Abstract:Abstract:Levan is a fructan mainly linked by β-(2，6)-glycosidic bonds with some β-(2，1)-linked branch chains．Some microbial levan exhibit biological activities such as antitumor，antidiabetic and immunostimulating activities，hypolipidemic effect，and function as prebiotics，which has a wide and potential application in the pharmaceutical and food industry． Because of low extraction yields from microbial fermentation and a very complex process for chemical synthesis of levan，enzymatic synthesis of levan has attracted tremendous interest．Levansucrase (EC 2.4.1. 10)，a betapropeller protein belonging to the glycoside hydrolase family 68 (GH68) with reaction mechanism of non-Leloir glycosyltransferase，catalyzes the synthesis of levan by transferring the fructosyl group of non-activated sucrose into the fructan chain．The molecular structure and regulation of gene expression of some microbial levansucrases have been elucidated．Meanwhile，the enzymatic synthesis of levan by levansucrase is widely studied．In this review，catalytic mechanism of levansucrase，molecular structure and regulation of gene expression of some microbial levansucrases，and the application of levansucrases in enzymatic synthesis of levan were summarized．

Abstract:Abstract:Mycobacterium tuberculosis，an intracellular bacterium，mainly lives in the phagosome of the macrophage．It utilizes competitive uptake of nutrients as well as active efflux of noxious compounds to maintain its survival．Thus，the ATP-binding cassette transporters involved in either process mentioned above are essential for its pathogenicity．There are 38 ATP-binding cassette transporters identified in the genome of M．tuberculosis．This sort of proteins facilitate the transport of various substrates ranging from ions，sugars，amino acids，oligopeptides and drugs．In this review，we summarized the substrate specificity and transport mechanism of these transporters and their relationship with virulence in M．tuberculosis．

Abstract:Abstract:［Objective］We investigated the genetic diversity and phylogeny of 28 rhizobial isolates from root nodules of soybean growing in the Hilly Area of Central Sichuan in China.［Methods］We used 16S rDNA PCR-RFLP and phylogenetic analyses of the 16S rDNA，glnII and symbiotic genes (nodC).［Results］Five 16S rDNA genotypes among the isolates were distinguished with restriction endonucleases HaeⅢ，HinfⅠ，MspⅠand TaqⅠ．In the 16S rDNA PCRRFLP analysis，all the isolates are divided into Bradyrhizobium group and Sinonrhizobium group at the 83% level，and Sinonrhizobium strains accounted for 75% of the isolates．The phylogenetic analyses of 16S rDNA，glnII and nodC show that 4 representative strains SCAUs1，SCAUs2，SCAUs7 and SCAUs4 were closely related to S．fredii USDA205Twhile the other 2 representative strains SCAUs3 and SCAUs5 were closely related to B. yuanmingense CCBAU10071T and B． diazoefficiens USDA110T．The 16S rDNA，glnII and nodC sequence similarity of 4 Sinonrhizobium representative strains were 98.3%－99.9%，98.2%－100% and 100%，respectively.［Conclusion］Soybean rhizobia isolated from the Hilly Area of Central Sichuan in China has rich genetic diversity，S．fredii was the predominant genus.

Abstract:Abstract:［Obiective］To screen new agro-antibiotics，rare actinomycetes were isolated by improved separation methods from soil samples and the chemical structure of the antifungal active product was elucidated.［Methods］Dry heating method was used for soil samples pretreatment and the improved HV separation medium for rare actinomycetes separation;agar block rapid screening was used for the rapid evaluation of rare actinomycetes biological activity．For the identificationof a strain numbered TJ430，morphology observation，cell chemical composition analysis，physiological and biochemical analysis，enzymology characteristics analysis，16 S rDNA sequence analysis，and DNA hybridization method were used．Bioactive crude extract from fermentation was purified by column chromatography and preparative chromatography; infrared spectroscopy and high resolution mass spectrometry was used for structure elucidation of bioactive ingredient.［Results］A total of 570 rare actinomycetes strains were isolated．Antibacterial activity of rapid screen showed that the numbed TJ430 strain showed excellent anti oomycetes and broad-spectrum antifungal activity．Strain identification results show that the strain is a S．cavourensis．The molecular formulas of the effective ingredient is C40 H66 N3O11，molecular weight is 765．Amino，methyl，methylene，carbonyl，covalent bond，isopropyl and other chemical groups should contained in the molecular．［Conclusion］The characterized antibacterial active ingredient has good development prospect．

Abstract:Abstract:［Objective］ The FarR protein was involved in the regulation of arginine biosynthetic pathway in corynebacterium，but the regulation mechanism of FarR protein and its relationship with the negative regulator ArgR have never been reported．In this work，we constructed two deletion mutants: C． crenatum △farR and C．crenatum △argR △farR，and investigated the FarR function and its relationship with ArgR through the determination of transcriptional levels of arginine biosynthetic genes in four strains，including C．crenatum △argR constructed in previous work.［Methods］We used marker-less knockout technology to construct C．crenatum △farR and C．crenatum △argR△farR，and compared the transcriptional levels of the arginine biosynthetic genes in three mutant strains with those of the wild type strain using real-time fluorescence quantitative PCR.［Ｒesults］The results of ＲT-qPCR indicate that，in the absence of ArgR，FarR acted as a positive regulator．When farR gene was knockout alone，the transcriptional levels of arginine biosynthetic genes appeared up-regulated，down-regulated or no influence．［Conclusion］FarR and ArgＲ are involved together in the regulation of arginine biosynthetic pathway of C．crenatum．

Abstract:Abstract:［Objective］To study the effects of co-overexpression of purF，purM，purN，purH and purD genes on adenosine production in Bacillus subtilis． ［Methods］First，an extra purF gene under control of the P43 promoter was integrated into the B．subtilis chromosome at the native purF locus by single crossover，resulting in simultaneous expression of purF，purM，purN，purH and purD．Then the transcriptional levels of the five genes in the engineering strain were tested by Realtime Quantitative PCR．The activity of PRPP amidotransferase was also detected．Finally，cell growth，glucose consumption and adenosine production in engineering strain along with original strain were examined.［Results］The transcriptional analysis showed that purF and its downstream genes purM，purN，purH and purD were simultaneously upregulated at different level．The PRPP amidotransferase activity of engineering strain was about 2.4-fold that of original strain．Shake flask fermentation showed the improvement in adenosine yield and conversion ratio from glucose to adenosine (17.5% and 26.1%，respectively)．Fed-batch fermentation by the engineering strain was conducted．Compared with the original strain，adenosine accumulation of engineering strain increased within the same fermentation time．However，the cell growth of engineering strain was retarded． ［Conclusion］The co-overexpression of purF and its downstream genes purM，purN，purH and purD could enhance the adenosine yield in the culture broth． This paper could facilitate future research by providing theoretical evidence and method of metabolic engineering．

Abstract:Abstract:［Objective］We isolated the bacterial strain XM2 from prunes (Prunus domestica L.) fruit surface．XM2 was identified and tested as an antagonist for postharvest biological control of black spot disease (Alternaria alternata) on pear fruits.［Methods］Strain XM2 was identified according to its morphological，physiological and biochemical characteristics and 16S rDNA sequence analysis．Inhibition tests were performed in wounds of pear fruits using different types of XM2 inocula，different concentration and inoculation time of XM2 and A．alternata．Effect of XM2 on mycelia morphology of A．alternata was observed under microscope.［Results］Strain XM2 was identified as Pantoea agglomeran．Biological control of XM2 against black spot disease was significantly better than the control，and the best inhibitory was observed when inoculated with suspension (97.73% of control effect)． Higher XM2 concentration and lower A．alternata concentration showed better inhibitory effect．Similarly，the earlier inoculation of XM2 than A． Alternata，the better inhibitory effect on disease development．Microscopic observation found that XM2 broke part of the mycelia，making cell contents spilled and hyphae distorted.［Conclusion］Pantoea agglomeran XM2 has the potential as an effective antagonist against postharvest pear blank spot disease.

Abstract:Abstract:［Objective］There are one-way or two-way cross-reactions among Streptococcus suis serotype 1，2，1/2 and 14，the reason to which was unknown． ［Methods］ The capsular polysaccharides of serotype 14 and 1/2 were purified on Sephacryl S-300 column and identified by phenol-sulphuric acid method and dot-ELISA．The molecular weight of the serotype 14 and 1/2 capsular polysaccharides was revealed as 487.38 kDa and 512. 72 kDa by high performance gel permeation chromatography，respectively.［Results］The monosaccharide composition of serotype 14 and 1/2 capsular polysaccharides was determined as Glc/Gal/GlcNAc/Rha/Neu5Ac (1:2.94:1.35:0.24:0.37) and Glc/Gal/GlcNAc/GalNAc/Rha/Neu5Ac (1:1.67:1.05:0.93:0.72:0.7)by pre-column derivatization high performance liquid chromatography，fluorescent labeling HPLC and NMR，respectively．These were compared with the composition of serotype 1 and 2 capsular polysaccharides．Glc，GlcN，Gal and Neu5Ac was contained in the capsular polysaccharides of serotype 1，2 14 and 1/2．But there is no prominent correlation between the monosaccharide composition and crossreactions．The cross-reactions among them could be induced by the structure of the capsular polysaccharides and/or the other components on the cell wall．

Abstract:Abstract:［Objective］Increasing the activity of aspartokinase (AK) from Corynebacterium pekinense.［Methods］The gene of AK was constructed and mutated by site-specific mutagenesis．The mutational recombinant plasmid was heterologously expressed in Escherichia coli BL21．The mutational AK was purified by Ni2 + -NTA column after ultrasonicating of the recombinant bacteria，and then identified by SDS-PAGE and Western blot．We compared the kinetic difference between R169H AK and WT AK by determining the enzymatic activities．Some other characteristics of R169H AK and WTAK were also studied.［Results］The mutant R169H was successfully constructed．The molecular weight of AK was 48kDa．Vmax of R169H AK was 226.3 U/mg·s－1，which was 2.3 times higher than that of WT AK．The optimum reaction temperature of R169H AK was 26℃，the same as that of WT AK．The optimum reaction pH of R169H AK was 9.0，slightly higher than that of WT AK．The half-life period of R169H AK under optimum temperature and pH were 5.5h，much higher than that of WT AK．Lysine，threonine and methionine had an active effect on the activity of R169H AK when they were in low concentration.［Conclusion］ The hydrogen bond between R169 and E92 was broken down in R169H AK，which could affect the degree of polymerization and further lowered the affinity of mutant AK with substrates and then decreased the inhibition inducing by the metabolites. Thus，the Vmax of mutant AK from R169H had increased by 2.3 times compared with that of WT AK.

Abstract:Abstract:［Objective］Marek’s disease virus (MDV)，GD06 strain，was isolated from healthy Three-Yellow chickens without MD vaccine．In this study，we characterized GD06 strain/［Methods］Meq gene and repeat short region of GD06 strain were amplified and analyzed，and the serotype was determined by indirect immunofluorescence assay．The growth characteristics of GD06 stain were evaluated in Specific Pathogen Free (SPF) chickens and chicken embryo fibroblasts．The pathogenicity of GD06 strain was assessed by challenge test using SPF chickens.［Results］GD06 strain was MDV-1 serotype with a naturally inserted reticuloendotheliosis virus long terminal repeat sequence．Similar to the vaccinated strains CVI988/Rispens and 814，the Meq gene of strain GD06 contained a 59-aa insertion in the proline-rich domain compared with very virulent strain Md5．On 96，120，144，168 and 192 hours post infection，the virus titers of GD06 were higher than that of CVI988/Rispens in vitro (P＜0.05)，and the viremia levels in GD06-infected group were significantly higher than that of CVI988/Rispens group on 21 and 42 days post inoculation (P＜0.05)．The result of challenge test demonstrated that GD06 remained nonpathogenic for chickens and induced little immunosuppressive effects.［Conclusion］GD06 is an MDV-1 non-pathogenic strain naturally integrated with reticuloendotheliosis virus and long terminal repeat sequence．

Abstract:Abstract:［Objective］MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells．In this study，we examined miＲNAs’effects on the replication of Coxsackievirus A16 ( CA16) in rhabdomyosarcoma cells.［Methods］We constructed target gene of miRNAs screening system． We used 3' untranslated region (UTR) dual luciferase reporter analysis to identify putative miRNA targets in the CA16 virus genome．First，12 segments of CA16 virus genome were inserted to the pMIＲ vector and the luciferase expression were assayed to identify the target gene of putative miRNA．The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated．Then，using online analysis programs we screened the miRNAs that may target to 5'-UTR．Furthermore，Western blot and real-time PCR test were used to study the effect of miRNAs on viral replication.［Results］The study showed that miR432* could stimulate the replication of CA16 virus．On the contrary，miR432* inhibitor could suppress CA16 virus replication.［Conclusion ］Cellular miRNAs could regulate the replication of CA16 virus in host cells．Our findings support the notion that the cellular miRNAs play an important role in the host and virus infection．

Abstract:Abstract:【Objective】To improve the transduction efficiency of baculovirus and exogenous gene expression level，we chose a mammalian cell-specific promoter-human extension factor 1α promoter ( EF1-α)，used virus pseudotyped tools-truncated vessicular stomatitis virus protein G (VSV-GED )，added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs)．【Method】We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE．The recombinant transfer vectors pWK-eGFP，pWK-ITＲ-eGFP and pWK (－)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1α promoter． Constructed recombinant plasmid transfected Sf9 insect cells，and observed the expression of green fluorescent protein by using the inverted fluorescence microscope．【Results】The fluorescence expression rate of BV-WK-eGFP，BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control，ITRs can effectively extend the expression time of eGFP，the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BVWK(－)-eGFP．The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP，BV-WK-ITR-eGFP was obviously shorten in OL cells，and reduced 24 hours compared to the negative control BV-WK(－)-eGFP，transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK(－)-eGFP，respectively．【Conclusion】The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus，WPRE could enhance the expression efficiency of the exogenous gene significantly，and ITRs extend the expression time．The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine．

Abstract:Abstract:［Objective］To efficiently study the virulence genes distribution of Avian pathogenic Escherichia coli (APEC)，we developed four multiplex PCＲ to detect adhesin-associated genes，invasin and toxin-associated genes，serum resistanceassociated genes and iron acquisition-associated genes． ［Methods］According to gene sequences published in GenBank，we designed and synthesized 18 specific primer pairs，which were used in the four multiplex PCR．Then，we determined the sensitivity of multiplex PCR using diluted bacterial or DNA templates．To verify the feasibility of these multiplex PCR， we determined the distribution of virulence genes in 100 APEC isolates using these multiplex PCR．［Results］According to the results of PCR，we can conclude that each of the 18 genes was exactly and effectively amplified in the four multiplex PCR．The sensitivities of these four multiplex PCR were 103 Colony forming units (CFU)，103 CFU，105 CFU，105 CFU bacteria and 1ng，1ng，10ng and 10ng DNA，respectively．Furthermore，the results multiplex PCR for virulence genes distribution in 100 APEC were same as the single PCR．[Conclusion] These results suggest that multiplex PCR developed in this study could efficiently detect the virulence genes of APEC，which was a useful and rapid technique for epidemiological investigation．

Abstract:Abstract:［Objective］ To clone a halogenase gene from halometabolite-producing Streptomyces sp．604F to facilitate identification of potential halometabolites and its biosynthetic gene cluster.［Methods］We used agar block method to detect the antimicrobial activity of Streptomyces sp． 604F． We further amplified the conserved regions of type I polyketide synthase (PKS I)，type II polyketide synthase (PKS II) and nonribosomal peptide synthetase (NRPS) encoding genes by degenerative PCR． We detected halometabolites in fermentation extracts of Streptomyces sp．604F analyzed by liquid chromatography-time of flight mass spectrometry (LC-Tof MS)．Next，we amplified halogenase gene fragment from Streptomyces sp．604F by using degenerative primers targeting reduced flavin adenine dinucleotide (FADH2)-dependent halogenase genes．Finally，we cloned the full halogenase gene and its flanking sequences through high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR).［Results］Streptomyces sp．604F showed promising antifungal activity against Candida albicans ATCC 10231，and its genome contained genes encoding PKS I，PKS II，NRPS and a halogenase with 1443 bp．The halogenase is a new non-tryptophan halogenase，and most closely related to halogenases catalyzing the chlorination of glycopeptides.［Conclusion］Streptomyces sp．604F possessed a new non-tryptophan halogenase，which may be involved in halogenation of glycopeptide-like metabolites．The cloning and analysis of this halogenase have provided guidance for searching target halometabolites，and laid the foundation for obtaining the biosynthetic gene cluster.