Abstract:Abstract: Benzoisochromanequinones antibiotics，a group of bioactive polyketide compounds with aromatic polyketide skeletal cores，are accumulated in streptomycetes． The biosynthesis of benzoisochromanequinones antibiotics has triggered great interest because they not only represent model biosynthetic mechanisms of aromatic polyketide skeletal structures，but also possess a variety of tailoring modifications rendering them highly structural and bioactive diversity． Here we reviewed important advances in biosynthesis of benzoisochromanequinones antibiotics in recent 25 years with focusing on the modification mechanisms of these compounds and on the prospects of the metabolic engineering and pharmaceutical discovery of benzoisochromanequinones antibiotics．

Abstract:Abstract:Recombinant adeno-associated virus (rAAV) is one of the most commonly used vectors for gene therapy．Despite the promising safety profile demonstrated in preclinical trials，the clinic efficacy of using rAAV was hampered by undesired response from the immune system． It is important to understand the mechanisms that lead to the induction of immune response against rAAV． Although a crucial role for innate immunity is shaping adaptive immune responses，the innate immune to rAAV was ignored in the past． Till now，at least three human cell types (dendritic cells，macrophages and endothelial cells) were discovered to be involved in sensing rAAV infection． The engagement of TLR9 by rAAV vector genomes triggers the activation of NF-κB signaling cascades，leading to the induction of pro-inflammatory cytokine genes．The viral capsid components are detected by TLR2，and this leads to the production of type I interferon mediated by interferon regulatory factors (IRFs) pathway． Self-complementary rAAV vectors induced higher TLR9 dependent innate immune response than single stranded rAAV． This review highlights the recent findings regarding the innate immune responses to rAAV vectors，the signaling pathways involved，and the impacts of innate immunity on the adaptive immune response to rAAV and its transgene expression．

Abstract:Abstract:［Objective］We studied a novel disease occurred among cultured Carassius auratus gibelio at a farm located in Yancheng City，Jiangsu Province． ［Methods］ The dominant bacteria were isolated from diseased fish． The pure culture of the isolated strain was analyzed using conventional physiological and biochemical tests，together with 16S rDNA gene sequencing． An experimental infection of Carassius auratus gibelio with the isolated strain was performed to fulfill the Koch postulates．K-B method was used for antibiotic susceptibility testing． ［Results］ The causal agent of the disease was finally proved to be one species of bacteria that was identified as Shewanella putrefaciens． Experimental infection with S．putrefaciens resulted in the same gross signs as naturally infected fish and the same bacteria were recovered in a pure culture from freshly dead fish． The LD50 of S． putrefacien was calculated as 2.1×103cfu/g．The result of drug sensitivity test showed that S． putrefaciens was sensitive to Pipemidic acid，Nalidixic acid，Fluperacid，Enoxacin，Florfenicol，Rifampicin， Minocycline， Fleroxacin， Enrofloxacin， Ceftriaxone， Cefalexin， Ceftazidine， Roxithromycin and Levofloxacin． ［Conclusion］ This is the first report on a new pathogen of Carassius auratus gibelio，revealing that S．putrefaciens as a potential new pathogen may pose a threat to the culture of Carassius auratus gibelio．

Abstract:Abstract: ［Objective］ We investigated the promoter activity of yigP gene and analyzed its transcriptional regulatory sequence．［Methods］We cloned promoter fragment into promoter probe plasmid which has a reporter gene lacZ，thus the promoter activity could be measured by detecting β-galactosidase activity． Then，the promoter region was minimized by cloning different truncated promoter fragments into promoter probe plasmid． Using site-directed mutagenesis technology，we introduced site mutations in some key sequences and investigated their effects on promoter activity．［Results］ The promoter region of yigP gene was determined，and also the -10 region and -35 region were identified． Meanwhile，a negative regulatory sequence just upstream of yigP promoter was discovered，and our results show that these sequences have important influence on the transcriptional regulation．［Conclusion］ We identified the transcriptional regulatory sequences of yigP gene，and this facilitated our understanding of gene transcription．

Abstract:Abstract:［Objective］ To learn about the function and gene phylogenetic relationship of the conserved regions in capsular polysaccharides synthesis (CPS) locus，［Methods］ based on the CPS locus sequence of Streptococcus suis serotype 1，2， 7 and 9 and the results to the cross-hybridization experiments，the CPS locus of Streptococcus suis was hypothesized as cassette-like structure which is similar to Streptococcus pneumoniae． PCR，sequencing and southern blotting was used to certify the hypothesis． ［Results］ The CPS locus of Streptococcus suis was cassette-like structure． The 5'-end of the CPS locus contained 4 regulatory genes which are highly similar in all the serotypes． The flanking genes of the CPS locus are conservative． The flanking gene aroA，which is at the downstream to the CPS locus，was selected as the objective gene to develop the PCR to amplify the serotype-specific regions． The orfY、orfX、cpsA、cpsB、cpsC、cpsD and aroA are conserved with high sequence identity in different serotypes．

Abstract:Abstract:［Objective］ We studied the resistance of Fusarium fujikuroi against carbendazim，to confirm whether the resistance was related to the β-tubulin gene．［Methods］ The isolated strains were identified based on TEF-1α gene sequence and morphological characteristics． The whole nucleotide sequence of β-tubulin gene was analyzed from different sensitive strains by the primers designed according to the nucleotide sequence of β-tubulin gene from the sequenced strain 7600 of F． verticillioides． Real-time quantitative RT-PCR (qRT-PCR) was applied to analyze the expression pattern of β-tubulin gene from different sensitive strains treated with or without carbendazim．［Results］ The full-length nucleotide sequence of β-tubulin gene ( Accession Number: JQ026022) was cloned from different sensitive strains of F． fujikoroi， which spanned 1671 bp with 4 introns，encoding 447 amino acids． This gene showed 100% nucleotide sequence homology between sensitive and resistant strains． The expression of the β-tubulin gene in 2 sensitive strains was significantly higher than 3 resistant strains when these strains were cultured on carbendazim-free media (p=0.05)．Carbendazim at the concentration of their EC50 caused the expression of the gene from the five strains increasing significantly (p=0.05) in comparison with that of strains treated without carbendazim． However，there was no significant difference between strains when treated with carbendazim．［Conclusion］ The resistance of F． fujikuroi against carbendazim was irrelative to β-tubulin gene.

Abstract:Abstract:［Objective］We constructed a recombinant Escherichia coli strain for butanol production by cloning the cDNA sequence of the key butanol synthetic pathway genes from Clostridium acetobutylicum ATCC824．［Methods］ We amplified the genes of thil，adhE2 and BCS operon by PCR with C.acetobutylicum ATCC824 genome as a template. We constructed the recombinant strain E.coli pBAT (BCS operon-adhE2-thil/pTrc99a/MG1655)．We used 0.1 mmol/l Isopropyl beta-D-thiogalactopyranoside (IPTG) to induce the recombinant E.coli pBAT for 5 h for recombinant protein expression. We measured acetyl-CoA acetyltransferase (THL)，β-hydroxybutyryl-CoA dehydrogenase (HBD)，3-hydroxybutyryl-CoA dehydratase (CRT)， butyryl-CoA dehydrogenase (BCD) and butyraldehyde dehydrogenase (BYDH)/butanol dehydrogenase (BDH) activities in E.coli MG1655 and E.coli pBAT． The fermentation of E.coli pBAT was done in flask in aerobic，micro-aerobic and anaerobic mode separately．［Results］ In the recombinant E． coli pBAT，THL activity was 0.160 U/mg protein，about 30 times higher than that of E.coli MG1655． HBD activity was 5 times higher than that of E.coli MG1655.CRT activity was 1.53 U/mg protein whereas not detectable in E.coli MG1655. BCD activity was about 32 times higher than that of E.coli MG1655.In addition，the results show that nbutanol could be produced under anaerobic and micro-aerobic conditions． The maximum n-buntanol concentration of 84 mg/l was detected in cultivation broth.［Conclusion］ The key genes of butanol synthetic pathway were expressed in E.coli and the recombinant strains would offer an alternative strategy for butanol biosynthesis.

Abstract:Abstract:［Objective］ To explore whether the S2P homolog，sll0862 in cyanobacterium Synechocystis sp． PCC 6803 is involved in stress response．［Methods］We compared the growth curve of sll0862 mutant and the wild type under high temperature or oxidative stress． We detected chlorophyll fluorescence under heat shock or oxidative stress by water-PAM (pulse amplitude modulated fluorometry).［Results］ Under normal condition of autotrophic growth，the growth curve of sll0862 mutant was similar with that of the wild type． However，after heat treatment at 48℃ for 30 minutes，the survival rate of sll0862 mutant was lower than that of the wild type． The sll0862 mutant hardly survived when incubated in 1 mmol/L H2O2，whereas the wild type is not affected． Meanwhile，different chlorophyll fluorescence under stress between the wild type and the mutant was observed using water-PAM．［Conclusion］ These results indicate that the S2P homology sll0862 plays an important role in response to heat shock and oxidative stress in cyanobacterium Synechocystis sp.PCC 6803，which provides foundation for further research of the sll0862 function and mechanism.

Abstract:Abstract:［Objective］Gamma-butyrobetaine hydroxylase is an enzyme that catalyzes the last step in the biosynthesis of Lcarnitine． We cloned，expressed and characterized a gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp.L-1，to facilitate the production of L-carnitine using microorganisms.［Methods］We cloned bbh gene by PCR，and then cloned the open reading frame of bbh into pET-15b vector and expressed by Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction． After His-Bind Resin purification，the characteristics of BBH were studied． The three-dimensional structure of BBH monomer was modeled by SWISS-MODEL Workspace and resting cells were used for L-carnitine transformation．［Results］We cloned a gamma-butyrobetaine hydroxylase gene bbh (GenBank: JQ250036) from Pseudomonas sp.L-1 and expressed the gene in Escherichia coli BL21(DE3)．BBH fusion protein was a homodimer，and the molecular weight of subunit was about 46. 5kDa． The optimal temperature and pH was 30℃ and pH 7.5. The enzyme was stable below 45 ℃．The enzyme was most stable at pH 6.0. We used resting cells of recombinant E.coli for L-carnitine biotransformation，after incubated at 30℃ and pH 7.0 for 31 h，the concentration of L-carnitine reached 12. 7 mmol/L.［Conclusion］The bbh gene from Pseudomonas sp.L-1 strain is remarkably different from that of reported one． The gamma-butyrobetaine hydroxylase expressed by this gene could effectively transform γ-butyrobetaine for L-carnitine production． Beside by reporting of a bbh gene from bacteria，this research also provided a new process for biotransformation of L-carnitine.

Abstract:Abstract:［Objective］Cloning and heterologously expressing the α-galactosidase gene (agaAGN14) from Arthrobacter sp．GN14 isolated from feces of black-neck crane (Grus nigricollis)．［Methods］The full-length agaAGN14 was cloned based on degenerate PCR and GC TAIL-PCR (thermal asymmetric interlaced PCR) ，ligated into pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells．The recombinantα-alactosidase (rAgaAGN14) was purified to electrophoretic homogeneity by Ni2 + -NTA metal chelating affinity chromatography，and then the enzyme characterizations were determined． Amino acids sequences of agaAGN14 (AgaAGN14) and α-galactosidases from Actinobacteria and gastrointestinal microorganisms were aligned and used for constructing a neighbor-joining phylogenetic tree． ［Results］The 2109-bp full-length agaAGN14 (66.8% GC content) encodes a 702-residue polypeptide (AgaAGN14; 77.5 kDa)．AgaAGN14 showed the highest identity of 53. 7% with α-galactosidases in public databases，and＜43% identities with α-galactosidases from gastrointestinal microorganisms.AgaAGN14 was put in a phylogenetic branch sharing the catalytic motifs KWD and SDXXDXXXR，and close to α-galactosidases from soil microorganisms and far from α-galactosidases from gastrointestinal microorganisms． The purified rAgaAGN14 efficiently hydrolyzed pNPG，raffinose，melibiose，stachyose，rapeseed meal and cottonseed meal; showed apparent optimal at pH 6.0 and 45℃，stability and activity (＞50%) at pH 6.0－9.0，and activities of 28%，30% and 80% at 10℃ ，20℃ and 37℃ ，respectively; exhibited Km，Vmax and kcat values of 0.41 mmol/L，18.28 μmol/min/mg and 25.36 s－1，respectively，using pNPG as the substrate at 45℃ and pH 6.5; strongly inhibited by Ag+，Hg2+ and SDS，partial inhibited by K+，Ca2+，Mn2+，Fe3+，Ni2+，Cu2+ and β- mercaptoethanol，and little influenced by Co2+，Pb2+，Zn2+，Mg2+，Na+and EDTA．［Conclusion］ The Arthrobacter strain isolated from feces of Grus nigricollis，and the sequence analysis，phylogenetic analysis，heterologous expression and recombinant enzyme’s biochemical characterizations of an α-galactosidase from Arthrobacter strain were first reported. rAgaAGN14 was a novel α-galactosidase．

Abstract:Abstract:［Objective］The intracellular α-amino acid ester hydrolase (AEH) from Xanthomonas rubrillineans was purified and characterized． ［Methods］ AEH was extracted by butyl acetate，and then purified to electrophoretic homogeneity by calcium phosphate gel precipitation，ammonium sulfate fraction precipitation，anion exchange with DEAE Sephadex A-50，cation exchange with CM cellulose 52，and Sephadex G 200 column chromatography． ［Results］ The subunit molecular mass of AEH was 70 kDa by SDS-PAGE． The optimal reaction pH for cefaclor synthesis was 6.8，and optimal temperature was 42℃．The enzyme was stable between pH 5.0 and 8.0，and at 35℃．The enzyme activity was enhanced by Mn2+ and Ca2+，however was strongly inhabited by Cu2+，Fe2+ and high concentration of acetone．The kinetic parameters that the enzyme hydrolyzed D-Phenylglycine methyl ester，D-Hydroxyphenylglycine methyl ester and cefaclor were determined，and the values of kcat/Km were 123.7±3.7，2.9±0.6 and 101.3 ± 2.1 mmol－1·s－1·L respectively．The kcat/Km values indicated that the enzyme hydrolyzed D-Phenylglycine methyl ester more efficiently than other substrates． The mechanism of enzymatic reaction with bi-substrates by AEH is Ping-Pong kinetics，and the kcat value that the enzyme catalyzed the synthesis of cefaclor is 547.3±38.2 s－1．［Conclusion］ The studies of AEH from Xanthomonas rubrillineans were rare， and our research may provide an important basis for industrial application of AEH for beta-lactam antibiotics synthesis．

Abstract:Abstract:［Objective］In order to study the microorganisms on the surface of the ancient stones in Yungang Grottoes and nearby rock samples，a rapid microbial detection assay was designed．［Methods］ The microbial community composition from the rock samples of the Yungang Grottoes and nearby was analyzed by PCR-DGGE． ［Results］ According to the phylogenetic analysis，the microbes from the rock samples of Yungang Grottoes were divided into four groups: Gammaproteobacteria，Sphingobacteria，Alpha-proteobacter and Actinobacteria． By aligning the sequencing results in GenBank，Gamma-proteobacteria，Firmicutes and Alpha-proteobacter were found in the rock samples near Yungang Grottoes．［Conclusion］ Our research successfully detected the microbial community composition on the surface of the rock samples in and near the Yungang Grottoes，which brought us significant theoretical basis for the future protection of the Yungang Grottoes． We also proved that the combination of DGGE and molecular cloning was a useful，rapid and accurate method for detecting the microbial community composition on stone relics．

Abstract:Abstract:［Objective］To investigate potential pathogens in waters of Xiamen from Jiulong River，and to provide useful information for the prevention and control of potential pathogen infections． ［Methods］ All samples were spread on Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar plates，and then incubated at 26 ± 1℃ for 24 ± 2 h． In total 158 TCBS strains were isolated from TCBS agar plates and pure-cultivated on 2216E agar plates． All strains were identified using the 16S rRNA gene- Restriction fragment length polymorphism (RFLP) ，16S rRNA sequence analysis，GenBank database Basic Local Alignment Search Tool (BLAST) and phylogenetic analysis． ［Results］ The results show that 158 TCBS strains from the sediments of Jiulong River estuary were classfied as 7 genus，which were Pseudomonas (28%)，Aeromonas (24%)，Pseudoalteromonas (19%)，Shewanella (13%)，Bacillus (11%)，Vibrio (4%) and Psychrobacter (1%)．The composition and distribution of TCBS bacteria groups varied with stations． Non-halophilic or haloduric bacteria groups were dominant in the upper area of Jiulong River estuary，and halophilic and haloduric bacteria were dominant in the lower area，which characterized a typical estuary feature． The salinity played a key role in the distribution of TCBS groups． Vibrios did not constitute a significant proportion (6%－19%) of the total TCBS strains at different stations，and most of the them distributed at the lower region． ［Conclusion］ There were a lot of potential pathogens in Jiulong River estuary． Aeromonas， a typical genus of halotolerant bacteria，was the potentially terrigenous bacteria contamination to the waters of Xiamen． Most Vibrio specieses were marine aborigines，which was not directly contaminated from the runoff of Jiulong River．

Abstract:Abstract:［Objective］In order to study the characteristic of nitrogen transport，the community structure and diversity of related microorganisms in aquaculture water of the Pearl River Delta． ［Methods］We established an artificial aquaculture v ecosystem to study the microbial community of 15N-stable isotope probing (15 N-SIP) labeled nitrogen transport microorganisms． The 15N-labeled DNA was separated by CsCl-ethidium bromide density gradient centrifugation，and was used to construct 16S rRNA gene clone libraries of bacteria and archaea． ［Results］ Phylogenetic analysis shows that 19 Operational Taxonomic Units (OTUs) from bacterial library were clustered in Proteobacteria and Planctomycetes．Proteobacteria (99.2%) was the dominant group，mainly consisted of Comamonas (15.7%) ，Nitrosomonas (12.4%)，Enterobacteriaceae (11.5%) and Nitrobacter (11.5%)．From archaeal library 9 OTUs were divided into 3 phyla:Thaumarchaeota，Crenarchaeota and Euryarchaeota． ［Conclusion］We successfully elucidated the microbial community of nitrogen transport microorganisms in aquaculture water of Pearl River Delta by using 15N-SIP． The data of the community will provide essential information for isolating nitrogen degrading microorganism，and provide scientific basis for creating a healthy aquaculture environmentl.

Abstract:Abstract:［Objective］To investigate the antiviral activity of porcine lung surfactant protein A (SP-A) to porcine reproductive and respiratory syndrome virus (PRRSV) in vitro．［Methods］ The SP-A gene was amplified by PCR from the plasmid containing porcine SP-A gene，and subcloned into pcDNA3. 1A-CD5 vector containing the human CD5 signal peptide to generate SP-A eukaryotic expression vector pcDNA-CD5-SPA/MH．The recombinant expression vector was transfected into HEK293T cells mediated with calcium phosphate． The expressed recombinant SP-A was identified by Western blot and purified from culture medium by Ni-NTA-Agarose beads． The binding activity of SP-A with PRRSV was identified by ELISA． The antiviral activity of SP-A to PRRSV was analyzed by viral titer reduction assays on MARC-145 cells and porcine alveolar macrophages (PAM)．［Results］ The results showed that the eukaryotic expression vector of SP-A gene could mediate SP-A expression in HEK293T cells，the expressed SP-A could bind PRRSV in a dose dependent manner． The PRRSV incubated in advance with SP-A showed the lower infective activity compared with no-SP-Aincubated PRRSV on both MARC-145 cells and porcine alveolar macrophages． The SP-A-treated PRRSV titers in MARC-145 cells and PAM cells were significantly lower than that of SP-A-untreated PRRSV at 72 h post-infection．［Conclusion］ Recombinant porcine SP-A significantly inhibit the infection of PRRSV to the host cells in vitro，which indicates that recombinant SP-A possesses anti-PRRSV activity．

Abstract:Abstract: ［Objective］To further enhance ectoine (1，4，5，6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) synthesis efficiency．［Methods］ We cloned Halomonas salina DSM 5928T teaABC gene of ectoine-specific transporter TeaABC by walking PCR and constructed ectoine absorption defective mutant H． salina DSM 5928T (teaABC－) by Red recombination technology．［Results］ The total concentration of ectoine and productivity in a 10 L fermentor were 9.10(±0.08)g/L and 9.93(±0.09)g/L.d．［Conclusion］ The ectoine absorption defect mutant H． salina DSM 5928T(teaABC－) compromised the negative feedback regulation of ectoine synthesis，which can significantly improve the efficiency of ectoine production.