Abstract:Abstract:Facultative methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to use methane and some other compounds containing C-C bond as their sole source of carbon and energy． Recently，facultative methanotrophs in the genera Methylocella， Methylocapsa and Methylocystis，which belong to the Alphaproteobacteria，have been reported that can grow on larger organic acids or ethanol for some species，as well as methane．In this paper，the research history of facultative methanotrophs was summarized systematically，some other facultative methane-oxidizing microorganisms were introduced， the metabolic mechanisms of utilizing multi-carbon compounds by facultative methanotrophs were analyzed，and the current problems and the future engineering applications were discussed．

Abstract:Abstract:Non-coding RNAs (ncRNAs) existing widely in many living organisms are functional RNA molecules，function directly as structural or regulatory RNAs in organisms．Although large and diverse populations of ncRNAs have been extensively studied and well understood in animals and plants，few reports could be found about ncRNAs in fungi．Recently，with the development of modern biological techniques，a number of ncRNAs have been identified in fungi， including snoRNA-derived RNAs，long non-coding RNAs，small interfering RNAs (siRNAs)，dsRNA Killer viruses，and novel classes of ncRNAs discovered in filamentous fungi．These ncRNAs play important roles in gene transcription and translation，RNA processing and modifying，chromatin structure，and even fungal pathogenicity．Therefore，studies on ncRNAs in fungi may shed light on the regulatory system of gene expression and the characteristics of fungal growth，and even provide some clues towards understanding pathogenic mechanisms of pathogenic fungi，which will contribute to the treatment of fungal diseases． Here，we reviewed the discovery of fungal ncRNAs，their origins and processing，classification，and biological functions，aiming to establish a theoretical foundation and basis for deep understanding of fungal ncRNAs in future．

Abstract:Abstract:［Objective］We explored 4 new methods to improve the isolation of actinobacterial resources from high salt areas.［Methods］Optimized media based on 4 new strategies were used for isolating actinobacteria from hypersaline beaches． Glycerin-arginine，trehalose-creatine，glycerol-asparticacid，mannitol-casein，casein-mannitol，mannitol-alanine，chitosan-asparagineand GAUZE' No.1 were used as basic media． New isolation strategy includes 4 methods: ten-fold dilution culture，simulation of the original environment，actinobacterial culture guided by uncultured molecular technology detected，and reference of actinobacterial media for brackish marine environment．The 16S rRNA genes of the isolates were amplified with bacterial universal primers． The results of 16S rRNA gene sequences were compared with sequences obtained from GenBank databases． We constructed phylogenetic tree with the neighbor-joining method．[Results] No actinobacterial strains were isolated by 8 media of control group，while 403 strains were isolated by new strategies．The isolates by new methods were members of 14 genera (Streptomyces， Streptomonospora，Saccharomonospora，Plantactinospora，Nocardia， Amycolatopsis，Glycomyces，Micromonospora， Nocardiopsis， Isoptericola，Nonomuraea，Thermobifida，Actinopolyspora，Actinomadura) of 10 families in 8 suborders．The most abundant and diverse isolates were the two suborders of Streptomycineae (69.96%) and Streptosporangineaesuborder (9.68% ) within the phylum Actinobacteria，including 9 potential novel species．［Conclusion］New isolation methods significantly improved the actinobacterial culturability of hypersaline areas，and obtained many potential novel species，which provided a new and more effective way to isolate actinobacteria resources in hypersaline environments.

Abstract:Abstract:［Objective］Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis．In most bacteria，ACC is composed of four subunits encoded by accA，accB，accC，and accD. of them，accA encodes acetyl-CoA carboxyltransferase α-subunit．Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress． This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance.［Methods］The Aa-accA was amplified by PCR from A．amylolytica N10 and expressed in E． coli K12 host．The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions． Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation． The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock．［Results］The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases．It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E.coli. Compared to the wild-type strains，transgenic E．coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels． The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9，respectively． Complementary expression of Aa-accA in an accA-depletion E.coli can recover the tolerance of K12ΔaccA to salt and alkali stresses to some extent． Similar to the transgenic E.coli，transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition.［Conclusion］ We found that Aa-accA from A． amylolytica N10 overexpression enhances the tolerance of both transgenic E.coli and tobacco BY-2 cells to NaCl and alkali stresses．

Abstract:Abstract:［Objective］We constructed an integrative recombinant Corynebacterium crenatum that could directly convert glucose in γ-aminobutyric acid (GABA)．［Methods］ Using overlap PCR，we obtained ΔargB fragment that lacked 491 bp of N-acetylglutamate kinase (NAGK) gene． The glutamate decarboxylase (GAD) gene attached tac promoter was amplified and inserted into ΔargB fragment． Using the upstream and downstream of ΔargB as homologous arms，we constructed pK18-ΔargB:: tacgad which was used for the integration of tacgad gene onto the C．crenatum genome．Through two homologous recombinations，we got an argB blocked strain C．crenatum ΔargB:: tacgad which could successfully express GAD． We also fermented the recombinant strain with glucose as substrate and the production of GABA was detected．［Results］In C．crenatum ΔargB::tacgad，NAGK was totally inactivated and no L-arginine was detected though L-glutamic acid was accumulated. As a result of the efficient expression of GAD，part of L-glutamic acid was transformed into GABA，and the final yield of GABA was about 8. 28 g /L.［Conclusion］We successfully constructed a recombinant strain that could efficiently produce GABA by one-step fermentation from glucose． This research provided a new approach for GABA production.

Abstract:Abstract:［Objective］In order to provide more natural antifouling compounds，marine bacterium Pseudomonas putida isolated from the sponge Haliclona sp． was explored to test its anti-diatom compounds.［Methods］The strain was identified by colonial morphology，scanning electron microscope (SEM) and 16S rDNA sequence analysis．The separation procedure was guided by bioactive (Anti-diatom) and chemical (TLC，DAD-HPLCand 1H NMR) analysis，and their structures were elucidated by spectrographic techniques．The anti-diatom activity of all purified compounds was assayed．［Results］Strain 272 isolated from the sponge Haliclona sp．was identified as Pseudomonas putida．Six diketopiperazine compounds were isolated from the culture of this strain and their structures were determined as cyclo(Leu-Pro) (1)，cyclo (Leu-Ala) (2)，cyclo(Phe-Ala) (3)，cyclo(Val-Tyr) (4)，cyclo(Ala-Tyr) (5)，cyclo(Ala-Trp) (6); Compounds 3 and 6 displayed significant anti-diatom activity with the inhibitory rate of 50% and 85% at the concentration of 50μg/mL，espectively． ［Conclusion］The anti-diatom compounds isolated from marine bacterium Pseudomonas putida were cyclo (Phe-Ala) and cyclo (Ala-Trp)．

Abstract:Abstract:［Objective］To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas.［Methods］The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E.coli and specific antibody was prepared．Then the growth curve and cytokinin contents of E.coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively． ［Results］The length of tRNA-ipt genes from PaWB as well as mulberry dwarf，periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp．All these genes encoded the proteins consisting of 291 amino acids．They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites．Four phytoplasma tRNAIPTs shared the 99.1－99.5%，amino acid sequence indentity with each other，shared 95.4－99.3% sequence identity with the same group phytoplasmas，whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas．The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection． The determination of growth curve suggested that the growth rate increase of E.coli was affected by the transformation of exogenous tRNA-ipt gene，which might be associated with the cytokinin accumulation.［Conclusion］This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria，which may play an important role in pathogenic processes of phytoplasmas and symptom development.

Abstract:Abstract:［Objective］The objective of the present study is to elucidate the effects of the application of cake fertilizer fermentation fluid with antagonistic bacteria and soil disinfectant chlorine dioxide on the occurrence of banana fusarium wilt disease and soil bacterium community． ［Method］Under the field cultivation conditions，the Biolog and T -RFLP method was used to investigate the soil bacterium diversity and community features in different treatments at different periods.［Result］The results show that both cake fertilizer fermentation fluid with antagonistic bacteria and soil disinfectant could reduce disease index of banana fusarium wilt disease significantly，the highest control effect could reach 60.82% with the combined application of these two methods． The result of Biolog eco plate shows that the application of cake fertilizer fermentation fluid with antagonistic bacteria could improve soil microbial AWCD(average well color development) and population uniformity，the use of soil disinfectant significantly reduced the soil microbial population’s abundance and the uniformity． Principal component analysis shows that the soil microbial population using carbon source had an increasing trend throughout the banana growing season，the main carbon sources in the early stage were amino acids，carboxylic acids，amphiphilic compounds and carbohydrates，and the increased main carbon sources in the later stage were carboxylic acids and amphiphilic compounds． Soil bacterial diversity analysis by T-RFLP shows that the treatments of cake fertilizer fermentation fluid with antagonistic bacteria had the highest bacterial TRFs (Terminal restriction fragment) fragments，which resulted from the increase of Flavobacterium，Pseudomona and Lactobacillus population in the soil.［Conclusion］The application of cake fertilizer fermentation fluid with antagonistic bacteria combining soil disinfectant could increase antagonistic microorganisms species，enhance soil microbial diversity，improve soil microbial ecological structure on the basis of reducing pathogen in soil，finally achieve the goal of improving the control effects of banana fusarium wilt disease．

Abstract:Abstract:［Objective］Toisolate and characterize bacteria that can be used todevelop microbial biosensor for methanol (MeOH) determination.［Methods］ We used selective medium and streak plate to isolate bacteria． Morphological，physiological characteristics and 16S rDNA sequence analysis were used to identify the strain．AnMeOHbiosensorwas then developed by immobilizing M211 along with dissolved oxygen (O2) sensor.［Result］AnMeOH utilizing bacteriumwas isolated from biogas-producing tank using methanol as the sole carbon source，and identified as Methylobacteriumorganophilium．Decrease of O2 concentration is linearly related to the MeOH concentration in the range from 0.02% to 1% ，with the MeOH detection limit of 0.27mg /L.The response time of the biosensor is within 20 min．Furthermore，the result of interference test and the detection of methanol sample are both satisfactory．［Conclusion］Good results are obtainedin interference test and the detection of methanol sample． The proposed methodseems very attractive in monitoring methanol.

Abstract:Abstract:［Objective］ To investigate the effects of Mycobacterium tuberculosis ESX-1 protein early secreted antigenic target of 6 kDa(ESAT-6) in modulating phagocytosis of RAW264. 7 cells.［Methods］RAW264.7 cells were transfected with recombinant plasmids pFLAG-ESAT-6 and pFLAG-EGFP by liposome． After screening with a high level of G418，the macrophage cell lines stably expressing flag-ESAT-6 or flag-EGFP proteins were obtained．The cell lines were further identified by PCR，RT-PCR and western blot．The phagocytosis of those cell lines was analyzed for ingested fluorescent beads by flow cytometry and for phagocytized Escherichia coli(E.coli) by colony count and confocalmicroscopy.［Results］We established successfully RAW-E6 cell line stably expressing flag-ESAT-6 and RAW-EGFP cell line stably expressing flag-EGFP． Flow cytometric analysis shows that the percentage of phagocytosis of RAW-E6 was higher than that of RAW264.7 and RAW-EGFP. Colony count and confocal microscopy test also show that RAW-E6 had higher phagocytosis ability than RAW264.7 and RAW-EGFP.［Conclusion］The secreted protein ESAT-6 can enhance the phagocytosis of macrophages，which provides new evidence to understand the pathogenesis of Mycobacterium tuberculosis.

Abstract:Abstract:［Objective］To investigate the effect of the Interferon-α-induced gp96 upregulation on the anti-HBV efficiency of Interferon-α(IFN-α).［Methods］The effect of IFN-α on the transcription and expression of heat shock protein gp96 was determined by real-time PCR，luciferase reporter assay and Western blot．The effect of over-expression or knock-down of gp96 by transfection or RNA interference，and treatment with IFN-α on HBV expression and replication was examined by ELISA and RT-PCR.［Results］IFN-α treatment led to increased gp96 expression in a time- and dose-dependent manner. The overexpression of gp96 enhanced HBV expression and replication，whereas downregulation of gp96 resulted in decreased HBV replication． Finally，we verified that blocking IFN-α-induced upregulation of gp96 significantly enhanced IFN-α-mediated HBV inhibitory effects.［Conclusion］ IFN-α-induced upregulation of gp96 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insight for enhancing the antiviral efficacy of IFN-α by simultaneous inhibition of gp96．

Abstract:Abstract:［Objective］Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis． At present，Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach． However，the responses are usually poor and the viral infection reoccurs． Therefore，exploring new antiviral agents and therapies is under urgent needs．［Methods］The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software，DNAMAN and RNA Structure． The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site． Based on the flanking sequence of this assumed cleavage site，a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA，which is catalytic subunit of the RNase P derived from Escherichia coli using PCR． We named this new targeting ribozyme M1GS-HCV /C52 and it antiviral activities were analyzed in cultured cells．［Results］In the in vitro cleavage assay，M1GS-HCV /C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site． Moreover，comparing to the blank control，this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCVinfected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant． An another M1GS ribozyme，M1GS-HCV /C52 * ，which has the same guide sequence but does not contain a 24nt-long bridge sequence，did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay．［Conclusion］We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA．Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells，thus provided a new promising approach for clinical anti-HCV therapeutic strategy.

Abstract:Abstract:［Objective］ Polysaccharides inhibiting cucumber mosaic virus (CMV) on tobacco were screened and their influence on tobacco defensive enzyme activities was explored．［Method］We detected the deactivating，preventing and treating effect of 21polysaccharides on CMV by half leaf method on Nitcotiana tabacum var．Samsun NN． We detected the variation of enzyme activity of Nitcotianatabacum var． NC89 handled by antiviral polysaccharide．［Result］Results show that Marasmiu sandrosaceus polysaccharide had good deactivating and preventing effect on CMV． The preventing rate could reach 83. 41% when the tobacco was dealt with the admixture of its 200-fold dilution and equivalent virus liquid for 30min．The inhibition rate could reach 93. 15% when the tobacco was inoculated after spraying with M．androsaceus polysaccharide for 24h．Moreover，the diversification of enzyme activity of tobacco-related was detected． Results show that peroxidase (POD)，polyphenol oxidase (PPO) andphenylalanine ammonia lyase (PAL) activities significantly enhanced．M．androsaceus polysaccharide sprayed on tobacco after inoculating CMV for 24h．Its enzyme activities increased．The peaksof POD，PPO and PAL activities ofthe treatment were 2.74，3.45 and 2.82 times of the value of comparison，respectively.［Conclusion］M. androsaceus polysaccharide can increase the resistance of tobacco on CMV by enhancing defense enzyme activity of tobacco.

Abstract:Abstract:［Objective］ To identify lactic acid bacteria (LAB) in commercial yogurts and investigate their antibiotic resistance.［Methods］LABs were cultured from 5 yogurt brands and the isolates were identified at the species level by 16S rRNA sequence． Genotyping was performed by repetitive extragenic palindromic PCR (rep-PCR)．The sensitivity to 7 antibiotics was tested for all LAB isolates by Kirby-Bauer paper diffusion (K-B method). Meanwhile，9 antibiotic resistance genes(ARGs)，including erythromycin resistance genes (ermA and ermB) and tetracycline resistance genes (tetM，tetK，tetS，tetQ，tetO，tetL and tetW)，were detected by PCR amplification in the identified LAB isolates．The PCR products were confirmed by sequencing． ［Results］ Total 100 LABs were isolated，including 23 Lactobacillus delbrueckii ssp．bulgaricus，26 Lactobacillus casei，30 Streptococcus thermophilus，5 Lactobacillus acidophilus，6 Lactobacillus plantarum，and 10 Lactobacillus paracasei. The drug susceptibility test shows that all 100 isolates were resistant to gentamicin and streptomycin，42 isolates were resistant to vancomycin，and on the contrary all were sensitive to cefalexin，erythromycin，tetracycline and oxytetracycline. Moreover，5 ARGs were found in the 28 sequencing confirmed isolates，ermB gene was detected in 8 isolates，tet K in 4 isolates，tetL in 2 isolates，tetM in 4 isolates，tetO in 2 isolates. erm A，tet S，tet Q and tet W genes were not detected in the isolates． Antibiotic resistance genes were found in 53.57% (15/28) sequenced isolates，2－3 antibiotic resistance genes were detected in 4 isolates of L.delbrueckii ssp． bulgaricus.［Conclusions］Some LABs were not labeled in commercial yogurt products. Antibiotic resistance genes tend to be found in the starter culture of L. delbrueckii ssp． Bulgaricus and S. thermophilus. All the LAB isolates were sensitive to erythromycin and tetracycline，even though some carried erythromycin and/or tetracycline resistance genes．We proved again that LAB could carry antibiotic resistance gene(s) though it is sensitive to antibiotics．