Abstract:[Objective] We studied the influence of solid-state and submerged cultures on the extracellular proteome of filamentous fungus Rhizopus chinensis CCTCC M201021, to understand the specificity of enzyme production by filamentous fungi in different cultures. [Methods] Using same media, we carried out solid-state agar-plate culture and submerged culture of R. chinensis. Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS were used to analyze the extracted extracellular proteins, and the differential proteins were identified. [Results] Protease activity of extracellular protein from the solid-state culture was much higher (9.2-fold) than that from submerged culture. The 2-DE gel maps indicate the differences in the extracellular proteomes between solid-state and submerged cultures, and most proteins (about 70%) were special from solid-state or submerged culture. After identification, the variety and expression level of extracellular proteins from the two different cultures were significantly different. Among these differential proteins, hydrolases were the majority, most of which were related with protein degradation. [Conclusion] Different cultures influenced the composition of extracellular proteome by R. chinensis, and some proteins were only expressed in a specific culture. Relatively more variety and higher expression level of some proteases under solid-state culture could be the reason, which resulted in the much higher protease activity of extracellular protein. These results suggested the possible inconsistencies of extracellular enzymes produced by filamentous fungi between the strain screening and submerged fermentation in industry production, when solid-state culture and submerged culture were used.

Abstract:[Objective] Effect of mycoplasma on gene function was studied in vitro. [Methods] FIGNL1, a critical gene to repair DNA double-strand breaks, was suppressed by siRNA in H446 and H1688 cells infected with or without Mycoplasma hyorhinis or after mycoplasma removal. Expression of targeted gene and cell cycle analysis was measured using real-time PCR, flow cytometry and other methods. [Results] No significant effect of M. hyorhinis was observed on the suppression of FIGNL1 expression by siRNA. After expression of FIGNL1 was suppressed in H1688 and H446 cells without Mycoplasma infection or after Mycoplasma removal, no significant change of cells proportion in S phase was observed between experimental group targeting FIGNL1 (T1) and negative control group compared with the blank group containing transfection reagent (mock). However, cell proportion in S phase of experimental and negative control group was increased approximately 1.38 and 0.51 folds, respectively comparing with blank group in H1688 cells infected with M. hyorhinis, 1.27 and 0.55 folds respectively in H446 cells infected with M. hyorhinis. [Conclusion] When the expression of FIGNL1 was suppressed in H1688 and H446 cells, M. hyorhinis can significantly induce S phase arrest because Mycoplasma can cause host cells DNA damage and FIGNL1 is a critical gene to repair DNA double-strand breaks. Mycoplasma is widespread and significantly influential on cells, we should be highly alert to it on function study of gene and tumor research.

Abstract:[Objective] Zygosaccharomyces bailii is the dominant strain in the brewing process of Maotai-flavor liquor. To explore the contributions Z. bailii made to the brewing procedure, its physiological characteristics and interaction with other functional strains in the brewing environments was studied. [Methods] One excellent phenotype Z. bailii strain was filtered from the fermented grains of Maotai-flavor liquor, and then comparing its physiological characteristic, metabolic function with ATCC 58445 and studying the combinated fermentation with Maotai-flavor characteristics bacteria Bacillus licheniformis. [Results] Z. bailii 15 with excellent phenotype from fermented grains was obtained, which could tolerate pH 2.0, 37 °C, 8% alcohol concentration (V/V) and more suitable for the brewing environment when comparing with ATCC 58445. Its ethanol production (33.58 g/L) was even to the Saccharomyces cerevisiae MT1 (34.29 g/L), which was far higher than the type strain (19.04 g/L). It can specificly produce 3,7,11-Trimethyl-2,6,10- Dodecatrien-1-ol, 1-Dodecanol, 2-Nonanol, 2-Ethylhexanol, Decanoic acid, Lauric acid, Octanoic acid, Ethyl octanoate, Acetophenone, 4-Tert-butylphenol. In the co-culture system, B. licheniformis inhibits the growth of Z. bailii at 37 °C, but has little effect on 30 °C. In addition, MT6 promotes the conversion of ethanol produced by Z. bailii 15, it can also affects the species and content of flavor greatly. [Conclusion] Z. bailii shows a significant performance in ethanol production and flavor production, which made it have a great contribution to the Maotai-flavor liquor production.

Abstract:[Objective] To achieve the efficient heterogenous expression of a bacterial maltogenic amylase in Bacillus subtilis and investigate the characterization of the recombinant enzyme. [Methods] We cloned the genes of xylose isomerase promoter region and its regulatory protein from Bacillus megatherium into an Escherichia coli / Bacillus sp. shuttle expression vector, and constructed an recombinant expression plasmid which harbored an encoding gene of maltogenic amylase from Bacillus licheniformis. Then we transformed the expression plasmid into Bacillus subtilis and optimized the induction conditions of the transformant to increase the production of the recombinant enzyme. [Results] The recombinant Bacillus subtilis which inductively expressed the maltogenic amylase was obtained. The optimal induction condition was as follow, that 1% inducer was added into fermentation medium after 9 h-incubation at 45 °C. The recombinant maltogenic amylase had a molecular size of 67 kD. From the study of the enzymatic properties of the enzyme, it was found that, with soluble starch as substrate, the reaction products were 60.42% maltose and few glucose. Furthermore, the recombinant enzyme showed an optimal activity at 45 °C with pH 6.5. Ca2+、Co2+ and EDTA could improve the efficiency of enzyme reaction. [Conclusion] With induction of xylose, the bacterial maltogenic amylase was efficiently expressed in the recombinant Bacillus subtilis, and the maximal yield reached 296.64 U/mL, which showed good application prospect in industry.

Abstract:[Objective] To evaluate the antibacterial activity of essential oils against Vibrio scophthalmi HZ-C1 isolated from farmed turbot Scophthalmus maxius in vitro and in vivo. [Methods] The paper disc diffusion method and microbroth dilution technique were employed to determine the antibacterial activity of 14 essential oils. Cell membrane disruption induced by Litsea cubeba oil was examined by transmission electron microscopy (TEM), and by measuring the release of intracellular lactate dehydrogenase (LDH) and 260 nm absorbing material. Meanwhile, antibacterial activity of Litsea cubeba oil in vivo was investigated using a turbot model of vibriosis by intraperitoneal injection. [Results] The 14 essential oils showed different antibacterial activities against V. scophthalmi HZ-C1, and cinnamaldehyde had the highest activity with a minimum inhibitory concentration (MIC) of 0.25 μL/mL, followed by thymol, eugenol, citral and Litsea cubeba oil with a MIC of 0.5 μL/mL. Litsea cubeba oil can disrupt the plasma membrane of V. scophthalmi HZ-C1 and cause the leakage of protease and nucleic acid. After immersing in a bath containing 200 μL/L Litsea cubeba oil, the mortality of turbots infected by V. scophthalmi was reduced from 50% to 0. [Conclusion] Essential oils rich in aromatic aldehyde, phenolic compounds and citral show high antibacterial activities against V. scophthalmi HZ-C1, and thus have the potential to replace the use of antibiotics for treating vibriosis in farmed turbot.

Abstract:[Objective] to improve acrylonitrile biodegradation efficiency, we studied acrylonitrile degradation characteristics of a nitrile-degrading bacterium Rhodococus rhodochrous BX2 isolated by our laboratory. [Methods] Single factor test and response surface methodology were used to optimize the biodegradation conditions. At the same time, the effects of various additional carbon and nitrogen resources on cell growth and acrylonitrile degradation were studied, as well as cell growth and degradation rate of strain BX2 in acrylonitrile synthetic wastewater. [Results] The maximum biodegradation rate (95.1%) was obtained under optimal degradation conditions (substrate concentration 403.51 mg/L, pH 7.44, temperature 34.46 °C). The cell growth and degradation effectiveness can be obviously improved by addition of carbon sources (glucose) or nitrogen source (ammonium chloride). The degradation rate of acrylonitrile in acrylonitrile synthetic wastewater reached 89.4% after 30 h. [Conclusion] the acrylonitrile biodegradation efficiency of Rhodococus rhodochrous BX2 in acrylonitrile synthetic wastewater was enhanced by optimizing biodegradation conditions and the addition of exogenous substance. This work provides technical support for the development and application of new techniques in biological treatment of acrylonitrile wastewater.

Abstract:[Objective] To obtain the carotenoid-producing yeast with potential application value, 379 strains of yeasts were isolated and screened from Fuxian Lake samples. [Methods] Carotenoids were extracted using acid-heating method, the content of carotenoids was determined by ultraviolet spectrophotometer, and the distribution features of the carotenoid-producing yeasts were analyzed by SPSS software. [Results] 318 yeast strains (83.91% of the tested strains) could produce carotenoids. The yields of carotenoid among most strains ranged from 10 to 300 μg/g, and the highest amount was up to 590.83 μg/g. These strains mainly belonged to the genera Rhodosporidium and Rhodotorula; and the carotenoid-producing abilities of basidiomycetous yeasts were higher than those of ascomycetous yeast strains. In this study, 9 strains with high carotenoid-producing abilities were obtained, namely Rhodosporidium diobovatum (3 strains), Rhodosporidium paludigenum (2 strains), Rhodotorula glutinis, Rhodotorula graminis, Sporidiobolus ruineniae, and Cystofilobasidium macerans (1 strain respectively). [Conclusion] Large numbers of the carotenoid-producing yeasts inhabited Fuxian Lake, and the “red yeasts” were higher yielding yeasts than others. The major groups of carotenoid-producing yeasts were Rhodosporidium and Rhodotorula.

Abstract:[Objective] Multidrug-resistant strains of Enterococcus faecalis are becoming increasingly common worldwide. Therefore, we aimed to find novel agents to control antibiotic-resistant E. faecalis. We isolated a lytic bacteriophage from hospital sewage. [Methods] Phage morphology was observed using transmission electron microscopy (TEM), and its genome was sequenced using the Ion Torrent sequencing platform. Genome annotation and comparative and evolutionary analyses were performed following assembly of the complete genome sequence. [Results] Lytic bacteriophage vB_EfaP_IME195 was successfully isolated from infected E. faecalis host cells. TEM analysis indicated that the isolated bacteriophage resembled members of the Podoviridae family. The complete genome of vB_EfaP_IME195 was 18 607 bp long, and was comprised of circular dsDNA (GenBank accession No. KT932700). The genome had a G+C content of 33%, and contained only 27 coding sequences. BLASTn analysis showed that vB_EfaP_IME195 shared the highest homology (82%) with E. faecalis phage vB_Efae230P-4. [Conclusion] The isolation and characterization of this novel lytic E. faecalis phage provides the basis for potential treatment alternatives for multidrug-resistant E. faecalis infection.

Abstract:[Objective] The aim of the present study was to find key factors of arginine metabolism on the stress tolerance. [Methods] Arginine metabolism was switched by argininosuccinate synthase (ASS) or argininosuccinate lyase (ASL) overexpression in L. lactis NZ9000 to enhance the stress tolerance. [Results] The recombinant strains exhibited higher growth performance, viability and fermentation performance compared with the control strain under environmental stresses. Analysis of the physiological data showed that the recombinant cells exhibited higher intracellular pH, intracellular NH4+ and ATP content, and H+-ATPase activity under acid stress, and the content of amino acid in arginine deiminase (ADI) pathway was significantly higher than the control strain. Further transcriptional analysis showed that the expression of the genes related to aspartate synthesis and arginine catabolism were up-regulated. [Conclusion] These results suggest that ASS or ASL overexpression in L. lactis NZ9000 can enhance arginine metabolism flux and up-regulated arginine metabolism flux improve the multiple-stress tolerance of cells. As arginine synthesis pathway widely exists in various kinds of microorganisms, results presented in this study provide new idea to enhance stress tolerance of tons of microorganisms especially industrial strains.

Abstract:[Objective] Our aim is to clone cDNA sequence of hepcidin gene (k-hepc) and find its expression pattern in koi (Cyprinus carpio). [Methods] In this study, the full-length cDNA sequence of hepcidin gene in koi was cloned by RT-PCR and RACE PCR and sequenced. The liver, spleen, kidney, intestine, brain, heart, muscle and gill were obtained 0, 4, 8, 12, 24 and 48 h after the koi were challenged with Aeromonas veroii and evaluated by real time PCR to find gene expression of k-hepc. [Results] The sequence of k-hepc cDNA (GenBank No. KC795559) is 755 bp in length, and ORF is 276 bp long. The deduced amino acid sequence of k-hepc gene consists of 91 amino acids including signal peptide, prodomain and mature peptide. The mature peptide contains 8 cysteines, and is able to form 4 disulfide bonds. The deduced amino acid sequence of k-hepc has 93% similarity to hepcidins of known common carp and 29%?93% similarity to hepcidins of other fish species. Expression of k-hepc is found in all the tissues tested by our lab. The relative expression levels in normal fish showed high basal values in liver and low values in gill. The expression of k-hepc mRNA was significantly increased in the liver and heart but not significantly induced in other tissues after bacterial-challenge. [Conclusion] The protein encoded by k-hepc is a member of hepcidin family. Expression of k-hepc is mainly affected by intrinsic regulation factors.

Abstract:[Objective] The aim of this study was to check the effect of water quality control by RAS (Recirculating Aquaculture System) and analyze the function of microbial community on different substrates during the period of Microbrachium rosenbergii over-wintering cultivation. [Methods] The microbial samples were collected from pond water, artificial aquatic plants which were composed of normal fiber membrane and nano fiber membrane in external biofilter after RAS being operated for 88 days. The V4 and V5 regions of 16S rRNA gene on three different substrates microbial were analyzed using DNA extraction, PCR amplification and quantification, high-throughput miseq sequencing technology. Sequence data was processed by read trimming and identification of V4?V5 sequences, followed by filtering and assigning the operation taxonomic units (OTU). Based on the OTU analyzed the microbial community diversity index and structure. Water quality was monitored every 3?4 days with the national standard method. [Results] The pond water quality maintained in a good condition that ammonia-nitrogen and nitrite-nitrogen kept at 0.17±0.08 mg/L and 0.28±0.15 mg/L, respectively. Bacteria composition and community diversity varied in three substrates and 64 species were identified which belonged to 64 genus and 9 phylum including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Nitrospirae, Acidobacteria and Chlorobi. On the level of genus, Comamonadaceae_unclassified was the most dominant genus in pond water which both appeared in the other two substrates. Inhella dominated in surface of normal fiber while Fusibacter was predominant in nano fiber membrane surface. Nano fiber membrane had the highest bacteria community diversity, followed by normal fiber membrane and then pond water. Water quality of over-wintering cultivation maintained at a relatively steady state after operating recirculating aquaculture system for 40 days. [Conclusion] It is viable to regulate and control water quality of over-wintering cultivation by using fiber membrane as a microbial substrate. With the development of new material science, it is necessary to develop a filter material suitable for aquaculture.

Abstract:[Objective] Bio-control Bacillus resource with cellulose-degrading research in special ecological environment of Qinghai-Tibetan Plateau was explored in this research. [Methods] Five Bacillus strains isolated from rhizosphere of birch in Huzhu Northern Mountain of Qinghai Province, were identified by molecular taxonomy methods including BOX-PCR fingerprints, as well as gyrB and 16S rRNA gene partial sequence analysis. Cellulose-degrading activity of these isolates were detected by CMC plate screening method, and antagonistic activity to pathogenic fungi and bacteria were checked by plate confrontation method. Furthermore, growth-promoting and stress-resistance activity of isolates including salt-resistance and low-temperature adaptability were tested. [Results] Five isolates were identified as Bacillus amyloliquefaciens and all of isolates presented distinguished cellulose-degrading activity and could form transparent zone (diameter ≥20 mm) of cellulose-degrading on CMC medium. Isolates presented distinct antagonistic activity and bio-control efficacy to Sclerotinia sclerotiorum, Fusarium oxysporum, Xanthomonas oryzae pv. oryzae and Erwinia amylovora. Strains could grow under the condition of 10 °C low-temperature as well as LB medium with 11% NaCl concentration, and bacterial cell suspension (concentration of 106 CFU/mL) of strains BS11 and BS12 could promote seed germination and seedling-growth of rice. [Conclusion] Five B. amyloliquefaciens strains own advantage with stress-resistance, cellulose-degrading and bio-control characteristic, thus possess application potential in ecological agriculture and animal husbandry field in Qinghai Province.

Abstract:[Objective] In order to study the taxonomic status and characteristic of a highly effective phosphate-solubilizing strain Y3-35 which was isolated from the saline-alkali soil. [Methods] The morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence of strain Y3-35 were analyzed. The method of halo ring was applied to isolate the phosphate-solubilizing bacteria, the method of Mo-Sb colorimetry was applied to measure phosphorus-dissolving ability. The single factor experiment and the orthogonal experiment were applied to find optimal phosphorus-dissolving condition of strain Y3-35. [Results] The result of identification revealed that phosphate-solubilizing strain Y3-35 can be classified as Pantoea aff. theicola. The phosphorus-dissolving ability of strain Y3-35 showed a negative correlation with pH. The result of the orthogonal experiment indicated that the optimal phosphorus-dissolving condition of phosphor bacteria Y3-35 was as follows: glucose 20.0 g/L, peptone 15.0 g/L, NaCl 2.5 g/L, 24 °C. The maximum phosphorus-dissolving ability could be up to 723.34 mg/L and the phosphorus-dissolving ability increased 251% than before. [Conclusion] The result indicates that Y3-35, an highly efficient phosphate-solubilizing bacteria, might act as a potential candidate for the application.

Abstract:[Objective] Ralstonia solanacearum, a devastating, soil-borne plant pathogen, causes a bacterial wilt disease in diverse plants. Studies on the metabolic mechanisms of fatty acids will facilitate the discovery of novel methods or biopesticides to efficiently control the bacterial wilt disease. [Methods] RSc2857 (RsfadD) gene was found in the genome of Ralstonia solanacearum GMI1000 through sequence alignment with Escherichia coli FadD, which was annotated to encode a fatty acyl-CoA synthetase (FACS). For complementation analysis, RsfadD gene was amplified by PCR, and was ligated into an expression vector pBAD24M, which was subsequently transferred into an E. coli fadD mutant JW1794. The growth of transformant was analyzed. RsfadD was also fused in-frame to pET-28b, and expressed in E. coli BL21(DE3). The hexahistidine-tagged RsFadD was purified by Ni-NTA, and the activity was analyzed in vitro. RsfadD deletion mutant was obtained by homologous recombination, and the mutant growth was also analyzed. [Results] RsfadD conferred the E. coli fadD mutant to grow on the minimal medium with fatty acids as the sole carbon source. In vitro enzymatic analysis demonstrated that RsFadD has FACS activity, and could utilize fatty acids of different chain lengths as substrates to form fatty acyl-CoAs. While the activity of RsFadD was lower than that of E. coli FadD. RsfadD deletion mutant grew well on nutritional medium, but grew weak on the minimal medium with fatty acids as the sole carbon source. [Conclusion] All of above suggested that RsfadD encodes a FACS, which plays an important role in fatty acids utilization. The weak growth of RsfadD deletion mutant on the minimal medium indicated other genes encoding FACS may exist in the genome. This study will contribute to further research about FACS and fatty acids utilizing mechanism in R. solanacearum.

Abstract:[Objective] To study bacterial community and diversity in pit mud with different-aged cellars used to produce Baiyunbian liquor. [Methods] Total DNA of microbes in pit mud (2 and 23 year) were extracted, then PCR-DGGE and high-throughput sequencing technology were used to analyze bacterial communities. [Results] Bacteria in the pit mud fell into 4 phyla: Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria, with dominant genera (≥1.0%) including Corynebacterium, Myroides, Sphingobacterium, Lactobacillus, Clostridium, Acetobacter, Alcaligenes, Enterobacter, and Acinetobacter. Furthermore, Dysgonomonas, Fluviicola, Proteus and Wohlfahrtiimonas were present in 2-year uniquely, whereas Staphylococcus, Hazenella, Weissella, Gluconacetobacter and Morganella were especially predominant in 23-year pit mud. [Conclusion] Bacterial diversity in pit mud of 2-year was higher than in that of 23-year.

Abstract:[Objective] To expound the necessity and importance of the storage process of Daqu, and to control the quality of Daqu, we researched the succession of bacterial community and the flavor components of Daqu. The relationships between different bacterial genera were also analyzed. [Methods] We analyzed the bacterial community structure of Daqu in different storage processes through MiSeq high-throughput sequencing technology; Co-occurrence pattern analysis method was used to analyze the relationships between different genera of bacteria; SPME-GC-MS was used to analyze the flavor compounds of Daqu in different storage processes. [Results] 3 710 OTUs were obtained through MiSeq technology, which classified to 160 genera, 29 phyla. Lactobacillus, Bacillus, Leuconostoc, Thermoactinomyces and Lactococcus were the dominant genera in Daqu, which is the guarantee for the normal fermentation. During the storage process, acids, alcohols, esters and other components which good for the flavor of Chinese liquor were accumulated, while the off-odor compounds such as 4-methyl phenol were constantly reduced. The significant pairwise linear regressions for bacteria and flavor components indicated that lactic acid bacteria was closely correlated to lactic acid, acetic acid and other acid components. Besides acids components, Bacillus was correlated to nitrogen compounds also. [Conclusion] During the storage process, the bacterial community structure of Daqu was adjusted and the flavor components were changed for better flavor. In addition to the environmental factors, the metabolic activity of bacteria is the main influence of the formation of flavor components. Therefore, the storage of Daqu is necessary.

Abstract:[Objective] To study the refolding between SLA-3-YDY protein derived from Yorkshire swine and peptides derived from foot-and-mouth disease virus in vitro. [Methods] A pair of primers was designed to amplify the extracellular domain of SLA-3-YDY and then the PCR product was cloned into pMD19-T Simple Vector. After cleaved by Nde I and Xho I, the positive clones were selected to be sequenced. Analyzed by biological soft, the cloned product with correct sequences was selected to be inserted into pET-21a(+) and transformed into BL21. After induction with IPTG, the interest of protein was detected by SDS-PAGE. The bacteria were broken ultrasonically, then inclusion body from the bacteria was isolated. The heavy chain of SLA-3-YDY, light chain sβ2m and the epitope-peptides Hu52 from foot-and-mouth disease virus (FMDV), were refolded at a ratio of 1:1:1 in diluted refolding buffer followed by purification in molecular sieve of superdex 200 to detect whether the complex was refolded. [Results] The PCR result shows that the SLA-3-YDY gene was amplified successfully. After sequencing and analysis, the sequence of the positive clone of SLA-3-YDY was consistent with the primary sequence. By cleavage, the interest of gene was proved to be successfully inserted into pET-21a(+) Vector. After induction with IPTG and SDS-PAGE detection, the interest of gene was expressed and the molecular weight was about 33 kD. The isolated inclusion body was also detected by SDS-PAGE, and it was shown that the molecular weight of the inclusion body was consistent with the interest of protein. By refolding in a dilution system, the heavy chains of SLA-3, peptides and light chain succeed to be refolded in vitro. Then, by using the molecular sieve column to separate and purify the refolded proteins and detection by SDS-PAGE, it was shown that the complex protein of SLA-3-Hu52-sβ2m were obtained (45 kD) finally. [Conclusion] The prokaryotic expressing vector of SLA-3 derived from Yorkshire swine was constructed successfully in this research, and the interest of protein was obtained so as to the SLA-3-Hu52-sβ2m complex was refolded finally, which will lay a base to study the structure and function of the SLA-3-YDY in future.

Abstract:[Objective] To analyze the tendency, genetic characteristics and evolution of hemagglutinin gene of the influenza A/H1N1(09pdm) virus circulating in China between 2009 and 2015. [Methods] Epidemiological data of A/H1N1(09pdm) and hemagglutinin sequences were analyzed. Epidemiological data were collected from the website of Chinese National Influenza Center and HA sequences of the influenza A/H1N1(09pdm) virus were filtered from The Global Initiative on Sharing All Influenza Data and National Center for Biotechnology Information. Phylogenetic trees and the mutations of amino acids were constructed and analyzed by biological software. [Results] A total of 4 activity influenza peaks emerged between 2009 and 2015. Compared with vaccine strain A/California/07/2009(H1N1), the HA gene sequence of 2009?2015 shared different nucleic acid sequence similarity decreased with years. The phylogenetic analysis showed that HA gene of the same year clustered nearly on the phylogenetic tree. The HA gene from 2011 divided into two clusters. The amino acid substitutions of HA protein were observed in all four epitopes. Position 203 in epitope Ca, position 163 in epitope Sa and position 185 in epitope Sb changed into new amino acid gradually. The positive pressure site was observed HA protein site 240 in each year except 2010 and 2012. [Conclusion] The influenza A/H1N1(09pdm) has become one of the main subtypes in China. The HA gene and amino acid changes gradually and this emphasize the importance of reinforcing virus surveillance.

Abstract:[Objective] To study the diversity of endophytic diazotrophic bacteria in flue-cured tobacco, explore the resource of endophytic diazotrophic bacteria, and enrich the gene pool of endophytic diazotrophic bacteria. [Methods] Pure culture, transcription factor amplification (BOX-PCR), 16S rRNA gene sequencing, and phylogeny analysis were applied to study the diversity and phylogeny of endophytic diazotrophic bacteria strains isolated from flue-cured tobacco leaf. The nitrogenase activities, phosphorus and potassium solubilizing activities and indole-3-acetic acid (IAA) production of the isolates were also evaluated. [Results] totally, 62 endophytic diazotrophic strains were isolated by using Ashby culture media. Based on the BOX-PCR patterns, 16 typical strains were selected for further 16S rRNA gene sequencing. Phylogeny analysis results based on the 16S rRNA gene sequences showed that the isolates were affiliated to 3 genera, including Bacillus, Pantoea, and Curtobacterium. Bacillus was the dominant species among all the isolates. 32.3% (20/62) of the isolates exhibited nitrogenase activities, 12.9% (8/62) showed abilities in producing IAA, 6.5% (4/62) had phosphate solubilizing activities and 4.8% (3/62) possessed potassium releasing activities. [Conclusion] Abundant culturable endophytic diazotrophic bacteria existed in flue-cured tobacco and showed a potential application prospect.

Abstract:[Objective] To screen and identify antagonistic bacteria against the soft rot of Pinellia ternata from it’s rhizosphere soil. [Methods] Antagonistic bacteria were isolated by serial dilution and dual culture, and then identified by respective morphological and biochemical characteristics and 16S rRNA gene sequences. [Results] Total 228 strains of bacteria were isolated from the rhizosphere soil of Pinellia ternata, and three strains named as GZDF2, GZDF3 and GZDF4 among them showed the strong antagonistic activities to pathogens of Pectobacterium carotovorum subsp. carotovorum and Fusarim oxysporum and Fusarium solani with diameters of inhibition zone on 23 mm and had a broad spectrum antibacterial activity GZDF2, GZDF3 and GZDF4 were identified as Brevibacillus brevis according to its’ morphological, physiological and biochemical characteristics and 16S rRNA gene sequences. Phylogenetic analysis based on gyrB and rpoB sequence showed that GZDF3 was geneticly different from other two strains. [Conclusion] Three strains (GZDF2, GZDF3 and GZDF4) isolated from rhizosphere soil of Pinellia ternata showed potential to develope for biocontrol agents.

Abstract:As a novel nanomaterial, fluorescent quantum dots (QDs) have been synthesized by a variety of approaches, including physical, chemical and biological (bionic) methods. Among these methods, biosynthetical technique has gained increasing interest for its environmentally friendly process and good biocompatibility of product. Based on the literatures, this review summarized the methods to manufacture fluorescent QDs by different biomatrices, such as bacteria, fungi, some other living organisms, and bionic. We also addressed the prospects of biosynthetical (bionic) methods.

Abstract:Mutualism is the relationship that exists in two organisms of different species in which each individual benefits from the activity of the other. In this paper, some typical symbiosis examples between ascidian and cyanobacteria are introduced to explain the discovery process of their symbiotic relationship, the transmission mechanism of cyanobacteria between ascidian generations, and the possible physiological function of both sides in the symbiosis relationship. For example, cyanobacteria symbionts can both provide nutriment and participate in defense for the ascidian host by means of carbon fixation, nitrogen recycling and metabolites production. Ascidian host can provide a part of nitrogen nutrients for the cyanobacteria symbionts’ growth and protect them from ultraviolet radiation. The study of symbiotic relationship between ascidian and cyanobacteria will help us understand the process of biological evolution, and provide research foundation for further utilization and development of symbiotic relationships between marine organisms.

Abstract:Rumen methanogens can generate CH4 using hydrogen, methanol, methylamine and other substances. CH4 emissions from ruminants not only cause the dietary gross energy loss, but also have a negative effect on environment. Therefore, many scholars are looking for methods to reduce methane emissions from ruminants. Among the strategies, the use of Direct-fed microbes (DFM) is one possible option to decrease rumen CH4 emission, which remains in the phase of initial exploration. We review several vital DFMs and the mode of action that can be modulated by the use of DFM, and study on the use of DFM for mitigation of ruminant CH4 emissions.

Abstract:Gene recombination through recombinases and auxiliary protein enzymes exists widely in bacteria and it has great significance to bacterial genetic diversity and evolution. At present, genetic recombinations in bacteria are classed into three types: homologous recombination, site-specific recombination and transposition recombination. In this article, recombinases, and related protein enzymes of bacterial recombination systems, the mechanisms of various recombinations, and applications of recombination systems to genetic manipulation were reviewed.

Abstract:Conjugation is the key and difficult point in microbiology teaching. For student’s intuitive understanding of conjugation, we set up a conjugating experiment using Escherichia coli and Shewanella oneidensis, on the basis of systematic introduction of its discovery procedure and molecular mechanisms. A problem oriented strategy was adopted in theory teaching, thereby student’s logic thinking was developed in questions, and the scientific rigor was imparted in discussions. An inquiry based method was applied in lab teaching, where student’s meticulous operating style was nourished via exploring conjugation efficiency under different environmental conditions. By this way, student’s flexible thinking abilities, questioning spirits, and broad humanistic wisdoms were cultivated, and their comprehensive innovation qualities were improved.

Abstract:[Objective] In order to investigate the amount of virus released from silkworm infected by Bombyx mori bidensovirus (BmBDV) at the different periods and to develop a method for rapid and accurate detection whether silkworm were infected by BmBDV or not. [Methods] A small fragment of ns1 gene of genomic segment VD1 and ns3 gene of VD2 were cloned to pMD18-T vector respectively, and then the standard curve of ns1 and ns3 was obtained. The release dynamics of BmBDV were investigated by detecting the amount of BmBDV in the faeces of 5th instar silkworm at different infection stage. [Results] The results shown that a lot of virus were detected in silkworm faeces at the 3 days post-infected about 1.38×106 copies/μg VD1, 6.54×105 copies/μg VD2. The amount of virus increased exponentially from 5 days post-infected. The amount of BmBDV reached a plateau at 7 to 8 days post-infected about 2.12×107 copies/μg VD1, 1.34×107 copies/μg VD2. The PCR results also indicated a similar dynamics. [Conclusion] The release dynamics of virus suggests that the replication cycle of BmBDV is about 60 h. The infection of BmBDV in silkworm can rapidly, simply and accurately be determined by detecting the virus in silkworm faeces using real-time quantitative PCR.