Abstract:[Objective] Acidophilic filamentous acitnomycetes are active in the turnover of organic matter in acid litters and soils, and are a source of antifungal antibiotics and acid-stable enzymes. The aim of this study is to delineate the diversity of neutrotolerant acidophilic streptomycetes in acidic soil environment, and to investigate the resources of species. [Methods] 367 actinomycetes with cultural characteristics of streptomycetes were isolated from 14 acidic soil samples collected in Yunnan Province, China, using the method of DDC (dispersion and differential centrifugation) and a selective medium. The isolates were color grouped on the basis of their properties of aerial mycelium, substrate mycelium and diffusible pigments. 97 representative isolates were picked from the color groups for micromorphological observation and for the test of pH range for growth. Among the neutrotolerant acidophilic streptomycetes, 16 representatives were further selected and were subjected to a molecular systematic study based on almost complete 16S rRNA gene sequences and DNA-DNA relatedness. [Results] The isolates were assigned to 12 color groups, and 80% of them were neutrotolerant acidophilic streptomycetes. The 16 representative strains formed eight distinct subclades within the genus of Streptomyces, and probably represented at least eight new genotypic species of Streptomyces. [Conclusion] The neutrotolerant acidophilic streptomycetes isolated in this study were placed in eight distinct evolutionary groups, indicating the good diversity and novelty of these microorganisms in acidic soils in Yunnan Province.

Abstract:[Objective] We developed a molecular method to mark and differentiate Lactobacillus rhamnosus strains and to analyze genetic diversity among isolates from different sources. [Methods] Ten strains of Lactobacillus spp. were isolated from 56 feces samples of elderly people above 90 years of age in regions of Hetian, Xinjiang and Bama, Guangxi, China. We used API 50CHL test chip for strain identification. These isolates belonged to Lactobacillus rhamnosus with 98.6%~99.9% satisfactory. Ten L. rhamnosus isolates and one reference strain ATCC7496 were analyzed by random amplified polymorphic DNA (RAPD) technique to differentiate L. rhamnosus strains at molecular level. We screened 5 random primers named P14, OPG28, OPG25, P7 and P4 and developed optimum RAPD amplifying system. [Results] The clear and stable DNA fingerprints of each strain were obtained; the amplicon size ranged from 100 to 2000 bp, and the band patterns showed polymorphism among different L. rhamnosus strains. Genetic similarity coefficients based on RAPD patterns varied from 0.581 to 0.935, which suggested genetic diversity and different genetic relationship among these strains. Eleven strains of Lr could be clustered into 5 groups (group A to E) at the level of 80% similarity. Seven L. rhamnosus strains isolated from Hetian, Xinjiang, were grouped in group B and C, 3 isolates from Bama, Guangxi, were grouped in group D and E. [Conclusion] RAPD method is feasible for molecular mark and differentiation of L. rhamnosus strains and high level of genetic diversity and different relationship are found among L. rhamnosus isolates.

Abstract:[Objective]The biodiversity of endophytic Bacillus isolated from Medicinal plants was studied. [Methods] Endophytic bacillus isolated from medicinal plants and reference strains were characterized by numerical taxonomy, 16S rDNA PCR-RFLP, BOX-PCR fingerprints and 16S rDNA sequence to describe the phenotypic and genetic diversity and phylogeny. [Results]The phenotypic characterization of these strains showed that most of these strains had high stress resistance. There were 13 clusters formed at 84% similarity by numerical taxonomy. The genetic diversity was highly revealed by 16S rDNA PCR-RFLP. The diversity of genomic level was described by BOX-PCR fingerprints. The results were correlated well with numerical analysis. Homology analysis was made by searching in Genbank with NCBI, and the phylogenetic tree was reconstructed. Sequence analysis of 16S rRNA gene showed that SCAU11 was highly related to B. sphaericus, SCAU78 and SCAU25 were identified as B. subtilis subspecies, SCAU39 was highly related to B. megaterium. [Conclusion]Overall, the study results demonstrated a high phenotypic and phylogenetic diversity of endophytic Bacillus isolated from Medicinal plants.

Abstract:[Objective] Studied fungi diversity in the guts of larval Hepialus gonggaensis using culture-independent and traditional culturing methods. [Methods]For the culture-independent method, the total DNA of fungus was extracted from the in-testinal contents and internal transcribed spacer (ITS) regions were amplified with fungal universal primers. A near-full length ITS gene library was constructed. Subsequently, the fingerprints of the microorganisms were analyzed by isolated plasmid and digestion with MspⅠ, HaeⅢand TaqⅠenzymes, respectively. Restriction fragment length polymorphism (RFLP) analysis based on the fungal ITS sequences indicated that the library established includes 23 operational taxonomic units (OTUS) and a phylogenetic tree depicted the linkage of the isolated fungi. [Results]Abundant fungi were in the intestines of H. gonggaensis larvae, but their abundance was very different. The dominant fungi belonged to Mor-tierellaone and Trichosporon and accounted for 46.34 % and 39.02 % of the total ITS clones, respectively. Only three genera of fungi were identified from eight isolated fungal populations by traditional culturing methods. [Conclusion] We could get more information by combined traditional culturing and molecular biology methods.

Abstract:[Objective]Bacterial foot rot, caused by Erwinia chrysanthemi pv. zeae, is one of the most important diseases in rice. Genetic and molecular characters of toxin producting for Erwinia chrysanthemi pv.zeae were conducted in this paper. [Methods] A plasmid-deficient strain, Ech7-mu1, was obtained by chemical mutation,and the relative specific molecular mark with toxin was screened from Random Amplified Polymorphic DNA (RAPD) by PCR.[Results].The wild strain Ech7 and the plasmid-deficient strain Ech7-mu1 could both produce toxin.We screened 260 random primers in PCR, and found that a specific fragment (2139bp) could be amplified with K10 primer from theminus-toxin strain Ech7-4 DNA, but could not from the wild strain Ech7 DNA. The amplified fragment DNA was cloned and sequenced, and specific primers were designed to amplify it. The 2139bp fragment DNA could be a specific molecular mark with 100% SCAR identity between wild strain and thetoxin mutant strain. Sequence analysis showed that there were five open reading frame (ORF), two of them were NADH-flavin reductase and N-acetyltransferase,respectively. Another ORF, located in the end region of 2139bp fragment, had 66% and 46% homologies with permeases of the drug/metabolite transporter (DMT) from Pseudo-monas aerginosa (ZP00136947) and Yersinia Pestis(ZP01177873). [conclusion] Toxin biosynthesis in E. chrysanthemi pv. zeae might be coded by chromosome, but not by the bacterial plasmid.The position of gene mutation in the mutant Ech7-4 might be at the 3′ region of toxin-relation SCAR DNA fragment.

Abstract:[Objective] To detect the influence of the CagT protein on interleukin-8 secretion and proliferation in SGC-7901 cells. [Methods] Helicobacter pylori cagT gene was amplified by PCR with the genomic DNA of H. pylori NCTC 11637 as template, then it was inserted into an expression vector pQE30. The recombinant plasmid was transformed into E.coli M15. Recombinant protein was expressed by Isopropylthio-b-D-Galacgoside (IPTG) induction and confirmed by Western blot. Fusion protein with 6×His tag was purified using Ni2+-NTA agarose. Interleukin-8 mRNA expression of SGC7901 cells was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). [Results] The GenBank accession number of the amplified sequence is EF114758. The sequence analysis for cagT showed that it shares 97%-99% homology with other strains of H. pylori in Gene bank. The molecular mass of the product is 32kDa, and its purity is 98% analyzed by SDS-PAGE. After the protein was dialyzed, it can stimulate SGC7901 cells to express interleukin-8 and the growth of cells was inhibited in a dose- and time-dependent manner. [Conclusion] It is indicated that we have obtained the correct cagT gene and expressed in E.coli M15. The protein can stimulate the cells to express cytokine interleukin-8 and inhibit proliferation of cells, which posed a basis for further research on its biological function.

Abstract:[Objective] Random outbreak of lysogenic bacteriophage from Bacillus thuringiensis was very harmful to the production of Bacillus thuringiensis insecticide. We clarified the background of the phage from Bacillus thuringiensis MZ1 at the molecular level to solve the problem of random outbreak. [Methods] After the strain MZ1 from a company in Meixian County of Guangdong Province was induced, we obtained phage particles. Then phage DNA was extracted and pep gene was cloned, expressed and analyzed. [Results] We obtained a lysogenic bacteriophage MZTP01with clear plaque and 1 mm diameter. Fragment D with 2362bp (Genebank No. AY639599) was obtained after the phage DNA hydrolyzed by HindⅢ/EcoRⅠ. Among fragment D, the pep gene with molecular weight of 47 kDa and length of 1101bp was cloned and expressed. Recombinant M15 (pQE30pep) was built and overexpressed in Escherichia coli with a 47kDa clear band. At the same place a clear band was observed by Western blot. Judging from the time course expression, we could conclude that PEP protein produced at 1 hour after induction and then increased gradually. PEP protein was mainly in the form of inclusion body in the recombinant and slowed the growth speed of host. Homologous comparison of PEP protein from phage MZTP01 with other PEPs from BLAST were that phage MZTP01 PEP protein had 100% homologe with that of Escherichia coli K12, and most of others took the similarity in the range between 37%~84%. [Conclusion] PEP protein had ability to hydrolyze casein with the enzyme activity of 0.3mg/ml trypsin. PEP protein may be a kind of trypsin.

Abstract:[Objective] Our aim in this study was to determin the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. [Methods]Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. [Results] The hemagglutinin (HA), nu-cleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. [Conclusion] Phylogenetic analyses re-vealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.

Abstract:[Objective] We studied the secretomics’ properties of Trichoderma reesei, an important industrial microorganism used for cellulase production. [Methods] We analyzed the amino acid sequences coded by 9997 ORFs in Trichoderma reesei genome with bioinformatics approaches, identified 294 possible secreted protein sequences, and classified them by functions. We also applied motif search methods to search key motifs in the function-unknown sequences and preliminary predicted their functions. Moreover, we analyzed the signal peptide sequences of the secreted proteins. [Results] There were 188 hydrolytic enzymes in Trichoderma reesei’s secretomics, including 114 glycosidases, 42 proteases, and 11 li-poidases. The glycosidases included 22 reported cellulases, and 15 chitinases, as well as 30 other protein sequences probably related to cellulose degradation. The homology of signal peptides of secreted proteins was low whereas se-quences near the digesting site of signalase were conservative. [Conclusion] This method gave insights into the whole secreted proteome of Trichoderma reesei and provided basis for further studies on secretomic features at a genome level.

Abstract:[Objective] We identified a microbial transglutaminase (MTGase) gene from Streptomyces hygroscopicus; cloned and expressed it in Escherichia coli. We also analyzed the active sites sequence of S. hygroscopicus MTGase through homologous sequence comparison. [Methods] Wild-type microbial transglutaminase zymogen (pro-MTGase) was purified from liquid culture of S. hygroscopicus(CCTCC M203062)。N-terminal amino acid sequence of this pro-MTGase was determined. According to the N-terminal sequence and the corresponding nucleotide sequence of MTGase from other three Streptomyces species, PCR primers of S. hygroscopicus pro-MTGase were designed and the completed gene of pro-MTGase was amplified and sequenced. The gene was sub-cloned into pET-20b(+) vector downstream pelB signal peptide to construct the expression vector pET/pro-MTG. [Results] The nucleotide sequence showed 92 % homologue with that of S. platensis and S. caniferus. Rosetta(DE3)pLysS carrying the expression vector was induced with IPTG at 24℃ and expressed pro-MTGase as extracellular soluble protein. SDS-PAGE showed the expressed recombinant pro-MTGase was about 44 kDa, similar to the wild-type pro-MTGase purified from S. hgroscopicus. Recombinant pro-MTGase was activated with trypsin and the enzyme activity reached to 0.24U/mL. [Conclusion] This is the first re-port of the gene encoding microbial pro-transglutaminase from S. hygroscopicus, and also this is the first report of ex-pression extracellular soluble pro-MTGase in E. coli in our country.

Abstract:[Objective]To study the biological function of phosphatidylcholine in bacteria, the borrelial pcs gene was inserted into ptac85 plasmid. Then E. coli Top10 pcs+ was constructed via the transformation of the recombinant plasmid. Phosphatidylcholine (30%) in total phospholipids was achieved when the bacterial cells were incubated in Luria-Bertani (LB) medium supplemented with 1% choline and induced by 0.5 mmol/L isopropy-β-D-thiogalactoside (IPTG) for 4~8 hours at 37℃. [Methods] Ampicillin inhibitionof E. coli Top10 pcs+ was tested at first, and then b-lactamase activity in periplasm was examined. Finally Western blot was used to detect the amount of b-lactamase in both bacterial periplasm and cytoplasm. [Results] Antibiotic tests showed that high concentrations of ampicillin inhibited the growth of E. coli Top10 pcs+ with an IC50 of 70~800 mg/mL. Active assays revealed that the b-lactamase activity in periplasm was only 1/5 of that for the control strain E. coli Top10/ptac85. Western blotting confirmed that the low activity of b-lactamase in E. coli Top10 pcs+ resulted from a lower amount of b-lactamase in its periplasm. [Conclusion]Our results demonstrated that the phospatidylcholine incorporated into bacterial membrane retarded secretion of Escherichia coli penicillin b-lactamase from cytoplasm into periplasm, which suggested that phosphatidylcholine might play a role in the regulation of protein secretion in those bacteria able to synthesize phosphatidylcholine.

Abstract:[Objective] Dextransucrase was a glucosyltransferases catalyzing the transfer of D-glucopyranosyl units from sucrose to synthesize α-glucans or oligosaccharides. [Methods] dexYG gene (GenBank Accession No. DQ345760), encoding the dextransucrase from Leuconstoc mesenteriodes 0326, was subcloned into expression plasmid pET28a(+). The recombinant plasmid was then transformed into E. coli BL21 (DE3). Kanamycin resistant transformants were selected and verified by restriction endonuclease assay. [Results] Dextransucrase could be efficiently expressed in engineered strain BL21 (DE3)/pET28-dexYG by Isopropyl b-D-thiogalactopyranoside (IPTG) induction, although the growth of E. coli host was inhibited during induction. Recombinant enzyme producing conditions such as induction time, IPTG concentration, incubation temperature, cell density (OD600) and pH value were studied．The optimum conditions for producing dextran-sucrase were as follows: incubation at 25℃, 0.5mmol/L Isopropyl b-D-thiogalactopyranoside (IPTG) induction at cell density (OD600) of 1.0 for 5h, pH 6.0. Under these conditions, the recombinant dextransucrase activity was increased from 5.39U/mL to 35.62 U/mL. The highest activity under the optimal culture conditions after 5h induction in medium with pH 6.0 was 3.5 times as that of in Luria-Bertani medium without pH-adjustment. Moreover, the pH value was one of the main reasons that caused the degradation of enzyme in the later stage of induction. [Conclusion] These results showed that dextransucrase could be efficiently heterologous expressed in E. coli and a strong dextransucrase activity had been de-tected.

Abstract:[Objective] To isolate the bacteriophage of Serratia marcescens(8039) from sewage, and to study on its biological characteristics. [Methods] We used Serratia marcescens(8039) as the host to isolate phage from raw sewage from the Sewage Treatment Center of Hospital. Phage SM701 was examined in electron microscope. The genome of SM701 was extracted and the size of its nucleic acid was identified with restriction enzyme analysis. Phage isolated was subjected to plaque forming unit (PFU) assay using double layer agar medium plate method and morphological properties of plaque was observed, and finally optimal MOI multiplicity of infection and one-step growth experiments were carried out. [Results] Phage SM701 specific to Serratia marcescens (8039) was isolated successfully from the raw sewage. SM701 had an isometric polyhedral head (about 64nm in diameter) and a long noncontractile tail (about 143nm long).The nucleic acid could be cut off by dsDNA restriction enzyme BamHⅠor HindⅢand its complete size was about 57kb. The plaque of SM701 was transparent about 1mm in diameter at the 12th hour. When MOI equaled 10, the number of phage offspring was higher. One-stepgrowth kinetics was determined according to the results of one-stepgrowth experiment, which showed that the latent period was about 30 min, the rise period was about 100 min, and the average bust size was about 63 pfu/cell. [Conclusion] Phage SM701 belonged to tailed family: siphoviridae and lytic bacteriophage. It was quite easy to observe and count the plaques due to existing color differences between lytic and nonlytic zone.

Abstract:[Objective] We compared the microbial communities of two denitrifying bioreactors acclimated by quinoline and indole under identical condition. [Methods] We acclimated two reactors by using the same seeding sludge and acclimating conditions. When both reactors reached steady stage with a high pollutant removal capacity after 6 weeks adaptation, we constructed the 16S rDNA clone libraries to analyze the structure of microbial communities of those 2 bioreactors. [Results] Despite the same seeding sludge and identical acclimating condition, molecular ecological analysis showed distinctly different communities of two reactors. All operational taxonomic units (OTUs) of 16S rDNA clone library of quinoline acclimated bioreactor were affiliated to Betapro-teobacteria, whereas in library of indole acclimated bioreactor, the percentage of Betaproteobacteria was only 56.3%. Results showed that microbial diversity in indole acclimated community was higher. Clones (73%) in quinoline acclimated community were Thauera related OTUs from the Rhodocyclaceae family. But OTUs from the families of Comamonadaceae, Alcaligenaceae and Rhodocyclaceae were dominant OTUs in indole acclimated community. The most dominant OTU from Comamonadaceae was 28.7％ in clone library. [Conclusion] The type of pollutants in the wastewater had a strong effect on the selection of population in microbial community. Our study was the first report comparing the microbial structure of two effective denitrifying communities which could efficiently degrade quinoline and indole.

Abstract:A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes（SS1, SS7, SS9） and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high ho-mology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was ob-served with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The in-duced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the patho-genicity of SS2.

Abstract:[Objective] To study the epidemiological status of reticuloendotheliosis virus (REV) infection in ducks. [Methods] Two hundred and twenty tissue samples of the Bursa, spleens and livers were randomly collected from 97 sick or dead ducks from 3 areas in Shandong Province, China. REV infections were tested by virus isolation in cell cultures, direct dot blot hybridization, regular PCR and nest-PCR with tissue-extracted DNA as the templates. PCR products of env gene fragments were cloned and sequenced, sequences of env gene fragments were compared and analyzed. [Results] REV infections were detected in 35/39 (90%) bursal samples, 54/84 (64%) spleens and 32/97 (33%) livers by Nest-PCR. The positive rate of bursal samples was significantly higher than spleen and liver samples (P<0.01). No positive sample was detected by virus isolation, direct dot blot hybridization or regular PCR from the same Bursa, spleen and liver samples. In sequence comparisons of amplified env gene fragments, Yinan-1 and Binzhou-1 had 99.8% of identity with reference SNV strain isolated from ducks in USA, and Linqu-1 had 100% identity with two other reference strains isolated from chickens in USA. However, they had lower homology with strains isolated from chickens in China. [Conclusion] More attention should be given to bursal samples in detection of REV. Since REV was existing only at a very low level in infected sam-ples and difficult to be detected, nest-PCR should be conducted as the more sensitive assay. Sequence homology com-parisons and phylogenetic tree analysis suggested that REV infections in these ducks may originally come from imported breeder ducklings without inspection for REV.

Abstract:[Objective] To clone and express the immunodominant domain gene of the chlamydial protease-like activity factor(CPAF) from Chlamydophila pneumoniae.The value of the recombinant protein was evaluated for diagnosing early infection. [Methods] According to bioinformatics analyses and references, we chose the immunodominant region epitope of chlamydial protease-like activity factor(CPAF) from Chlamydophila pneumoniae, then amplified the genes of the epitope by PCR, and then ligated the genes into a pGEX6p-2 vector. We purified the expressed recombinant protein with glutathione S-transferase (GST) agarose gel FF, then identified it by SDS-PAGE. By immuning New Zealand rabbits evaluated the immunogenicity of the recombinant protein, and analyzed its antigenicity with Western blot. The specific IgM antibodies in 300 clinical sera samples and C. pneumoniae reference sera, the antigen of C. pneumoniae in 120 spu-tum and throat swabs were detected with the developed indirect ELISA. At last, we investigated the cross-reactivity against Chlamydia trachomatis with the developed ELISA method to detect anti-C. trachomatis positive antisera and se-cretions in genitourinary tract. [Results] We attained successfully a 51.3kDa recombinant protein. Western blot assay proved the recombinant protein could only specifically react with human anti-C. pneumoniae antisera. The titer of the specific IgM antibodies in the immuned New Zealand rabbits was above 1:8000. The developed ELISA detected anti-C. pneumoniae IgM positive and negative reference sera, the sensitivity and specificity were both 100% (40/40).The con-cordance rate between the indirect ELISA and the MIF to 300 clinical sera samples was 98.3%. The concordance rate of antigen detection between the new ELISA and the PCR reagent to 120 sputum and throat swabs was 88.3%. When de-tecting anti-C.trachomatis positive antisera and secretions in genitourinary tract with the developed ELISA, we didn’t found any cross reaction against C.trachomatis. [Conclusion] The prepared recombinant protein of the CPAF immu-nodominant region epitope gene from C. pneumoniae shows excellent antigenicity and can highly benefit on developing new indirect ELISA as methods to detect IgM antibodies and antigen of C. pneumoniae for diagnosing early infection.

Abstract:[Objective] We evaluated relative quantification by real-time RT PCR of a target gene transcription. [Methods] On the basis of (1+E)-△△Ct mathematical model and the E=10[-1/slope]-1 equation, the detected Ct data of the real-time RT PCR was analyzed by the new DNA subtraction assay. DNA was used as standard for the initial amount of bacteria. RT and RT— samples for real-time PCR detection were prepared to quantify the DNA that simultaneously existed with RNA isolated from the bacteria samples. The detected quantitative data were subtracted from total nucleic acid simultaneously contained RNA and DNA. Enzymatic digestion with DNaseⅠwas not included in this protocol. [Results] The gene expression of staphylococcal enterotoxin A (sea), 16S rRNA and RNAⅢof Staphylococcus aureus were detected. These two different analysis methods, DNA subtraction method and absolute quantitative method, led to similar results (p>0.05). [Conclusion] This is a time-saving and efficient method. Additionally, for further studies it would be conceivable to extend the detection of genes expression from S. aureus to other prokaryote.

Abstract:[Obiective]To develop a new method to cline large DNA fragment by combining whole genome sequence information and the principle of plasmid rescue. [Methods]First, we amplified a fragment downstream the region to be cloned and cloned the fragment into a suicide plasmid to construct targeting plasmid, which was then targeted into chromosome by homologous recombination. Then, genomic DNA was isolated, digested with appropriate enzyme, re-ligated and transformed into host bacteria to rescue the plasmid and the large DNA fragment. The rescued DNA fragment was sub-cloned into new plasmids for special purposes. [Results]Using this method, we successfully cloned the 11kb virB operon of Brucella and constructed complementary plasmid. The transcription of the disrupted virB operon was restored with the complementary plasmid, validating the feasibility of the strategy. [Conclusion] this recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria.

Abstract:[Objective] To investigate whether the recombinant baculovirus (Bac-CMV-EGFP) can effectively transduce into rhesus Bone-marrow derived Mesenchymal Stem Cells (rBMSCs) in vitro, and whether there are some efficiency to the rBMSCs of viability, proliferational and differentiational capacity after recombinant baculovirus transducing. [Methods] The rBMSCs were cultured in vitro. After passaged more than three times, the rBMSCs were transduced with various dose of baculovirus (Multiplicity Of Infection, MOI, the MOI is 50, 100, 200, 300, and 500 vector genome (vg)/cell, re-spectively). We used flow cytometry to detect different transductive efficiency of various dose of baculovirus to rBMSCs. Under a suitable dose of baculovirus (300v.g/cell), we studied cell viability, proliferation and differentiation capacity, and compared results with the control. [Results] Baculovirus could be transduced into rBMSCs in vitro. The transductive ef-ficiency reached about 80% when the MOI was 300v.g/cell, at 25℃, and incubated for 4 h. Furthermore, under a higher transductive efficiency of baculovirus, there were no obvious influence to the rBMSCs of viability, proliferation and dif-ferentiation capacity compared with that of the control. [Conclusion] The baculovirus can be safely and effectively transduced into rBMSCs in vitro, without any negative efficiency to cell viability, proliferation and differentiation capacity.

Abstract:[Objective] We chose a sponge-associated marine-bacterium Pseudoalteromonas piscicida NJ6-3-1 to study whether its antibacterial activity were regulated by quorum sensing. [Methods] We studied the relationship between the antibacterial activity and bacterial density under various growth conditions. To simulate the natural competitive environment, we monitored the antibacterial activity at low cell density when the species were co-cultured with Staphylococcus aureus. [Results] Antibacterial activity correlated closely with cell density. Marine bacterium NJ6-3-1 started producing antibacterial compounds when cell density reached the threshold value of OD630=0.4. Some signal molecules existing in the metabolites of S. aureus could induce the production of antibacterial substance by marine-bacteria NJ6-3-1 even cell density below the required threshold. [Conclusion] The results provide preliminary evidence to support the hypothesis that the antibacterial activity of NJ6-3-1 was regulated by the quorum sensing system of intraspecies and interspecies.

Abstract:Apoptosis is a conserved mechanism that plays an essential role in eukaryotes. Malfunction of apoptosis contributes to many human diseases including cancer and AIDS. Compared with yeast, a monocellular eukaryote, which is the model organism for apoptosis research, investigation on filamentous fungi apoptosis has particular advantages, which remained unrecognized until recent years. Filamentous fungi, especially Aspergillus nidulans and Aspergillus Fumigatus, are expected to become new model species. Research on filamentous fungi apoptosis also has important value in agriculture and medical care, providing new ideas for the biocontrol and therapies of human mycosis. This review describes progress regarding the phenotypes, exogenous /endogenous inducers and signal pathways of apoptosis in filamentous fungi.

Abstract:Three main mechanisms for stress tolerance response of O. oeni were addressed in the review. Activation of MLF and membrane-bound H+-F0F1-ATPases activity were utilized to maintain the intracellular environment and to control the energetic status of Oenococcus oeni; Cell membrane compositions and fluidity were adjusted to compensate for the various stresses environmental effects; In addition to a small heat shock protein Lo18, many other stress proteins and corresponding genes were also found to be involved in the Oenococcal adaptive stress responses. CtsR-dependent gene regulation systems were demonstrated in Oenococcus oeni. A better understanding of Oenococcus oeni stress adaptive response mechanisms should play an important role in screening of highly tolerant strains, mastering the characterization of malolactic starters and rationalizing industrial processes of preparing wine malolactic starter. Furthermore, Oenococcus oeni stress genes also could be used for constructing engineer strains with new properties.