Abstract:Abstract:A multi-vitamin auxotrophic yeast of Torulopsis glabrata was the most competitive strain for industrial production of pyruvate． Given its genomic characterizations and physiological functions，it was an efficient way to redirect carbon flux to the target metabolites through manipulating nutritional and environmental conditions，intracellular cofactor form and level． In this review，we summarized the progress on the elucidation and manipulation of physiological function of T.glabrata. Furthermore，we also evaluated the potential of T．glabrata as cell factory for production of fine chemicals．

Abstract:Abstract:［Objective］We characterized bacterial diversity in a deep-sea hydrothermal plume seawater in the southwest Indian Ocean to increase our understanding of the impact of the microorganisms on the ocean ecosystem and to survey microbial resources in this special environment． ［Methods］The deep-sea hydrothermal plume seawater in the southwest Indian Ocean was concentrated in situ by 1000 folds，enrichment cultures were established with the concentrated sample，and isolates were purified． Bacterial 16S rRNA gene libraries were constructed from both the concentrated seawater sample and from the enrichment culture and analyzed． The 16S rRNA genes from the isolated strains were also analyzed．［Results］A total of 104 16S rRNA genes were obtained，in which 50 were from the concentrated plume seawater，40 from the enrichment culture，and 14 from the isolated strains． These sequences are affiliated with γ-proteobacteria (74)，α-proteobacteria (14)，β-Proteobacteria(5)，Bacteroidetes (4)，Firmicutes (2)，Planctomycetes (2)，Verrucomicrobia (2) and Actinobacteria (1)，and fall into 29 different operational taxonomic units (OTUs)． Twenty-six sequences share less than 97% identity with the best-matched sequences in the public database，with the lowest being 86%．［Conclusions］There is rich bacterial diversity in the deep-sea hydrothermal plume seawater in the southwest Indian Ocean，where γ-proteobacterial groups were dominant，followed by α-proteobacterial groups．A number of species remain uncultured．

Abstract:Abstract:In many Pseudomonas，RsmA mediates the production of a set of secondary metabolites or virulence factors．［Objective］Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1．［Methods］ We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA． With the insertion of gentamycin resistance cassette ( aacC1)，the deletion mutant PA-RG was created and verified with PCR． To complement and overexpress the rsmA gene，pME10R and pME32R were also constructed． By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1and phz2'-'lacZ pMEZ2，we introduced them into the wild type strain PAO1 and the mutant PA-RG，respectively． Activities of beta-galactosidase were determined with Miller method． ［Results］In glycerol-alanine medium，overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain． In addition，beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain． However，beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG． ［Conclusion］ The regulation mediated by RsmA on two phenazine loci is specific and differential．

Abstract:Abstract:［Objective］To identify preliminarily the specific fragment SCF73's function in Verticillium dahlia virulence．［Method］The specific fragment SCF73 exposed to be existed in the high-virulent V． dahliae strain VDG1 and not in the mild one VDG2． The SCF73 fragment was obtained from comparatively aligned genome sequences of the two strains and its existence was confirmed using PCR method． According to SCF73's DNA sequence，a homologous recombination plasmid was constructed to knock out the fragment． The Agrobacterium tumefaciens-mediated transformation technique was used to initiate the mutant ΔSCF73，followed by antibiotic resistance screening，and PCR verification． The mutant's ability to secrete carbohydrate hydrolase was analyzed using pectin，cellulose and starch media and its virulence to the susceptible cotton cultivar Gossypium hirsutum cv． Junmian1 was assessed．［Result］ SCF73 (27.1 kb) contains 5 genes，two of them have glycosyl hydrolase activity． Although the mutant ΔSCF73's carbohydrate hydrolase secretion was not significantly different from the control VDG1，virulence of the mutant to cotton plants decreased significantly accompanied with disease outburst delay．［Conclusion］The specific fragment SCF73 plays an important role in the virulence of V．dahlia towards its cotton host plants．

Abstract:Abstract:［Objective］A Corynebacterium pekinense PD-67 mutant with aromatic amino acid transport system gene(aroP) in-frame deletion was constructed to decrease the uptake of L-tryptophan and reduce the intracellular pool of L-tryptophan，further to deregulate the feedback regulation of L-tryptophan and increase the extracellular accumulation． The effects of aroP knock-out as well as anthranilate synthetase (EC4. 1. 3. 27; AS) gene overexpression on L-tryptophan accumulation of the mutant were investigated． ［Methods］The aroP gene was cloned from C． pekinense PD-67 chromosome and ligated to integration vector，and then deleted about 600bp fragment by restriction endonuclease digestion． The mutant C．pekinense PD-67-ΔaroP was screened by homologous recombination． The mutant phenotype can be reversed by complementation with aroP gene from the expression vector． AS gene was cloned and ligated to expression vector to construct a recombinant plasmid． The plasmid was transformed into PD-67ΔaroP to generate the engineering strain PD-67ΔaroP/pXAS．The fermentation characteristics of the mutant and the engineering strain were investigated． ［Results］The aroP gene in-frame deletion was screened and confirmed by PCR analysis and the AS gene expression was confirmed by determination of enzyme activity． The aroP knock-out resulted in increase of L-tryptophan accumulation by 65% compared with that of the parent strain，while the expression of AS gene resulted in increase of L-tryptophan yield on cell mass by 25. 6% in engineered strain． ［Conclusion］The aroP gene knock-out of the strain PD-67 improved L-tryptophan accumulation． The expression of AS gene could further improve L-tryptophan yield on cell mass in engineered strain．

Abstract:Abstract:［Objective］ A sigma factor is an important component of RNA polymerase complex and is essential for initiation of RNA synthesis． The sigma factors fall into 2 categories: primary sigma factor is essential for bacterial growth and the alterative sigma factor is activated under different environmental conditions． Sigma F (SigF) is one of the sigma factors of Mycobacterium tuberculosis，affecting its virulence and pathogenesis． In contrast，the ortholog of the nonvirulent，fast growing strain Mycobacterium smegmatis has been suggested without similar physiology roles． Here，we studied the functions of M． smegmatis SigF． ［Methods］sigF knockout Mycobacterium smegmatis strain was constructed by specialized transduction． The wild type，knockout and complementary stains were challenged by oxidative stress and antibiotics． ［Results］The knockout sigF stain was susceptible oxidative stress，compared to wild type． Furthermore，there was no defect in resistance to antibiotics including isoniazid between the knockout sigF strain and wild type strain． In addition，SigF is required for carotenoid pigment production in M． smegmatis． ［Conclusion］ Our data suggested that SigF is important to detoxify the reactive oxygen species，probably through photo-oxidative stress response pathway，which is independent on the pathway that is required for the isoniazid activation.

Abstract:Abstract:［Objective］There are a large number of tandem repeats in FLO1，which are highly dynamic components in genome leading to the unstable flocculation profiles in Saccharomyces cerevisiae． The effects of complete or partial deletion of repeated DNA sequence A in FLO1 on the flocculation characteristics and genetic stability in yeast were studied to provide theoretical guide for construction genetically stable flocculation gene with minimal size． ［Methods］ We constructed the derived gene FLO1a with complete deletion of repeated DNA sequence A in the central domain by fusion PCR，and isolated the derived genes FLO1a1 － FLO1a5 with partial deletion of repeated DNA sequence A at different sites using E．coli DH5α carrying the FLO1 gene as selective model． We analyzed the physiological characteristics and genetic stability of flocculation in yeast strains YSF1，YSF1a，and YSF1a1 － YSF1a5 containing FLO1，FLO1a and FLO1a1－FLO1a5 respectively． ［Results］ No obvious flocculation was observed for yeast strain YSF1a，but various levels of flocculation were observed for strains YSF1a1 － YSF1a5． Flocculation of YSF1a3，YSF1a4 and YSF1a5 were more tolerant to environmental changes than that of strain YSF1，and displayed more genetic stability． ［Conclusion］Repeated DNA sequence A is important for the function of flocculation protein．

Abstract:Abstract:［Objective］In order to investigate the regulatory mechanisms of Mortierella alpina desaturase genes by low temperature and exogenous unsaturated fatty acids (UFAs) at the transcriptional level．［Methods］We performed timecourse studies of fatty acid desaturase gene expression by real-time PCR and determined fatty acid desaturase gene promoter activity using promoter-reporter constructs． ［Results］Relative expression results in real-time PCR showed that the mRNA levels of three fatty acid desaturase genes (Fad6，Fad12 and Fad3) were rapidly and transiently enhanced after 1 h of shifting to low temperature，in contrast，high concentration of exogenous oleic acid (OA) which was a monounsaturated fatty acid containing a D9 double bond suppressed the transcription of these genes and the transcriptional response appeared to be rapid and transient． Also，there was no absolute correlation between mRNA abundance and production of corresponding fatty acids． The pFAD6 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by addition of UFAs to the media，and α-linolenic acid (ALA) containing three double bonds appeared to have a more effective inhibition than linoleic acid (LA) and OA．［Conclusion］These results indicate that there may be post-transcriptional control and other modes of regulation of UFAs synthesis in M． alpina when facing different stimuli such as low temperature and exogenous unsaturated fatty acids besides the regulation in the transcription of fatty acid desaturase genes at the initial stage． Also，there may be an unknown endproduct (changes in fatty acid compositions) feedback regulation in the transcription of fatty acid desaturase genes to maintain cellular UFAs' homeostasis． In a word，we assessed mechanisms of transcriptional regulation in M． alpina fatty acid desaturase gene expression for the first time and we wish to make it possible to obtain a better understanding of the mechanisms and get some theoretical knowledge to offer some guidance to the industrial production of UFAs by transgenic technology and microbial fermentation technology．

Abstract:Abstract:［Objective］In order to reduce CO2 emission and lower the cost of microalgal industrial production at the same time，a technology was developed to combine CO2 sequestration with microalgal cultivation． ［Methods］The technology was based on rapid pH drift and high pH adaptability of two microalgal species，Chlorococcum alkaliphilus MC-1 and Spirulina platensis． A simple structure，CO2 leakage prevention covering-box，was designed to collect CO2 escaped from culture medium when CO2 gas was injected into the culture． ［Results］In the small-scale outdoor cultivation of MC-1，CO2 escaped from culture was all absorbed by culture with the help of covering-box． Results from the pilot-scale cultivation of S． platensis (HS 331) combined with the CO2 addition technology show that the cost of carbon source was remarkably reduced and deposition of CaCO3 and MgCO3 was effectively avoided． In addition，the average productivity was rather high ［9.54g/(m2·d1)］．In the large-scale cultivation of S． platensis，the annual yield was increased by 20% and high quality product was obtained with the CO2 addition technology． In addition，66% of NaHCO3 was saved，more than 58% of carbon cost was reduced，and about 45 tons of CO2 was sequestrated． ［Conclusion］ The technology was successfully applied in industrial production of S． platensis and the cost of producing S． platensis was dramatically reduced． The study would provide new insight into carbon dioxide sequestration．

Abstract:Abstract:［Objective］ To construct recombinant adenovirus containing canine interferon-γ (cIFN-γ) gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells (MDCK)．［Methods］The cIFN-γ gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-γ expression vector，from which the cIFN-γ expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-γ． The pAd-cIFN-γ plasmid was linearized by digestion and transfected into human embryonic kidney ( HEK) 293T cells to generate the replication-defective cIFN-γ recombinant adenovirus (Ad-cIFN-γ) ． To analyze its anti-canine parvovirus activity，the MDCK cells were pre-infected by Ad-cIFN-γrecombinant adenovirus，and then infected by canine parvovirus． The antiviral activity of the Ad-cIFN-γ recombinant adenovirus against parvovirus was analyzed． ［Results］ The recombinant adenovirus containing cIFN-γ gene was constructed by the ligation method． The recombinant adenovirus could mediates recombinant cIFN-γ secretory expression in MDCK cells． The Ad-cIFN-γ recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus． These results indicate that the Ad-cIFN-γ recombinant adenovirus has the potent antiviral activity against canine parvovirus． ［Conclusion］ The Ad-cIFN-γ recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus．

Abstract:Abstract:［Object］ Autophagy is a lysosomal degradation pathway in which eukaryotic cells dispose intracellular aggregates or defective organelles to maintain cellular homeostasis． Autophagy not only plays a key role in the growth，development，mature and differentiation of cells，but also is associated with pathogenesis，virus infection and immunity．To clarify the mechanism of Hepatitis B virus (HBV) infection and cell immune response，we investigated the relationship between autophagy and IFN factors in the HBV infected cells． ［Methods］ We inhibited the autophagy by the RNA interference knockdown of Beclin1 and Atg7，the essential autophagic genes，examined the number of autophagosomes by fluorescence microscopy and examined the expression of interferon factors by Real-Time PCR． ［Results］Autophagy was inhibited after transfected siBeclin1 or siAtg7. After inhibiting the autophagy，the expression of interferon factors were decreased，but cell apoptosis was not induced． ［Conclusion］ When the autophagy was inhibited，interferon signaling pathways were impaired in the HBV infected cells． The finding indicated that HBV induced-autophagy enhanced the interferon signaling pathways，and then increased the native immune response．

Abstract:Abstract:［Objective］ To investigate the effects of canine interleukin-2 (cIL-2) and cIL-7 genes on enhancing the immunogenicity of canine parvovirus (CPV) VP2 DNA vaccine． ［Methods］The bicistronic vectors of cIL-2 and cIL-7 genes were constructed using the eukaryotic expression vector containing internal ribosome entry site (IRES)． The cIL-2/clL-7 dicistronic vector plus previously constructed vectors，including CPV VP2 DNA vaccine vector，cIL-2 vector and cIL-7 vector，were used to co-immunize mice with different combinations，consisting of VP2 alone，VP2 + cIL-2，VP2+cIL-7 and VP2+cIL-2/cIL-7. The VP2-specific antibody levels in immunized mice were measured by ELISA at different time post-immunization． The proliferation indices and interferon-γ expression were measured by lymphocyte proliferation assay and ELISA，respectively． ［Results］The cIL-2/cIL-7 bicistronic vector was correct and could mediate cIL-2 and cIL-7 gene expression in eukaryotic cells． Immunization results revealed that the antibody titers and the neutralizing antibody levels of the mice co-immunized with VP2+cIL-7/cIL-2 vectors were significantly higher than that with either VP2+cIL-2 vectors or VP2+cIL-7 vectors (P＜0.05)．The lymphocyte proliferation indices of VP2+cIL-7/cIL-2 vector-immunized mice were also higher than that of other two groups although not statistically significant． However，the IFN-γexpression levels of VP2+cIL-7/cIL-2 vector-immunized mice were significantly higher than other immunized mice (P＜0.05)． ［Conclusion］ The cIL-2 and cIL-7 genes showed the significant synergic effects on enhancing the immunogenecity of CPV VP2 DNA vaccine．

Abstract:Abstract:［Objective］ To screen，identify bacterial strains with high capability to degrade cellulose from bacteria associated with Bursaphelenchus xylophilus and to clone related genes． ［Methods］ First，we collected B. xylophilus samples from pine wood nematode disease areas in Nanyang，Henan province，China． Then，we obtained the bacterial strains with high cellulase activities by primarily screening according to Congo red plate methods． The bacterial strain was classified by phenotypic and genotypic characteristics． We designed degenerate primes according to the known endoglucanase gene sequences in GenBank to carry out PCR，and analyzed the cloned sequence． ［Results］We obtained seven bacterial strains with high cellulase activities． Among them，the bacterial strain numbered C8 showed the highest cellulase activities． The bacterium was classified to be Enterobacter genus． The full length of a cellulase gene cDNA (1104 bp) (GenBank JQ845065) coding region was successfully cloned． The homogeneous analysis demonstrated that the deduced nucleotide and amino acid of the gene separately shared 97% and 92% with the cellulase from E． aerogenes KCTC 2190，and 82% with the endo-1，4-D-glucanase gene from Klebsiella pneumoniae，and 82% with the a cellulase gene from unculturable bacteria． ［Conclusion］It was a novel cellulose gene cloned from B． xylophilus associated bacteria．

Abstract:Abstract:［Objective］Riboflavin，called generally vitamin B12，is the precursor of cofactor flavin adenine dinucleotide (FAD) and flavin mononucleotide，which plays crucial roles in the biosynthesis of organisms． Bacteria need to synthesize riboflavin to maintain their survival and proliferation if they cannot obtain flavin from the surroundings．3，4-Dihydroxy-2-butanone-4-phosphate synthase (DHBPs) is one of the key enzymes in biosynthesis of riboflavin． In the presence of Mg2+，DHBPs can degrade ribulose-5-phosphate (Ru5P) into formate and 3，4-dihydroxy-2-butanone-4-phosphate (DHBP)． Potentially，these enzymes related to biosynthesis and metabolism of riboflavin，including DHBPs，may serve as the target for new antibacterial drug． In order to determine the three-dimensional structure and screen small molecule of inhibitor of DHBPs，we expressed and purified DHBPs from Streptococcus pneumonia (S.pn)，and characterized the activity of this enzyme． ［Method］DHBPs gene was amplified by PCR，and over-expression plasmid pW28-DHBPs was constructed． pW28-DHBPs was transformed into Escherichia coli BL21 (DE3) to express DHBPs; the recombinant protein was purified by nickel column and ion-exchange column． The enzymatic activity was tested using spectroscopy． ［Results］The plasmid of pW28-DHBPs was verified by restrictive enzyme digestion and sequencing． Soluble DHBPs was expressed in E． coli BL21，and purified with 95% purity． The result of size exclusion chromatography indicates that DHBPs was dimer in solution． Additionally，the recombinant protein has activity to hydrolyze Ru5P into formate and DHBP in the conditions of pH 7. 5 and 25℃ and in the presence of Mg2+．［Conclusions］DHBPs could be highly expressed in soluble form in E． coli BL21 strain，and the recombinant protein has activity to hydrolyze Ru5P．

Abstract:Abstract:［Objective］We studied the physiological，biochemical properties and metabolism of Enterococcus strain CJ-1 from high-altitude soil in Namtso，Tibet． ［Methods］Strain CJ-1T was isolated from the soil of Namtso alpine meadow soil by Hungate anaerobic technique． Through physiological，biochemical and phylogenetic analysis，we identified the strain CJ-1. ［Results］Strain CJ-1 was Gram-positive and facultative anaerobe，1－1.5μm in diameter． CJ-1T was atrichia nonmotile cocci，and always occurred in pairs． CJ-1T occurred in the presence of 0%－7% NaCl (optimum at 5%)，pH 5.0－8.5 (optimum 7.0) and temperature between 10℃ and 50℃ (optimum at 25℃)．CJ-1 could metabolize many carbon sources including cellobiose，melezitose and ribose． Metabolites of cellobiose were lactic acid，acetic acid，propionic acid，butyrate，CO2，and little H2 ． The mol% G + C content of the genomic DNA was 39.2 mol%．16S rRNA gene sequence analysis of the above strain showed the highest similarity of 95. 9% with its closest phylogenetic neighbor Enterococcus aquimarinus． The strain can degrade cellobiose which act as intermediate metabolites in the methane fermentation process． ［Conclusion］CJ-1T can degrade cellobiose． CJ-1T represents a novel species in the genus Enterococcus．