBcAg from 1-71 and 89-144 amino acid residues） were amplified from PYTAI- HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1^32 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 Ugase. The fusion gene fragments HBc.Ag1-71-TGF-β1^32 HBcAg89-144 were recloned to pET28a（＋） and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a （＋）/ CTC was transformed and expressed in E. coli BL21 （DE3） under induction of IPTG. After purification with Ni^＋2 NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS： Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass （Mr） of the expressed product by pET28a （＋）/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine. CONCLUSION： A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.

The effects of soil properties, cropping systems and geographic location on soil prokaryotic communities in four maize production regions across China

The diversity of prokaryotic communities in soil is shaped by both biotic and abiotic factors.However,little is known about the major factors shaping soil prokaryotic communities at a large scale in agroecosystems.To this end,we undertook a study to investigate the impact of maize production cropping systems,soil properties and geographic location(latitude and longitude)on soil prokaryotic communities using metagenomic techniques,across four distinct maize production regions in China.Across all study sites,the dominant prokaryotes in soil were Alphaproteobacteria,Gammaproteobacteria,Betaproteobacteria,Gemmatimonadetes,Acidobacteria,and Actinobacteria.Non-metric multidimensional scaling revealed that prokaryotic communities clustered into the respective maize cropping systems in which they resided.Redundancy analysis(RDA)showed that soil properties especially pH,geographic location and cropping system jointly determined the diversity of the prokaryotic communities.The functional genes of soil prokaryotes from these samples were chiefly influenced by latitude,soil pH and cropping system,as revealed by RDA analysis.The abundance of genes in some metabolic pathways,such as genes involved in microbe–microbe interactions,degradation of aromatic compounds,carbon fixation pathways in prokaryotes and microbial metabolism were markedly different across the four maize production regions.Our study indicated that the combination of soil pH,cropping system and geographic location significantly influenced the prokaryotic community and the functional genes of these microbes.This work contributes to a deeper understanding of the composition and function of the soil prokaryotic community across large-scale production systems such as maize.

Establishment of method for seroepidermiological detection of human herpes virus 8 infection by using the fusion protein in the prokaryotic expression system as antigen for testing

To establish a sensitive and specific method for seroepidermiological detection of human her- pes virus 8(HHV-8)infection,three potent antigenic proteins encoded by open reading frames(ORFs) K8.1,65 and 73C in genome of HHV-8 were produced as glutathione S-transferase fusion protein in the prokaryotic expression system and was used as antigen for testing.The recombinant fusion protein ex- pressed in the prokaryotic expression vector E.coli BL21 was purified by glutathione Sepharose 4B affin- ity chromatography and was quantitated with SDS-PAGE.All these 3 fusion proteins produced in the pro- karyotic expression system showed good immunogenicity as demonstrated by Western blotting and could be recognized by mixed sera of patients with Kaposi′s sarcoma(KS).The immuno-reactivities of the single or compound fusion protein were determined by means of ELISA and compared with the traditional immu- nofluorescence assay(IFA)to determine their sensitivity and specificity of the test.It was demonstrated that the sensitivity of mixed-antigen ELISA method was significantly higher than that of IFA(81.8% vs 34.4%),while the specificity of the former was demonstrated to be 97.9%.The coincidence of the de- tection rate between these two methods was considerably high,approaching up to 90.0%.These results suggest that the mixed antigen ELISA assay appears to be a sensitive and specific method for sero-epide- miological detection of human herpesvirus 8 infection.

Prokaryotic diversity and community composition in the surface sediments of the Changjiang River Estuary in summer

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.

Vertical microbial profiling of water column reveals prokaryotic communities and distribution features of Antarctic Peninsula

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.

Prokaryotic Expression of Pseudomonas Aeruginosa Lipase Gene

[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.

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

[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.

Construction of a High-efficient Expression Vector of Δ^(12) Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

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.

Study on the Knockout and the Soluble Prokaryotic Expression of VP5 Protein Transmembrane Region of IBDV

[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.

Cloning and Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene

Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.

Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr.

[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.

Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

Cloning, Analysis and Prokaryotic Expression of DsSP Gene from Dunaliella salina

[Objective] The purpose of this study was to clone a starch phosphorylase gene from Dunaliella salina and to preliminarily analyze its basic properties and protein expression. [Method] RT-PCR and RACE （rapid amplification of cDNA ends） method was used for gene cloning; basic properties of the gene were analyzed using bioinformatics method; prokaryotic expression vector PGS21a-DsSP was constructed and transformed into E. coil BL21; the fusion protein was purified and detected by GST-SefinoseTM Kit and Western Blot, respectively. [Result] A starch phos-phorylase gene （GenBank accession No. KF061044） named DsSP was successfully isolated from D. salina. Basic properties, subcellular localization, secondary structure and tertiary structure of the protein were analyzed and predicted. The fusion protein was found in the supernatant and inclusion bodies. The supernatant protein was successfully purified. Western Blot analysis showed that the fusion protein was successfully expressed in E. coil BL21. [Conclusion] This study laid experimental foun- dation for further clarifying the function and mechanism of DsSP.

Sequence Analysis of Transcription Factor AtWRKY35 and Construction of Prokaryotic Expression Vector

As members of a super gene family, WRKY transcription factors are widely distributed in higher plants. ln this study, bioinformatic analysis of WRKY35, a member of the WRKY gene family, was carried out. Results indicated that tran-scription factor WRKY35 harbors a WRKYGQK core domain and a Cys2His2 or Cys2His/Cys zinc finger in the 5’ end without transmembrane domain. After PCR amplification and restriction digestion, WRKY35 gene fragment was ligated to prokaryotic expression vector PET28. This study provided basis for expression anal-ysis of WRKY35 protein and subsequent functional identification of WRKY35 gene.

Study on the Factors Influencing Prokaryotic Expression of ScMYB Protein

[Objective] This study aimed to find out the factors influencing the prokary- otic expression of ScMYB protein and optimize the expression conditions. [Method] First, the prokaryotic expression plasmid ScMYB：：pEASY-E1 was constructed; and then the effects of various induction conditions on its expression were studied, in- cluding concentration of inductor IPTG, induction time, induction temperature and OD600 of the bacteria solution. [Result] All the induction time, induction temperature, and OD600 except the concentration of IPTG have significant influence on the expres- sion of target protein. [Conclusion] The expression of target protein reaches the high- est level under the conditions of induction temperature 37 ℃, induction time 4 h and OD600 0.6-1.0.

Prokaryotic Expression and Polyclonal Antibody Preparation of SDG711 C-terminal from Rice

[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and to prepare its polyclonal antibody. [Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained and confirmed by Western-blot analysis. [Result] The polyclonal anti- body was successfully prepared and could efficiently detect the expressed antigen. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein in rice.

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.