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Abstract:[Objective] This study is aimed to constitutive express lipase in Pichia pastoris and establish an efficient method for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Methods] The GAP gene promoter was amplified from the plasmid of pGAPZαA and inserted into the inducible expression vector pPIC9K-proRCL resulting in constitutive expression of the lipase gene-proRCL. Based on the homologous recombinant region of AOX1 gene, proRCL from Rhizopus chinensis CCTCC M201021 was recombined into chromsome of P. pastoris GS115 by double-crossover integration event resulting in constitutive expression of the single-copy lipase gene from R. chinensis. [Results] The activity of lipase reached its peak (130 U/ml) after fermentation of 144 h. An efficient method was established for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Conclusion] The method was very convenient with advantages of shortened screening cycle from 12 d to 3 d without the interferences of multi-copy mutants. This method also established the foundation of the screening of library by directed evolution.

Abstract:

Abstract:[Objective] In order to provide microbiology basis for protection, restoration and reconstruction of meadow steppe ecosystem in Inner Mongolian Region. To investigate the response of grassland soil microbes and enzyme activity in different grazing intensity. [Methods] Soil samples were collected from six different grazing intensity, the variation of different grazing intensities on the soil microorganisms, soil microbial biomasses (Carbon and nitrogen), soil enzyme activity and the interrelationship among them were analysised. [Results] The reseults showed that the number of microorganisms in different grazing areas had the same changing trends: bacteria>actinomycetes>fungi. The number of soil microorganisms and soil microbial biomass (Carbon and nitrogen) were higher in grazing areas than no-grazing areas. In 0?10 cm soil depth, the trend of the activities of catalase, invertase and protease was performed increased first and then decreased along with the increasing of grazing intensity, Moreover the activities of these enzymes in grazing areas was higher than no-grazing area. Compared with 0?10 cm, the descending range of the number of bacteria and fungi and the microbial biomasses (Carbon and nitrogen) were increased along with the increasing of grazing intensity in 10 cm?20 cm soil depth. The number of soil microorganism, soil microbial biomass, the activity of soil enzyme were higher in soil depth 0?10 cm than 10?cm?20 cm in the vertical distribution. Correlation analysis indicated that the number of soil microorganism was significantly correlated with soil microbial biomass. The soil enzyme activitiy was positively related to the number of soil microorganism and soil microbial biomass. The activities of catalase and invertase were extreme-significantly correlated with the number of bacteria and actinomycetes (P<0.01), and significantly related to soil microbial biomass C?(P<0.05); the activity of the protease was extreme-significantly correlated with the number of fungi and soil microbial biomass C、N (P<0.01), and significantly related to the number of bacteria (P<0.05). [Conclusion] Moderate grazing incereases soil microorganisms, soil microbial biomasses and soil enzyme activity. There were positive correlations between soil microorganisms, soil microbial biomasses and soil enzyme.

Abstract:[Objective] To provide useful basis for enhancing the biosynthesis of 2-phenylethanol (PEA), we studied the variation of physiological and biochemical characteristics of Saccharomyces cerevisiae sp. strain R-UV3 treated on varied concentrations of PEA. [Methods] Morphological observation was performed by a transmission electron microscope. Membrane permeabilization, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential after staining with propidium iodide, dihydroethidium and rhodamine 123 respectively were investigated at the single-cell level with flow cytometry. The expression of aro10 gene was determined by real-time fluorescence quantitative PCR. [Results] With the increasing PEA concentration (0?4.0 g/L), the membrane permeabilization, glycometabolism and expression of aro10 gene in the yeast cell decreased and mitochondrial membrane potential increased. ROS increased when PEA concentration was below 3.0 g/L and decreased when PEA concentration was beyond 3.0 g/L. The physiological and biochemical characteristics of the yeast, such as membrane permeabilization, glycometabolism and expression of aro10 gene, varied notably as PEA concentration increased from 2.4 g/L to 3.0 g/L. [Conclusions] The aqueous PEA concentration should be controlled at 2.4?3.0 g/L when in situ PEA removal technology was performed.

Abstract:[Objective] To construct a constructive expression vector pCambia-hph-Pr1 containing a Pr1 ORF of Pythium guiyangense for transformation of the fungus to get a genetic recombinant strain with enhanced virulence against mosquito larvae. [Methods] The Pr1 gene expression vector was constructed using a binary vector as basic frame, and Pr1 gene was placed between promoter PgpdA and terminator TtrpC. The recombinant strains were obtained by Agrobacterium-mediated transformation system, with P. guiyangense mycelia as acceptor, using vector pCambia-hph-Pr1 containing hygromycin B resistance gene as marker. The biological characteristics and virulence to Culex larvae of the transformant were observed. [Results] No distinct difference was found between wild-type stain and transformant in terms of growth rate and zoospore yield. A transformed strain of the fungus with significantly improved virulence was achieved. It caused an average larval death rate of 32.9% which was approximately double that of wild-type stain. Besides, the larvae exposed to transformant grew obviously slower. [Conclusion] A genetically stable strain with higher virulence was obtained by constitutive expression of Pr1 protease through Agrobacterium-mediated transformation.

Abstract:[Objective] The aim of this study is to explore the quantity and diversity of culturable endophytic bacteria in cucumber leaves sampled at blossoming and fruiting stages. [Methods] Leaf surface disinfection, strain isolation, 16S rDNA amplification and phylogenetic analysis were used in this study. [Results] It is found that there is a significant difference in the bacterial quantity and species at two stages. At blossoming stage, the isolated bacteria belong to 14 genera, including Curtobacterium sp., Arthrobacter sp., Microbacterium sp., etc. The isolating quantity is (2.6±0.18)′106 CFU/g fw, with Curtobacterium sp. being the dominant strain. However, at fruiting stage, the isolated bacteria belong to 11 genera, including Pantoea sp., Pseudomonas sp., Bacillus sp., etc. The isolating quantity is (5.2±0.42)′105 CFU/g fw, with Pantoea sp. being the dominant strain. [Conclusion] The species of endophytic bacteria at these two stages were quite different. The quantity of endophytic bacteria at fruiting stage was examined to be four-fold higher than that at blooming stage. These data indicate a significant diversity in culturable bacterial species at two different growth stages. Some strains obtained from this work provide possibility for further endophyte-cucumber association mechanism study and even field application perspectives.

Abstract:[Objective] Generation of C. gloeosporioides T-DNA insertion mutant library through Agrobacterium-mediated transformation was conducted in order to get more insight into the molecular mechanisms underlying the pathogenicity of C. gloeosporioides. [Methods] Agrobacterium containing the binary vector pSULF-gfp was used for transformation of C. gloeosporioides. Chlorimuron-ethyl resistant transformants were screened out and subjected to biological, morphological observation and PCR test. Detached copper-colour rubber leaves were used for pathogenicity assay. [Results] The transformation efficiency was up to 150?400 transformants per 106 conidia and 3721transtormants were generated, 25 transformants defective in pathogenicity were screened out from 3721 transformants. All of the transformants tested remained mitotically stable, maintaining their Chlorimuron-ethyl resistance after tenth generations of growth in the absence of Chlorimuron-ethyl. [Conclution] ATMT can be used to rapidly generate a large library of the fungal transformants, which offers highly efficient means for characterizing the genes that are important for the pathogenicity of C. gloeosporioides and should facilitate further molecular studies of this important plant pathogen.

Abstract:[Objective] The aim of this study was to screen acetylesterase-producing strain and to investigate its characteristics. [Methods] Anaerobic culture technique was used in screening and acclimatization of strain, and alkali lignin was a sole carbon source. The strain was identified by analyzing the sequence of 16S rDNA, Gram staining, Eosin Methylene Blue test, Methyl red test, and citrate utilization test. Acetylesterase was determined by ethyl p-nitrobenzoate. [Results] Acetylesterase-producing strain RB1 was isolated from rumen fluid of beef. It was identified as Escherichia coli. The growth curve showed that 0?42 h was lag phase, 42?60 h was log phase, 60?66 h was stationary phase, and 66?86 h was death phase. The optimum temperature for the acetylesterase was 40 °C, and the optimum pH was 8.0. The highest enzyme activity was 0.52 U/mL under optimum temperature and pH, and corn stover powder as a carbon source. [Conclusion] Strain RB1 was an acetylesterase-producing strain with application potential.

Abstract:[Objective] In order to investigate the relationship between the fatty acid composition and content and cell stress resistance in intracellular and cell wall. [Methods] In this study, three methods had been used for extracting the fatty acid from walls of chalmydospores, which were acid-heat method, soxhlet extraction and organic solvent, respectively. The fatty acid composition and content were detected by GC afterwards. [Results] Acid-heat extraction presented the best performance, by which the relative contents of saturated fatty acids in yellow, yellowish green and black chlamydospore walls were 26.92%, 17.23%, 23.71%, respectively, while unsaturated ones were 60.46%, 61.52%, 70.64%, separately. Further determination with the above same method, the relative total contents of 3 types of chlamydospores saturated fatty acids (precipitating wall plus supernatant) were 28.87%, 21.00%, 24.04%, separately and unsaturated ones with 55.43%, 55.87%, 63.89%, respectively. The content of stearic acid in saturated fatty acids in chlamydospore wall were yellow > yellowish green > black; the content of cis-5,8,11,14,17 eicosapentaenoic acid (EPA) methyl ester in unsaturated fatty acids in chlamydospore wall: black > yellowish green > yellow. [Conclusion] In all three colors of chlamydospore, either the total content of unsaturated fatty acids in spore or spore wall, the black showed the highest, which indicated that improving the content of unsaturated fatty acids in chlamydospores contributes to their dormancy and overwintering.

Abstract:[Objective] The influence of low pH on the adhesion ability and the surface properties of Bifidobacterium bifidum KLDS2.0603 were determined in this paper. [Methods] After Bifidobacterium bifidum KLDS2.0603 was treated by PBS of different low pH, the adhesion ability was assayed by gram staining microscopic examination and plate colony counting method. The autoaggregation ability and surface hydrophobicity of Bifidobacterium bifidum KLDS2.0603 were also assayed. [Results] The adhesion abilities of Bifidobacterium bifidum KLDS2.0603 treated by PBS with different low pH were decreased. The adhesion abilities of treatment groups were significantly lower than control group except the group of pH 5.0. The surface hydrophobicity of Bifidobacterium bifidum KLDS2.0603 treated by PBS of pH 3.0 and 3.5 was significantly increased. Except for experimental groups of pH 1.0, 1.5 and 5.0, the autoaggregation ability of Bifidobacterium bifidum KLDS2.0603 was significantly reduced. [Conclusion] The adhesion ability of Bifidobacterium bifidum KLDS2.0603 was reduced in the low pH environment. The autoaggregation ability and surface hydrophobicity of Bifidobacterium bifidum KLDS2.0603 were also affected. The adhesion ability was positive correlation with the autoaggregation ability and surface hydrophobicity excluding the groups of the pH 3.0 and 3.5 for Bifidobacterium bifidum KLDS2.0603.

Abstract:[Objective] A simple and efficient method for screening lactic acid bacteria which would produce phenyllactic acid (PLA) would be developed. [Methods] Strain R53 was incubated in MRS ( de Man , Rogosa , Sharpe ) broth supplemented with phenylalanine by the anaerobic culture, and PLA in the fermentation broth was determined by HPLC. [Results] 31 strains were obtained from swine's digestive tract, of which strain R53 from duodenum showed the highest PLA-producing ability (321.7 mg/L). Strain R53 showed the ability to inhibit the on the three radicals. The results showed that the radical-scavenging ratios on ·OH, O2ˉ ,and DPPH were 11.2%, 52.7% and 63.2% respectively. In addition, strain R53 could degrade cholesterol and the degradation rate was 32.5% in vitro. [Conclusion] Strain R53 was identified as Lactobacillus plantarum through its culture characteristics, morphological observation, biochemistry experiment and combining with the phylogenetic analysis of 16S rDNA. L.?plantarum R53 had the ability of phenyllactic acid-producing, cholesterol-reducing, and free radical-scavenging.

Abstract:[Objective] Polyphasic Taxonomy Method was used to identify 16 strains of Lactic acid cocci from Tibetan kefir. [Methods] Traditional physiological and biochemical method、16S-23S rRNA Intergenic spacer region polymorphism、Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene sequences analysis were carried out in this investigation. [Results] 16 strains were identified as Enterococcus, Lactococcus and Pediococcus, specificly 14 Enterococcus durans strains, 1 Pediococcus acidilactic strain and 1 Lactococcus lactis subsp. lactis strain. 16S rRNA gene sequences analysis was agreed with the previous results of three methods. [Conclusion] The results showed that polyphasic taxonomy method combined with traditional physiological and biochemical identification method, 16S-23S rRNA Intergenic spacer region polymorphism, DGGE and 16S rRNA gene analysis was suitable to accurately identify Lactic acid cocci.

Abstract:[Objective] To research the effect of enzymatic hydrolysate of Lentinula edodes on immune function and intestinal flora in mice. [Methods] Firstly, the immunosuppression model was produced by cyclophosphamide (CY) modeling method. And then one group was filled with no enzymatic hydrolysate by stomach and another group with enzymatic hydrolysate. Lastly, curative effect, immune index, the total number of intestinal bacteria, E. coli, Lactobacillus and Fungi in mice were determined. [Results] Enzymatic hydrolysate of Lentinula edodes could make immune suppression of mice, such as active degree, feces, fur and weight, return to normal levels. It also could improve the spleen and thymus index (P＜0.05), promote phagocytic activity of macrophages (P＜0.01). Meanwhile, it could improve the number of Lactobacillus which is the main beneficial bacteria in mice intestine (P＜0.01) . And it could restrain the growth of pathogenic bacteria such as E. coli (P＜0.01). [Conclusion] The enzymatic hydrolysate of Lentinula edodes could improve the immune function and the intestinal microflora environment. It could adjust intestinal function of mice. The enzymatic hydrolysate of Lentinula edodes had high edible and medicinal value.

Abstract:Pollution of the aquaculture environment has been a serious problem for the future development of aquaculture industry. Because of the significant importance of microorganisms in the environment of aquaculture, this paper provides an overview of modern microbial recognition technology from the technical aspects. According to the targets, the microbial recognition technology was divided into two types: one targetting nucleotides of microorgansims, the other targetting the surface antigens of microorganisms. The application of the two technologies was also summarized in the present paper, which could provide a reference for the improvement of aquaculture environment and the control of the disease in the aquaculture animals.

Abstract:By reforming the conventional biology experimental teaching model, we constructed the school-level biology experimental teaching demonstration center. This paper discussed the open and innovative practical teaching system of three levels and four modules which was established by increasing the proportion of designing and innovative experiments and strengthening the construction of open laboratory and practice base. Relying on the demonstration center, the experimental teaching level was increased and students’ ability of practice and creation was stimulated.

Abstract:Genome is a fast developmental frontal subject in the field of life science. In this article some conclusions were drawn from teaching reform and practice. The teaching work responded very well by some reforms that include optimizing contents of course, combining multimedia and traditional writing as teaching means, several teaching methods such as inspiration, discussion and task derivation, and bilingual teaching and so on. Our reform will provide a reference for the teaching work of Genome in other schools.

Abstract:[Objective] This work aims to investigate differential expression proteins at different development stages of Volvariella volvacea using isobaric tags for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) proteomics approach. [Methods] Firstly, the proteins extracted from V. volvacea at different development stages were analyzed by SDS-PAGE. Secondly, the tandem mass spcetrometry data obtained from 2D LC-MS/MS were used to search using MASCOT search engine. After that, principal component analysis, hierarchical clustering and Gene Ontology were used to analyze the detected results. [Results] Results showed that 1 039 protein groups, included a total of 2 335 unique peptides, were identified. Among them, 1 030 protein groups were provided quantitative information. Contrast to mycelia, 64 up-regulated and 150 down-regulated significantly differential proteins were found in fruiting bodies at different development stages of V. volvacea. Bioinformatics analysis revealed that iTRAQ-coupled 2D LC-MS/MS was a unique method for isolating and identifying protein groups of V. volvacea at different development stages. [Conclusion] It is helpful to insight into the molecular mechanism of the fruiting body formation and development of V. volvacea and other macro-basidiomycetes in the future.

Abstract:[Objective] The cluster analysis was applied to the optimization of the Flavomycin flask fermentation. [Methods] The data of Flavomycin flask fermentation from different batches was clustered by using system cluster, K-means cluster and fuzzy C-means cluster. The result showed that fuzzy C-means cluster was better than the other two. [Results] Then the classes were separated and the excellent samples were distilled, moreover, the range of control parameter of Flavomycin flask fermentation was optimized. [Conclusion] The experiment results indicated that cluster analysis was effective in the optimization of fermentation process.