Abstract:Abstract: Anaerobic ammonium oxidation (Anammox) is a microbial oxidation process of ammonium, with nitrite as the electron acceptor and dinitrogen gas as the main product, and is performed by a clade of deeply branched Planctomycetes, which possess an intracytoplasmic membrane-bounded organelle, the anammoxosome, for the Anammox process. The wide distribution of Anammox bacteria in different natural environments has been greatly modified the traditional view of biogeochemical cycling of nitrogen, in which microbial denitrifier is considered as the only organism to respire nitrate and nitrite to produce nitric and nitrous oxides, and eventually nitrogen gas. More evidences indicate that Anammox is responsible for the production of more than 50% of oceanic N2 and plays an important role in global nitrogen cycling. Moreover, due to the close relationship between nitrogen and carbon cycling, it is anticipated that Anammox process might also affect the concentration of CO2 in the atmosphere, and influence the global climate change. In addition, the simultaneous transformation of nitrite and ammonium in wastewater treatment by Anammox would allow a 90% reduction in operational costs and provide a much more effective biotechnological process for wastewater treatment.

Abstract:Abstract: [Objective] In order to study the biodiversity of actinomycetes isolated from salt lakes in Hami, Xinjiang, and the characteristics of enzymes thereof[Methods] Soil samples in salt lakes Hami were isolated with 4 isolation media containing 5% and 10% NaCl (w/v) by dilution-plate method. The activities of lipase, galactosidase, amylase, esterase and cellulose from isolated strains were qualitatively detected by using five selective media. Based on morphological characteristics, test of salt tolerance, antibacterial activitity, enzymatic characters and sequencing of 16S rRNA gene, strains were selected for phylogenetic analysis. [Results] A total of 63 actinomycetes were isolated from salt lake in Hami, of which 47 strains were halophilic actinomycetes. The antibacterial activity results showed that 23 strains had antibacterial activity toward Bacillus subtilis and other pathogens. Three strains produced proteinase, 46 strains produced amylase, 14 strains produced esterase, 34 strains produced galactosidase, and 5 strains produced cellulase. Analysis of 16S rRNA gene sequence indicated relatively rich genotypic diversity among these actinomycetes. [Conclusion] There were abundant actinomycetes resources in the salt lakes in Hami, Xinjiang. The strains had very promising enzyme activities.

Abstract:Abstract: The soluble methane monooxygenase (sMMO) from Methylosinus trichosporium IMV 3011 catalyzes the conversion of methane to methanol. [Objective] To identify the novel species Methylosinus trichosporium IMV 3011 and to describe its evolution status. [Methods]With the aid of the information from GenBank, we designed several sets of primers for PCR amplification and sequencing, the 16S rDNA and complete of genes sequence for soluble methane monooxygenases were gene sequenced and analyzed with biology software. [Results] We obtained a 5319 bp of full-length DNA of soluble methane monooxygenases and a 1290 bp of 16S rDNA. Software analysis for six open reading frames and the deduced amino acid sequences of soluble methane monooxygenases has shown that 99.0% to 82.7% identity to the counterpart of Methylosinus trichosporium OB3b, 99.4% to 81.8% identity and 99.8% to 89.2% similarity to the predicted amino acid of mmoX genes in compared five strains. The multiple alignments of MMOX amino acid residues reveal that there is high conservation in MMOX, especially in two Fe binding regions. [Conclusion] These results indicated that strain IMV 3011 should be a true member of Methylosinus trichosporium, and it is closer to the species Methylosinus trichosporium OB3b.

Abstract:Abstract：[Objective]The purpose of this research is trying to uncover the homology between two velogenic genotype III Newcastle disease virus (NDV) isolates with NDV strain Mukteswar, which was commonly used as vaccine in China. [Methods]The full-length genome of NDV isolates, JS/7/05/Ch and JS/9/05/Go, were determined by RT-PCR and then analyzed. [Results]The full-length genome of 2 genotype III velogenic NDV isolates shared 99.7% nucleotide identity with that of Mukteswar. The results of alignment of 6 viral genes showed that JS/7/05/Ch and JS/9/05/Go shared nucleotide and amino acid identities of 99.6 %～99.9% and 98.8%～99.8% with that of Mukteswar, respectively. Furthermore, the IVPI score of JS/7/05/Ch and JS/9/05/Go was 2.18 and 1.33, remarkablely higher than that of Mukteswar, while the 3 NDV strains shared the consensus cleavage site of virulent NDV strains (112RRQRRF117). Virulence of NDV is mainly determined by the amino acid sequence of the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of lentogenic strains. However, 3 NDV strains with same F protein cleavage site showed different virulence. The entire genomic sequence of JS/7/05/Ch, JS/9/05/Go and Mukteswar was further analyzed. Three strains shared some gene-start signal, gene-end signal, intergenic region and six highly identical viral genes. Most amino acid differences among JS/7/05/Ch, JS/9/05/Go and Mukteswar were found in the predicted HN and L protein, and the predicted NP, P, V, W, M and even F protein had few amino acid differences. [Conclusion]First, it is concluded that field isolates JS/7/05/Ch and JS/9/05/Go are derived from Mukteswar and more attention must be paid to the virulence enhancement of vaccine strains. Second, it is proposed that the differences in the amino acid sequence of HN and L protein may give rise to the significant virulence differences between two NDV isolates and Mukteswar.

Abstract:Abstract: [Objective] A strain of highly pathogenic duck hepatitis virus was isolated in south China from a Pekin duck flock in 1999. No cross reaction with type 1 and 3 duck hepatitis virus was found by serum neutralization. We suggested the strain should be classified as a new serotype of duck hepatitis virus and named it as DHV-N G strain in our previous study. We wanted to reveal the evolution of this virus in molecular level and gene sequence differences between it and DHV-1 strains.[Methods]We used RT-PCR and 5'-/3'-RACE to amplify the complete genome sequence of DHV-N G strain and compared it with other picornaviruses [Results]The genome of DHV-N G consists of 7774 nucleotides excluding a poly(A) tail of 12 nucleotides. It contains a single large open reading frame encoding a polypeptide of 2251 amino acid residues. The length of 3'UTR of DHV-N G is 366 nucleotides, which is 52 nucleotides longer than that of DHV-1 C80 strain. Significant amino acid variation was found in the protein VP1, especially at the position of 140-221 comparing with DHV-1. The DHV-N G genome shares 72.8%-73.4%, 96.3%-96.5% and 78.3% similarity with DHV-1 strains, Korea’s and Taiwanese DHV-N strains, respectively. [Conclusion]The genome structure of DHV-N G strain is obviously different with that of DHV-1 strains. The homologies of genome among the DHV-N group are variable, therein DHV-N G strain is more homologous with Korea’s strains than with Taiwanese.

Abstract:Abstract: [Objective] Xylanase is ahemicellulase. It can reduce xylan, which is the abundance resource on our earth. It is important in industries to obtain recombinant strains secreting xylanase to degrade hemicelluloses and produce xylose as desired products. We constructed an integrative vector of Candia utilis for xylanase expression. [Methods] On the basis of plasmid pBR322, an integrative expression vector pGLR9K for Candida utilis was constructed that contained strong promoter of GAP, 18S rDNA sequence for homologous recombination and mutated L41 gene as CYH resistant marker. Recombinant expression vector pGLXR was constructed by adding xylanase gene to the pGLR9K and then was transferred into C. utilis. [Results] Both intracellular and extracellular xylanase activities were detected in transgenic strains. [Conclusion] This system can be used for exogenous genes expression in transgenic C. utilis.

Abstract:Abstract：【Objective】In order to systematically investigate the cry gene resources from Bacillus thuringiensis (Bt) in different ecological regions in Sichuan Basin and further clone novel cry genes.【Methods】We used the methods of microscopic and scanning electron microscopic observation, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and test of insecticidal activities to research Bt strains collected in this basin.【Results】We screened 791 Bt isolates from 2650 soil samples, and found cry1, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes in this basin. Strains containing cry1 genes were the most abundant in our collection (66%), and 21 different cry1-type gene combinations were found. Furthermore, several novel holotype cry genes were found and the full-length sequences of 3 novel cry genes were designated as cry54Aa1, cry30Fa1, and cry30Ga1 by B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee. The results of insecticidal activities showed that Sichuan Basin harbored Bt isolates with insecticidal activities. SDS-PAGE assay of 80 strains without PCR products indicated that these strains may harbor potentially novel cry proteins gene.【Conclusion】The diversity and particularity of cry gene resources in Sichuan Basin have important meanings in theories and practices.

Abstract:Abstract: [Objective] To provided a reliable and sensitive method and a series of specific absorption spectra data of different carotenoids and bacteriochlorophylla in anxoygenic phototrophic bacteria, and reveal mechanism of regulation of photosynthetic pigments by light and oxygen in Rhodobacter azotoformans 134K20. [Methods] The metabolic diversity of photopigments regulated by oxygen and light was investigated by means of UV-VIS spectra and thin layer chromatography. [Results] The highest cell yield of strain 134K20 was obtained under aerobic conditions in the light. Nine types of photopigments including three yellow pigments, one red pigment, one purple pigment, two green pigments, and two blue pigments were synthesized, and yellow pigments synthetic genes were expressed on the higher level anaerobically in the light. Under aerobic conditions, the synthetic genes of two new red and one new purple pigment were triggered and expressed on the higher level, but the biosynthesis of yellow, blue and green pigments were inhibited by oxygen, another nine pigments including two yellow pigments, three red pigments, two purple pigments, one green pigment, and one blue pigment were synthesized. One yellow pigment was only produced in dark aerobic culture, the other pigments were the same as in dark aerobic cultures. [Conclusion] PpsR photopigment suppression regulation system regulated expression of photosynthetic genes via light and oxygen in Rhodobacter azotoformans 134K20. The yellow and red pigments belong to carotenoid series. The first yellow pigment belongs to spheroidene series. The other two yellow pigments are new carotenoids. Yellow pigments are capable of dissolving in different organic solvents. Red pigments belong to new spheroidenone series. The three red pigments are different in polarity, peak shape and peak value. Fine structures of red pigments only are appeared in hexane. The purple pigmments with polarity are identified as bacteriopheophytins. The blue and green pigments are four kinds of bacteriochlorophyll a intermediates. Diethylether and methanol is suitable for carotenoids extraction. The identification of bacteriochlorophyll a intermediates can be easily performed by polarity analysis.

Abstract:Abstract: The ability of iron acquisition in Candida albicans has an effect on its growth and pathogenesis. Ferric reductases are important components of high affinity iron acquisition system in C. albicans. [Objective] The aim of this study was to elucidate the function of FRP1 (Ferric reductase protein). [Methods] FRP1 gene expression was detected under low-iron and high-iron conditions by Northern blot. We used the method of PCR-directed gene disruption to construct frp1 null mutant and then the phenotypes of frp1Δ/Δ mutant were characterized. [Results] Low-iron condition induced FRP1 gene expression. frp1Δ/Δ mutant showed no growth on low-iron media compared to wild-type strains on solid plates. [Conclusion] FRP1 protein is probably the main ferric reductase under low-iron condition.

Abstract:Abstract：[Objective] To reveal the diversity of marine bacteria in disparate sites of the Northern Yeallow Sea. [Methods] Bacterial community structure and diversity within seawaters and sediments at two stations in the Northern Yellow Sea were investigated and assessed by the 16S rDNA clone library and the denaturing gradient gel electrophoresis (DGGE) techniques. [Results] The 16S rDNA clone library analysis indicated that the bacteria were abundant in the waters and sediments, and most of them were unknown. Phylogenetic analysis showed that the proteobacteria were the dominant group both in the sediments and waters, γ-proteobacteria and δ-proteobacteria were dominant in the sediments, α-proteobacteria were dominant in the water. However, all subphyla of proteobacteria presented phylogenetical divergence at the two sites. The clustering analysis on the DGGE patterns revealed that the dominant groups within the waters and sediments in shore were similar to each other, whereas that in the offshore were different. [Conclusion] The microbial diversity differed due to the geographical location and living medium; and the microbial distribution depended on the environment factors.

Abstract:Abstract: [Objective] Purified avian influenza virus (AIV) hemagglutinin (HA) gene fragment was inserted into green fluorescent protein (GFP) expression vector, pEGFP-C1. The role of signal peptide in the HA-GFP expression in 293T cells was investigated by cutting the signal peptide or placing it in different locations of HA-GFP fusion gene. [Methods] The expression of GFP in 293T cells was examined directly with fluorescence microscope and flow cytometry analysis. [Results] After transfected with pEGFP-C1, M1, M2 and M3 plasmid, under fluorescent microscope the HA-GFP fusion protein was successfully expressed. Fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C1, whereas signal peptide could obviously reduced the expression of GFP-HA compared with recombinant plasmid without signal peptide. In addition, we found that there was no significant effect of the location of signal peptide on the expression of HA-GFP fusion protein.

Abstract:Abstract: [Objective] To improve the immune response to HBsAg DNA vaccine and clear HBV, we investigated co-stimulatory molecule OX40L as adjuvant effect on the humoral and cellular immune responses to HBV DNA vaccine by immunizing mice with HBV DNA vaccine plus OX40L. [Methods] We immunized the C57BL/6 mice with pcDS2 alone, or with OX40L and candidate DNA vaccine against HBV (pcDS2) together by intramuscular injection. The immunization was performed on week 0, 2, 4. The concentration of the anti-HBs (IgG) and isotypes (IgG1, IgG2a), the stimulated index of T lymphocyte proliferation, and the expression of IL-4 and IFN- in CD4+ T cell and IFN- in CD8+ T cell, specific in vivo cytotoxic T lymphocyte (CTL) activity were detected at week 6. [Results] The concentration of the anti-HBs IgG induced by pcDS2 plus OX40L groups was much higher than that induce by pcDS2 alone, and the levels of IgG isotype of IgG2a were generally higher than IgG1 in all groups of mice immunized with different plasmids. Compared to mice immunized with pcDS2 alone, the pcDS2 plus OX40L group increased the stimulated index (SI) of T cell proliferation and elicited a higher level of IFN- and IL-4 in CD4+ T cells and a higher level of IFN- in CD8+ T cells. In all groups, OX40L plus pcDS2 induced significantly robust in vivo CTL response. [Conclusion] The co-immunization of OX40L and HBV DNA vaccine can enhance the humoral and cellular immune responses, especially CTL activity.

Abstract:Abstract：[Objective] and [Methods] Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis in fish. V. anguillarum strain VIB72 was defined as having high virulence whereas strain CW1 was defined as having low virulence on the basis of their different LD50 values to zebra fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. [Results] After screening, 59 subtracted library clones were isolated which were specific for strain VIB72, and the DNA sequences of these clones were determined. Seventeen fragments showed high homology to the genes of known functions in other bacteria. This includes soluble lytic murein transglycosylase, mobilization protein (MobA, MobC), transposase (IS66), resistance-related protein (metallo-beta-lactamase and acetyltransferase family), toxin protein (DT-201 and alveicin A immunity protein), ATP-dependent endonuclease of OLD family like protein, SocE and GTP-binding protein HflX (high frequency of lysogenization). These fragments may represent parts of putative pathogenicity islands (PAIs) in V. anguillarum. The remaining fragments showed no significant homology to any known genes. [Conclusion] The results indicated that SSH was successful in identifying genetic differences and putative virulence genes among different strains of V. anguillarum.

Abstract:Abstract: [Objective] To develop a rapid detection method for Salmonella in food by using specific Salmonella-phage O-I. [Methods] One hundred bacteria strains and 120 food sample isolates were infected using fluorescently labeled O-I phage genome with SYBR? gold stain (a nucleic acid dye, 1×working solution), then were observed under epi-fluorescence microscopy. The sensitivity of the method was tested. [Results] Among the 100 strains infected with O-I/ SYBR? gold stain, 40 Salmonella strains exhibited rod fluorescence. Other bacteria including 10 Proteus, 20 Shigella, 20 E. coli and 10 Staphylococcus did not exhibit this feature The sensitivity of detecting Salmonella was 10 CFU/100 μL. The detection for 120 food samples by using the O-I/ SYBR? gold stain had similar results to those by using the biochemical method. [Conclusion] Fluorescent-labeled O-I phage could rapidly, sensitively and specifically detect Salmonella species in food samples.

Abstract:Abstract: [Objective] A loop-mediated isothermal amplification (LAMP) technology with two loop primers was developed for rapidly detecting Enterobacter sakazakii in powdered infant formula. [Methods] Sequences of 16S-23S rRNA of Enterobacter sakazakii (ATCC29544) were used as target sequences, to design outer primers, inner primers and loop primers. We judged the results of detection, through visible to the naked eye of the white precipitate. [Results]The sensitivity of the LAMP assay was 0.101 CFU/mL; the detection limit of artificial contamination was 1.1 CFU/g. The detection could be finished about an hour from dealing with the sample to report the results with DNA kit. Compared with LAMP, the sensitivity of the PCR assay was 101 CFU/mL and the detection limit of artificial contamination was 1100 CFU/g in three hours. To apply the same method of extracting DNA, from dealing with the sample to report the results, it took about three hours. [Conclusion] Therefore, this LAMP-based assay is sensitive and fast. These results indicate that LAMP can provide a rapid yet simple test for the detection of Enterobacter sakazakii in powdered infant formula.

Abstract:Abstract: 【Objective】To study the diversity of lactic acid bacteria (LAB) and dominant LAB in fermented cabbage. 【Methods】 Culture-dependent and –independent (16S rRNA gene clone libraries were constructed) methods were used to determine the composition of LAB in fermented cabbage. 【Results】 Ninety LAB isolated from fermented cabbage were identified as species of Lactobacillus and Leuconostoc, whereas 115 clones of the 16S rRNA gene sequence from fermented cabbage DNA were identified as Lactobacillus, Weissella, Pediococcus and Leuconostoc. 【Conclusion】The significant difference of the LAB compositions by the two methods implies that some specialized nutrients may lead to a distinctive selection of the dominant organisms. Lactobacillus plantarum appeared as the dominant species in fermented cabbage by both methods.

Abstract:Abstract：【Objective】Willow Yellow is a plant disease caused by phytoplasma. We identified the taxonomic status of Willow Yellow phytoplasma(WY). 【Methods】We used specific conservative primers amplified 16S rDNA and ribosomal proteins gene(rp)gene. Obtained sequences were then analyzed and homology trees were constructed. The nested - PCR products were also analyzed by restriction fragment length polymorphism (RFLP). 【Results】We obtained 16S rDNA of 1246 bp and rp gene of 1212 bp, and sequence analysis showed a high identity with members of the 16SrI-C group phytoplasmas, all above 99. 0%, and had the highest identity with Wheat Blue Dwarf phytoplasmas, 99.8 % (16S rDNA) and 99.6 % (rp) respectively, RFLP pattern are also the same as 16SrI-C phytoplsama.【Conclusion】Willow yellow phytoplasma is a member of 16SrI-C.

Abstract:Abstract: [Objective] We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria that produced ACC (1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why they can promote the growth of plants. [Methods] Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity. [Results] In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99 %. Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/μg. [Conclusion] A strain producing ACC deaminase was identified and tested.

Abstract:Abstract: [Objective]To study the source and trend in evolution of ALV-J strains from layers in China. [Methods]The genomic DNA extracted from chicken embryo fibroblasts (CEF) infected by ALV-J strain SD07LK1 isolated from layers was used as template to amplify the ALV-J proviral DNA by PCR. Nine continuous and overlapping fragments were amplified with nine pairs of primers according to published sequences, then cloned into the T vector and sequenced. [Results]The complete sequence of the whole genome of ALV-J Chinese strain SD07LK1 was first established. [Conclusion]Comparisons of SD07LK1 sequence with that of the other Avian leukosis viruse strains, by using DNAstar software, demonstrated that the genes gag and pol of ALV-J were relatively conservative, the nucleotide identity of all the strains was over 95.0%. However, the gene env identity was only in the ranges between 88.6 and 94.0%.

Abstract:Abstract: [Objective] The wide application of live attenuated vaccine strains is limited because of drawbacks of residual virulence, similar antigenenicity to virulent strain and the difficulty to differentiate vaccination and natural infection. In this study, we modified the vaccine strain to prevent the drawbacks. [Methods] By using homologous recombination, we replaced the BP26 gene by the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5ΔBP26, was generated. The wild type strain and M5ΔBP26 were used to infect macrophage and mice to compare their intracellular survival capability. According to the conservative sequence of dnaK and the deleted region of BP26, primers were designed to develop a duplex PCR for discriminating the wild type strain and M5ΔBP26. [Results] A new tagged strain, M5ΔBP26, was successfully constructed. The tagged strain could survive in both macrophage and mice, indicating the feasibility as live attenuated vaccine strain. Results from mice infection showed that, at 2 weeks p.i., 102.9 CFU of Brucella were isolated from M5 infected mice, whereas only 101.1 CFU of Brucella were isolated from M5ΔBP26 infected mice (P<0.01). At 3 weeks p.i., 102.2 CFU of Brucella whereas no M5ΔBP26 were isolated. These results indicated that infection capability of M5ΔBP26 was decreased. Based on the sequence differences between M5ΔBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5ΔBP26, by which it can be differentiated from wild type and virulent strains. [Conclusion] The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.