Abstract:Abstract:The O-antigen of Gram-negative bacterium plays an important role in the signal identification，adhesion，immune evasion and other processes．There are three O-antigen polysaccharides biosynthesis mechanisms according to the type of the flippase that is involved． The Wzy-dependent mechanism is more commonly seen．In Wzy-dependent mechanism，the wzz gene is involved in regulating the length of O-antigen polysaccharide chain which can affect antigenicity of the pathogen and immune response of the host． Based on the crystal structure of Wzz ( regulator of the Oantigen polysaccharides length)，different length of O-antigen chain can be obtained through molecular modification of the gene wzz．Conjugating O-antigen or its mutants of a pathogen to a carrier protein could help to develop a vaccine that have both a good target specificity and a strong immunogenicity．Therefore，it is important to understand the function，structure and mechanism of Wzz for the development and production of glycoconjugates vaccine．

Abstract:Abstract:Biofilm is a prevalent lifestyle in nature and closely related to the life and production of humans．Bacillus subtilis is an important industrial strain and a good model for biofilm research．Combined with our current studies，we reviewed significant progress on biofilm formation by Bacillus subtilis，including main process，characteristics of biofilm formation，research models，and regulatory pathway for biofilm formation．In addition，further research focuses are addressed．

Abstract:Abstract:［Objective］In order to identify the function of lah-3 in osmotic regulation，we generated lah-3 deletion strain and analyzed its phenotype by osmostress treatment．［Methods］We used homologous recombination to replace lah-3gene by hph gene and treated these cells with 4% NaCl and 1 M sorbitol to analyze the phenotype．Northern blot was used to detect the expressions of osmoresponsing genes．Western blot was used to examine the phosphorylation level of LAH-3 and OS-2 and the expression of OS-2．［Results］In the deletion strain of transcription factor lah-3 gene，the expressions of osmoresponsing genes gcy-1，stl-1 and pck-1 were significantly reduced． Besides，the phosphorylation level of LAH-3 protein increased under the osmostress treatment． The phosphorylation of LAH-3 was not mediated by OS-2．The deletion of lah-3 did not affect the os-2 expression and the phosphorylation level of OS-2 upon osmostress．［Conclusion］LAH-3 involved in osmoresponsing was independent of OS-2 MAPK pathway．

Abstract:Abstract:［Objective］Cryogenic autolysis of Volvariella volvacea is an unusual phenomenon of abnormal metabolism．The aim of this study was to describe this molecular feature of abnormal metabolism at the genome-level．［Methods］We used 21 fungal species for the phylogenomic analysis and then selected 9 representative species in basidiomycetes for the comparative genomic analysis．［Results］The phylogenomic analysis shows that V． volvacea was located at the bottom of the cluster consisting of grass-degrading fungi．Phylogenetic tree shows that basidiomycetes and ascomycetes fungi have independent evolutionary trajectories．Therefore，nine representative species in basidiomycetes were chosen for the comparative genomic analysis．The result shows that compared to other grass-degrading fungi，V．volvacea has the tendency of contraction．The comparison of the number of gene families on a different scale shows that there was a significant expansion of 3 large size (＞ 200) gene families (fam1，fam4 and fam6) in V．volvacea with their total number significantly more than other species，representing that the molecular feature of V．volvacea is correlated with its abnormal metabolism．［Conclusion］ The significant expansion of 3 gene families (＞ 200) in V． volvacea indicates the enhancement of their function in specific gene families，which is most likely associated with cryogenic autolysis of V． volvacea．

Abstract:Abstract:［Objective］ To establish a convenient halophilic protein expression and purification system based on the haloarchaeal-type PhaP and polyhydroxyalkanoate (PHA) granule．［Methods］We cloned a strong haloarchaeal promoter and the phaP-tag into the haloarchaea- Escherichia coli shuttle vector pWL502，and then used the constructed vector to express the PhaP-tagged haloarchaeal proteins in the phaP-deleted strain Haloferax mediterranei ΔphaP．We purified the PhaP-fusion proteins，which were associated with PHA granules，by sucrose density gradient centrifugation．We also inserted a haloarchaeal intein-containing fragment between phaP and multiple cloning sites，and modulated the intein splicing activity by site-directed mutagenesis.［Results］We successfully constructed two expression vectors，pPM and pIP，in which PhaP was used as N-terminal and C-terminal fusion tag，respectively．The haloarchaeal proteins were effectively expressed by both vectors． The PhaP-tagged proteins were easily purified through the strategy of PHA granulemediated protein purification．In addition，we found that the intein-containing fragment Hbt21 from Halobacterium sp．NRC-1 had maintained splicing activity in H． mediterranei，and its C-terminal cleavage could be blocked or attenuated by mutating the conserved asparagine (N182) or serine (S183)，respectively．［Conclusion］ We have established a convenient and economical halophilic protein expression and purification system． We have also identified the splicing active sites of a haloarchaeal intein，which showed potential for removing the PhaP-tag from the purified proteins．

Abstract:Abstract:［Objective］ The paucity of genomic information limits the metabolic engineering of non-model microalgae Chlorella sorokiniana．Our study aimed to elucidate the fatty acid，triacylglycerol and starch biosynthetic pathways in the microalgae C．sorokiniana based on de novo transcriptomic analysis．［Methods］ We cultured C．sorokiniana with different nitrogen concentrations (KNO3:8g/L and 2g/L)，then sequenced the transcriptomeusing Illumina Hiseq2000 platform．We used Trinity to de novo assemble the reads so as to obtain transcripts，aligned all the transcripts with Nr database，UniProtKB/Swiss-Prot database and COG database to annotate the function and classify using BLASTx algorithm，and assigned the transcript with metabolic pathway by aligning with KEGG database． Then we used ＲSEM to calculate FPKM value，and used it for preliminary analysis of different gene expression in the related pathways．［Results］Over 49M high quality raw reads were produced with the length of 100bp，We used Trinity to assembled these reads into 49885 transcripts with an N50 of 1941bp，ranging from 300bp to 14100bp． 26479 transcripts were annotated through BLASTx similarity search，2357 transcripts were assigned with EC number，and 207 metabolic pathways were assigned in total．Based on these analyses，we reconstructed the fatty acids，triacylglycerol and starch biosynthetic pathways in C.sorokiniana．We also identified preliminarily different geneexpression in the pathways．［Conclusion］ Using RNA-seq technology，we reconstructed the metabolic pathways involving in the fatty acid，triacylglycerol and starch biosynthesis in non-model microalgae C.sorokiniana without genomic data，which is consistent with those in model microalgae Chlamydomonas reinhardtii，and compared the gene expression level under different conditions．These information is very useful for the metabolic engineering of C．sorokiniana and other microalgae to enhance the production of lipids．

Abstract:Abstract:［Objective］The ε-poly-L-lysine-degrading enzyme (Pld) derived from Streptomyces sp．M-Z18 was purified and characterized． Furthermore，Pld was used to produce the low polymerization of ε-poly-L-lysine (ε-PL)．［Methods］ Pld was purified to electrophoretical homogeneity through HiTrapTM Butyl HP hydrophobic chromatography after pretreated by ultrasonic and NaSCN dissolving． Subsequently，enzymatic characteristics，kinetic parameters and the time profile of ε-PL degradation by the purified Pld were studied． Meanwhile，we examined the effect of ε-PL with different degrees of polymerization on the minimal inhibitory concentration of bacteria and fungi．［Results］Pld was purified to homogeneity with a final fold of 80.4 and an overall yield of 59.3%. The optimal temperature and pH for the purified Pld were 37°C and 7.0，respectively．Moreover，the Km with L-lysyl-p-nitroanilide as substrate was calculated to be 0. 621 mmol/L，and the Vmax was 701.16 nmol /min·mg．Pld was stable in the range of pH 7.0－10.0，and temperature up to 50°C，respectively． Time profile of ε-PL degradation by the purified Pld indicated that Pld catalyzed endo-type degradation of ε-PL. The experiments of minimal inhibitory showed that ε-PL with high degree of polymerization (30－35) had a superior antibacterial effect on bacteria and the low degree of polymerization ε-PL (8－20) had a better antibacterial effect on yeasts．However，ε-PL with various degrees of polymerization had a poor antibacterial effect on mould． ［Conclusion］The present result showed that an endo-type Pld from ε-PL-producing strain was purified． Meanwhile，it is proved that ε-PL with different degrees of polymerization have exhibited significant different antibacterial effects on microorganism.

Abstract:Abstract:［Objective］The object of this study is to reveal the composition of active microorganism and their metabolic activities in flooded paddy soils with long-term fertilization (Mineral nitrogen，phosphorus，and potassium，NPK) and without fertilizer (Control check，CK) by environmental transcriptomics．［Methods］ Flooded soil microcosms were incubated in the laboratory for two weeks，then total RNA were extracted from the soil for transcriptome sequencing．Resulting fastq files were uploaded to the Metagenomics Analysis Server (MG-ＲAST) for taxonomic analysis，gene annotation and function classification．［Results］Transcripts from diverse active microorganism，including bacteria (＞95%)，archaea，eukaryotes and viruses，were detected in both flooded paddy soils of CK and NPK treatments．Most of the transcripts (active genes) of bacteria and archaea were derived from Proteobacteria (more than 50% of total bacterial transcripts) and Thaumarchaaeota (about 70% of total archaeal transcripts) respectively in both treatments．Transcriptional activity of Acidobacteria in NPK treatment paddy soil was significantly higher than that in CK treatment paddy soil．As for other phyla of bacteria and archaea，there were no significant differences of transcriptional activity of them between CK and NPK treatment paddy soils．The highest expressed gene in both CK and NPK treatment paddy soils is ABC transporter encoding gene which related to the transmembrane transport of substances．Based on gene function category of COG (Clusters of Orthologous Genes)，Subsystem and KEGG (Kyoto Encyclopedia of Genes and Genomes) database，we found that the main metabolic activities of microorganisms in both CK and NPK treatment paddy soils were related to energy production and conversion，carbohydrate metabolism，protein metabolism and amino acid metabolism，and the dominant KEGG pathways were oxidative phosphorylation and aminoacyl-tＲNA biosynthesis． ［Conclusion］Composition of active microorganism in CK and NPK treatment paddy soils was generally similar，except Acidobacteria whose transcriptional activity was significantly different between these two treatment paddy soils．It was also very similar between CK and NPK treatment paddy soils considering the metabolic activities of microorganisms in them，for dominant metabolic processes in these two soils were both related to energy obtaining and protein metabolism．So，dominant metabolic activities of microorganism in flooded paddy soils used in this study were not altered significantly under long－term inorganic fertilization.

Abstract:Abstract:［Objective］The objectives of this study were to use Roche 454 GS FLX system to develop SSR markers for Armillaria luteo-virens． These datasets will be valuable for detecting genetic diversity and population structure of this species．［Methods］We collected Armillaria luteo-virens samples from Yushu in Qinghai province，China．Total RNA was isolated by using the TRIzol reagent，after that we constructed cDNA library and performed one quarter plate of the whole run 454 pyrosequencing．We selected 98 primer pairs randomly from the 321 SSRs to evaluate their application and the polymorphism across 66 individuals (Armillaria luteo-virens) representing 3 wild populations．［Results］Roche 454 sequencing yielded 197，121 reads with a total nucleotide size of 88，585，965bp．27 of 98 SSRs loci were polymorphic．Numbers of alleles (Na) ranged from 2 to 8．Expected heterozygosity (HE) ranged from less than 0. 001 to 0.810 at locus ALV65，while observed heterozygosity (HO) from 0 at loci AIV64 and AIV92 to 0.900 at loci ALV8．We found no evidence of linkage disequiliburium，however 10 of 27 SSR markers showed significant deviation from Hardy-weinberg equilibrium．［Conclusion］These remaining 17 pairs of Armillaria luteo-virens SSＲ markers will be valuable for future research on detecting population structure and conservation of this species．

Abstract:Abstract:［Objective］ Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) was used as solid carbon source and biofilm carrier to remove nitrate from recirculating aquaculture system (RAS)．Dynamics of microbial community structure in biofilm coating on carbon source packed into denitrification reactor were investigated．［Methods］ Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial community in biofilm from denitrifiation reactor．Bacteria degrading PHBV were isolated from the reactor using pure culture method．［Results］Nitrate decreased remarkably in the RAS connected with dentrification reactor．In contrast，Nitrate increased continuously in the conventional RAS without dentrification reactor．According to the phylogenetic analysis，the microbes in the biofilm samples from denitrification reactor were divided into Proteobacteria (β-proteobacteria，γ-proteobacteria and δ-proteobacteria)，Firmicutes and Bacteroidetes．The major advantageous populations were Acidovorax and Bacillus in the 40-day reactor．The advantageous populations in the 150-day reactor were in order of Clostridium，Desulfitobacterium，Dechloromonas，Pseudoxanthomonas and Flavobacterium．Pure cultures of bacteria degrading PHBV isolated from denitrification reactor were classified into Acidovorax，Methylibium，Pseudoxanthomonas and Dechloromonas． ［Conclusion］Nitrate could be removed effectively from RAS using PHBV as carbon source．Advantageous bacteria and their dynamic changes were ascertained in biofilm from denitrification reactor packed with PHBV．

Abstract:Abstract:［Objective］Alcanivorax dieselolei B-5 is an important oil-degrading bacterium．We studied its substrate range and degradation of halogenated compounds．［Methods］Growth capability of B-5 was examined with different halogenated substrates as sole carbon source．A putative haloalkane dehalogenase (HLD) gene named dadA was found from the genome of strain B-5 and analyzed by sequence alignment，phylogenetic analysis and homologous modeling．After heterologous expression in Escherichia coli and purification，the activity of DadA towards 46 substrates was determined．［Results］Strain B-5 was capable of utilizing various halogenated compounds (C3 -C18) as the sole carbon source．DadA had typical catalytic pentad residues of HLD-II subfamily，but it was independent from other members of this subfamily according to phylogenetic analysis． Activity assay showed that DadA has higher specificity and narrower substrate range than other characterized HLDs and it only showed activity toward 1，2，3-tribromopropane，1，2-dibromo-3-chloropropane and 2，3-dichloroprop-1-ene among 46 tested substrates．［Conclusions］ Strain B-5 and its HLD DadA can degrade halogenated aliphatic pollutants although．

Abstract:Abstract:［Objective］Although much is done in the coding genes of Newcastle disease virus (NDV)，limited papers can be found with non-coding sequences．In this paper，the evolution tendency of non-coding sequences was studied．［Methods］NDV strain LC12 isolated from duck with egg drop syndrome in 2012，and others 35 strains genome cDNA of different NDV genotype were sought and obtained from GenBank． Analytical approaches including nucleotide homology，nucleotide alignment and phylogenetic tree were associated with the leading sequences，trailer sequences，intergenic sequences (IGS)，and coding gene between 5 ’and 3’UTＲ nucleotide，respectively．［Results］The location and the length of the non-coding sequences highly conserve，and the variation trend of non-coding sequences is synchronous with the entire genomes and coding genes． ［Conclusion］The molecular variation of the coding gene was indistinguishable with the non-coding gene in view of the NDV genome.

Abstract:Abstract:［Objective］We studied the molecular characteristics of the full-length genome of duck hepatitis A virus type 1 causing pancreatitis in Muscovy ducklings．［Methods］We determined the entire genomic sequence of duck hepatitis A virus type 1 strain MPZJ1206 using reverse transcription polymerase chain reaction assay and analyzed the bioinformatics of the viral genome sequence.［Results］ The genome length of strain MPZJ1206 comprised 7703 bases，with a G + C content of 43.05%．The genome of MPZJ1206 contains a single，long open reading frame encoding a polypeptide of 2249 amino acids，with a genomic organization similar to those of other isolates of duck hepatitis A virus type 1．MPZJ1206 is identical with previously isolates by 93.5%－99.6% in nucleotide sequence and 97.9%－99.6% in amino acid sequence and shares genetic distance no more than 7%．Phylogenetic analysis based on genome sequence indicates that MPZJ1206 shares a close genetic relationship with two strains isolated in 2011．［Conclusion］Although pathotype caused by MPZJ1206 strain is significantly distinct from those induced by classical isolates of duck hepatitis A virus type 1，the genome of MPZJ1206 shares high homology with those of previous isolates. The change of pathotype may result from an alteration in viral tissue tropism of MPZJ1206.

Abstract:Abstract:［Objective］ Investigation of ammonia-oxidizing archaea (AOA) in nature environments is important to understand the global nitrogen cycling．However，little is known about the AOA community in plateau wetland．Therefore，we studied the composition and diversity of AOA in Zoige plateau wetland swamp soil．［Methods］ Total DNA was extracted from the swamp soil of three typical wetlands including A' xi pastoral area，Maixi pastoral area and Fenqu pastoral area locate in Zoige plateau wetland，and amoA gene was amplified with universally AOA amoA gene primers and then cloned．Then 80 positive clones for each clone library were chosen for further restriction fragment length polymorphism (RFLP) analysis，and the typical RFLP types were selected for sequencing and clustered into operational taxonomic units (OTUs) at 98% cutoff using the Mothur software． The MEGA 5.0 software was used for the amoA gene phylogeny analysis．［Results］ A total of 240 positive clones for all 3 libraries were used for RFLP analysis，and 15 specific amoA sequences were sequenced and clustered into 7 OTUs at 98% cutoff． Among them，OTU6 was detected in all of the 3 libraries and included 27% of the total specific clones． The phylogeny analysis showed that the 15 amoA sequences were grouped into 3 subgroups consisted of Zoige Wetland Clade 1 (4 OTUs)，Zoige Wetland Clade 2 (2 OTUs) and Zoige Wetland Clade 3 ( 1 OTU)．BLAST analysis showed that all OTUs were affiliated with the phylum Crenarchaeota． Correlation analysis showed that the Shannon diversity index (H') was significantly correlated with ammonia，nitrate/nitrite (P＜0.05)．［Conclusion］AOA in the Zoige plateau wetland swamp soil are all belonged to the Crenarchaeota，and their diversity is significantly correlated with soil ammonia，nitrate/nitrite content．