Abstract:[Objective] Higher alcohols is an important part of liquor trace aroma components, but higher alcohols will have negative effects on wine flavor as well as the quality when its content is too high. Higher alcohols in liquor are mainly produced by yeast during alcoholic fermentation and alcoholic fermentation is mainly processed by Saccharomyces cerevisiae, it means that looking for higher alcohol synthesis functional proteins is of great importance for liquor production. [Methods] Two-dimensional electrophoresis was used to compare the differences of intracellular proteins between the Saccharomyces cerevisiae mutant ARTP5, whose higher alcohols production is much less than the origin strain, and the origin strain CF4. Comparative analysis of the two strains’ intracellular proteomic differences helps to find higher alcohols synthesis functional proteins. [Results] Compared with the origin strain CF4, the higher alcohols production of mutant strain ARTP5 was reduced by 20% and it indicates that 45 spots (expression ratio≥2) were expressed differently. Among them, 29 proteins were identified by MALDITOF MS-mass spectrometry, including proteins in carbohydrate and energy metabolism, stress response, protein translation and folding, amino acids metabolism, higher alcohol metabolism progress, etc. It is amazing to find out that the protein ILV5 involved in branched chain amino acid metabolism was up-regulated and the protein ADH1 involved in higher alcohol synthesis was down-regulated in strain ARTP5 compared with those in strain CF4, this could possibly be the reason that the higher alcohols production decreased in strain ARTP5. [Conclusion] The finding of the higher alcohols synthesis related protein ILV5 and ADH1 has important significance for the revealing of higher alcohol synthesis mechanism in Saccharomyces cerevisiae alcohol fermentation process.

Abstract:

Abstract:[Objective] The biosynthesis of ε-poly-L-lysine is controlled by the ε-poly-L-lysine synthetase. This paper aims to study the distribution and sequence features of Pls. [Methods] Pls proteins were predicted in the completely sequenced genomes via identification of the substrate-recognition and condensation domains and amino acid residues that determine the substrate specificity. [Results] One hundred and thirteen Pls were identified from 110 genomes, mostly distributed in Actinobacteria, with two identified in Gram-negative bacteria. Most Pls from closely related species display a high degree of identity. [Conclusion] Pls may be widely distributed in Actinobacteria. The adenylation, thiolation, and condensation domains of Pls are conserved while the transmembrane domains and linkers show otherwise.

Abstract:

Abstract:[Objective] Since hyaluronic acid (HA) biosynthesis pathway and the related genes in Streptococcus equi subsp. zooepidemicus have been thoroughly studied, it is of great significance of exploring a kind of strategy to identify new genes related to the synthesis of HA. [Methods] strains phenotype defect were screened using the randomly incorporation of the suicide vector pSET4s::sacB in host genomes and a mutant library was constructed. Integration loci were detected through ligation mediated PCR (LM-PCR) and verified by gene deletion and complementation, as well as complete genomic sequencing. [Results] A mutant library containing 150 mutants strains phenotype defect was constructed. We detected that the temperature sensitive vector was incorporating at 458 960 loci in Streptococcus zooepidemicus genomes and disrupting gene lacC which encodes 6-phosphate kinase. ΔlacC was obtained through markerless gene deletion system, and the phenotype analysis showed that ΔlacC still displayed the mucoid phenotype that was not the same as M1. The further complete genomic sequencing showed that base G in the locus of 206 613 in M1 mutant genome was lost, which was located in hasA gene encoding hyaluronate synthase and leading to frameshift mutation of hasA. What’s more, when complementation of hasA in M1 mutant, interestingly, M1 mutant rescued the capsule synthesis ability and displayed mucoid phenotype. [Conclusion] The result indicated that the loss of capsule synthesis ability of M1 mutant was the result of the loss of hasA gene function, which had nothing to do with the lack of lacC gene function. This study preliminarily established the strategies to high throughput screening new genes related to synthesis of HA in Streptococcus zooepidemicus, which laid a foundation for mining new genes in future.

Abstract:[Objective] We optimize the growth conditions of Bacillus subtilis BE-91 and its shake flask fermentation conditions producing extracellular β-mannanase. [Methods] Single-factor experiments were used to optimize the main factors (such as carbon source, nitrogen source, pH, temperature and so on) affecting the growth of B. subtilis BE-91 and its extracellular β-mannanase production in shake flask fermentation. Based on one-factor-at-a-time experiment, orthogonal tests were applied to obtain the optimal combination. [Results] The optimum growth conditions of B. subtilis BE-91 showed as follows: 0.3% beef extract, 0.2% yeast extract, 0.1% glucose, 0.4% konjac powder, 0.5% NaCl，initial pH of 6.0, and culture temperature of 35 °C. The optimum conditions for extracellular β-mannanase production were as follows: 0.7% konjac power, 0.4% soybean peptone, 0.1% (NH4)2SO4, 0.5% NaCl，initial pH of 6.0 and fermentation temperature of 35 °C. Under the optimized conditions, the highest extracellular β-mannanase production of B. subtilis BE-91 was 432.4 IU/mL in 10 h. Its extracellular β-mannanase production was more than 5-fold higher than that from other relevant strains reported, and the time appeared 14?86 h shorter than others. [Conclusion] B. subtilis BE-91 has advantage in short cultivation time and high extracellular production of β-mannanase, so it is the specious resources with potential use in the enzyme preparation industry.

Abstract:[Objective] For the purpose of control marine diesel pollution by biological treatment technology. [Methods] Diesel act as sole carbon source, using enrichment and screening methods to select diesel fuel-degrading bacteria from Shenzhen harbor area; bacterial consortium is constructed by mixing and orthogonal experiments; single-factor experimental is used to study the effect of environmental factors on diesel biodegradation by the consortium; using chromatography-flame ionization detection (GC-FID) to analyze the changes of diesel components before and after degradation, physiological, biochemical experiment and 16S rRNA gene sequence analysis are used to identify the strains. [Results] Sixteen diesel fuel-degrading bacteria were isolated and the highest degrading rates were up to 40.8% in 7 days. Bacteria consortium CQ1 was composed of C1-8, C2-10 and C3-13 strains, and the dosage was 0.5%, 2.0% and 1.0% respectively. Compared to the single strain, the degrading rate of CQ1 was increased by more than 10%. The best degrading effects would be up to 60% in 9 days under the optimum conditions that 30 °C, pH 7.6, shaking speed 220 r/min and diesel concentration 20 g/L. The result of GC-FID showed that most n-alkanes C11?C27 can be degraded by bacteria consortium CQ1 and the degradation rate of C21?C27 were up to 100%. In addition, the results showed that C1-8 belonged to Microbacterium sp., C2-10 belonged to Ensifer sp., C3-13 belonged to Corynebacterium variabile. [Conclusion] CQ1 has bright application prospects of bioremediation for offshore diesel pollution.

Abstract:[Objective] We aimed to isolate anthracene-degrading bacterium from saline-alkli soil and analyze its degradation characteristics. [Methods] Extinction dilution was used to isolate and purify the bacterium. Flow cytometry was applied to monitor bacterial growth. Anthracene and metabolites were analyzed by GC-MS. [Results] An anthracene degrading bacterial strain was isolated from highly saline soil. Based on its 16S rRNA gene sequence analysis, the bacteria was identified as Demequina salsinemorus. It can use anthracene as sole carbon and energy source. The highest degradation efficiency was 92%. Within a certain concentration range, the bacterial growth rate became faster and the degradation rate increased when the concentration of anthracene was decreasing. After adding exotic carbon source, bacterial growth rate increased significantly. However, the degradation efficiency decreased. 9,10-anthracenedione and phthalic acid were identified as the major metabolites. The phthalic acid path way was proposed as the degradation pathway. [Conclusion] A bacterial strain with a high anthracene-degrading efficiency was isolated. Our results may have important practical significance for petroleum-contaminated soil remediation.

Abstract:[Objective] This study aimed to prepare high viable count Pseudomonas putida bacteria powder, and improve the viability of drying and elongate the preservation period. [Methods] We selected cold blast drying technology to prepare bacteria powder, and optimized carriers and protective agents. [Results] Viability of drying was 65% on behalf of cold blast drying, which was significantly superior to spray drying (24%). Carriers and protective agents with orthogonal experiments were applied to obtain optimal formulation of preparation bacteria powder. The result was?as follows: as bacterial cell carriers, diatomite and corncob powder pretreated by Ammonia were mixed with ratio of 1:2. 7% mannitol, 5% sodium glutamate and 1% glycerin were mixed as protectives (W/W). Viable cell count was 1.03×1011 CFU/g in instant preparation. The viability was 71.67% and 40.54% after 60 d storage at 4 °C and 30 d storage at room temperature (RT) respectively. [Conclusion] Cold blast drying had little damage to bacteria because of its relatively low temperature (10?40 °C). Corncob powder pretreated by Ammonia, mannitol and glutamate had positive effect on preservation of bacteria powder. This method was successfully applied to resolve the difficulties of long-term preservation of gram negative non sporing bacteria.

Abstract:[Objective] A novel β-glucosidase encoding gene bgl17, which was functional screened from a metagenomic library constructed from the gut of Globitermes brachycerastes, was expressed in Escherichia coli BL21 and its characteristic were studied. [Methods] The recombinant enzyme Bgl17 was purified, and the stability and kinetic were identified. And the hydrolysates were analyzed by thin layer chromatography. [Results] Bgl17 was belong to glycoside hydrolase family 1 (GH1), and the optimal temperature and pH of Bgl17 with p-nitrophenyl-β-D-glucopyranoside (pNPGlc) were 70 °C and 5.0, respectively. The specific activity of purified Bgl17 was 115.69 U/mg and 297.39 U/mg with pNPGlc and salicin as substrate, respectively. The Km and Vmax of Bgl17 were 0.81 mmol/L and 227.27 μmol/(mL·min) with pNPGlc, respectively. Bgl17 can remain 50% activity at 50 °C for 1 hour and 50% residual activity was detected after 1 h at pH 5.0 and 6.0. [Conclusion] The β-glucosidase Bgl17 showed the high activity with salicin, which might be beneficial to the degradation of lignocellulose. It showed the potential for industrial applications because of high thermostability. The optimal temperature of Bgl17 was much higher than that of termites’ living environment, which would contribute to the study of cellulose degradation mechanism of termites.

Abstract:[Objective] To clone the mitogen-activated protein kinase (MAPK) gene from Polyporus umbellatus and carry out the bioinformatics and expression mode analysis. [Methods] RACE technology was carried out to clone the full length cDNA of MAPK gene. The characteristics of physiochemical properties and conserved domains of the predicted MAPK protein were determined using bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using BioEditor and MEGA 5.0 software. Real time quantitative PCR was used for gene expression analysis. [Results] The full length cDNA of MAPK was 1 293 bp in length and encoded a 386-aa protein with a molecular weight of 43.872 kD and an isoelectric point (pI) of 6.68. The PuMAPK clustered with Basidiomycete group according to the phylogenetic analysis. Real time quantitative PCR (qPCR) analysis revealed that transcripts were the most abundant in the beginning of sclerotial formation (20–30 days) with 7.86 fold over that in the mycelium, but the transcripts decreased sharply with the sclerotial development. [Conclusion] Molecular characterization of PuMAPK will be useful for the further functional determination of the gene involving in the development of P. umbellatus sclerotium.

Abstract:[Objective] In order to identify the community structure of the cultivable bacteria inhabiting the intestinal tract of adult oriental fruit flies (Bactrocera dorsalis) from laboratory-reared, laboratory sterile sugar-reared, and field-collected populations. [Methods] 16S rRNA gene PCR-DGGE molecular methods was employed to study the bacterial diversity. At the same time, the common species of cultivable bacteria were identified by colony morphology, physiological and biochemical characteristics tests. [Results] Six hundred culturable bacteria in the intestinal tract of adult B. dorsalis from the aboved three populations were classified into 53 unique phylotypes. All sequenced bacteria strains were grouped into three families: Enterobacteriaceae, Enterococcaceae and Bacillaceae. The Enterobacteriaceae was dominated in all the populations. The closely related sequences (>97% sequence similarity) which had been retrieved from culturable bacteria of three populations were grouped as one common species. We determined the common species: five strains of bacteria as Enterobacter, two strains of Klebsiella, one strain of Citrobacter, one strain of Pantoea, two strains of Enterococcus, and four strains of Bacillus. [Conclusion] The present study significantly contributes to the available information on bacterial isolates and the biological control of B. dorsalis.

Abstract:[Objective] To screening mycorrhization helper bacteria (MHB) which were facilitation to the quantity of mycorrhiza and the growing of mycorrhizal seedlings. [Methods] Pinus armandii was an experimental tree species, and eleven bacteria were isolated from rhizosphere soil of Tuber indicum as experimental bacteria. T. indicum inocula and different concentration bacteria were inoculated on P. armandii seedlings. The MHB were ascertained by statistics and analysis of the quantity of mycorrhiza, plant height and ground diameter of the P. armandii mycorrhizal seedlings synthesized by T. indicum. [Results] Pseudomonas sp. JCM 5481 (P143), Streptomyces sp. EN31 (S191) and Variovorax paradoxus (V633) of 2.4×109 CFU/ml had highly significant role in promoting (P<0.01) the quantity of mycorrhiza, plant height and ground diameter, as well as Pseudomonas chlororaphis (P11) and P. corrugate (P127) of 0.8×109 CFU/ml had significant role in promoting (P<0.05) the quantity of mycorrhiza, plant height and ground diameter. Different concentration setting of four Pseudomonas showed each strain has its suitable concentration respectively. [Conclusion] five MHB were obtained in the experiment, and indicated that the concentration of bacteria was a key factor to get MHB.

Abstract:[Objective] To explore new pathways for biocontrol of anthracnose by investigating the inhibitory effect of Bacillus subtilis C-D6 against appressorium formation of Colletotrichum capsici. [Methods] The antifungal activity of B. subtilis C-D6 against C. capsici was evaluated by dual-culture method. Optimal medium for production of antifungal active substances was determined in shake flask culture. The antifungal proteins were purified by ammonium sulfate precipitation, followed by Sephadex G-75 column chromatography and anion-exchange chromatography. The molecular mass of the purified proteins were determined by SDS-PAGE. [Results] B. subtilis C-D6 showed high antagonistic activity and inhibitory effect of appressorium formation against C. capsici. The optimum medium for the production of antifungal substances of C-D6 strain was YPD and 14 h after inoculation, the culture filtrate had a significant inhibitory effect on appressorium formation of C. capsici. An antifungal protein, with a molecular mass of 32 kD, was purified from culture filtrates of C-D6 strain and the purified protein exhibited obvious inhibitory activity against appressorium formation of C. capsici. [Conclusion] Our results suggest that B. subtilis C-D6 could be a potential bioagent for control of anthracnose caused by C. capsici.

Abstract:[Objective] Koumiss had beneficial therapeutic effects on cardiovascular disease, digestion disease, tuberculosis, diabetes and diarrhea, especially Kluyveromyces marxianus had antibacterial effect on Listeria monocytogenes. There was limited knowledge about antimicrobial compounds of K. marxianus in Koumiss, so, we evaluated the effect of antimicrobial compounds of K. marxianus in Koumiss on immune function and caecal microflora in mice challenged with pathogenic Escherichia coli O8. [Methods] Kunming strain mice (128 heads, 20±2 g) were divided into 4 groups, the control group, the challenged control group，K2 group, and K8 group. Mice in the challenged control group were oral administrated sterile PBS by gavage for 7 d, then were administered intraperitoneally E. coli O8 at 4 d. Mice in K2 group were oral administrated K. marxianus pH 2.0 and K8 group were oral administrated K. marxianus pH 8.0, then were administered intraperitoneally E. coli O8. The pathological section of small intestine of mice were observed by HE staining at 0, 4, 7 d. The thymus index and spleen index were weighed and calculated. Immunoglobulins in serum were monitored by enzyme-linked immunosorbent assay (ELISA). T subsets were analyzed by ?ow cytometry. The caecal microflora was calculated by the spread plate method. [Results] Mice in the challenged control group appeared clinical symptoms and pathological changes of small intestine after they were challenged with E. coli O8 at 4 d. Mice in K2 and K8 groups had better mental state, and less died mice than the challenged control group, K2 and K8 could improve pathological changes of small intestine after mice were challenged with E. coli O8. Compared with the control group, the thymus index was decreased at 7 d by the challenged control group, while the spleen index was increased at 4 d and 7 d by K2 and K8 groups (P<0.05). Compared with the control group, IgA was decreased at 7 d by the challenged control group, while IgA and IgG were increased at 4 d, IgG and IgM were increased at 7 d by K2 group, IgM was increased at 7 d by K8 group (P<0.05). Compared with the control group, CD8+ was decreased and CD4+/CD8+ was increased at 4 d and 7 d by K2 group, CD8+ was decreased at 4 d by K8 group, CD8+ was decreased and CD4+/CD8+ was increased at 7 d by K8 group (P<0.05). Compared with the control group, E. coli was increased and Bifidobacterium was decreased at 7 d by the challenged control group (P<0.05), while E. coli was decreased, Enterococcus was decreased, and Lactobacillus was increased at 7 d by K2 group (P<0.05), Enterococcus was decreased, Lactobacillus was increased at 7 d by K8 group (P<0.05). [Conclusion] These results suggested that antimicrobial compounds of K. marxianus in Koumiss named K2 and K8 could ease clinical symptoms after mice were challenged with pathogenic E. coli O8, enhance their immune function, influence their caecal microflora as Bifidobacterium and Lactobacillus increased, E. coli and Enterococcus decreased.

Abstract:[Objective] We studied the characteristics of chimeric foot-and-mouth disease viruses (FMDVs) bearing the S-fragment of O/GSLX/CHN/2010. [Methods] Based on 2 existing full-length infectious cDNA clones with the same lineage to O/GSLX/CHN/2010, 2 chimeric FMDVs bearing the S-fragment of O/GSLX/CHN/2010 were constructed and rescued by reverse genetic manipulation. Virus replication and virulence in suckling mice of the chimeric viruses, parent genetically engineering viruses and FMDV O/GSLX/CHN/2010 were evaluated. [Results] FMDV O/GSLX/CHN/2010 replicated slowly and its virulence in suckling mice was weak. Whether in virus replication or virulence in suckling mice, the chimeric viruses and their parent genetically engineering viruses were better than FMDV O/GSLX/CHN/2010 significantly. [Conclusion] S-fragment of FMDV O/GSLX/CHN/2010 cannot determine the weak phenotype of O/GSLX/CHN/2010 alone, which lay the foundation for further studies of the attenuation mechanism of O/GSLX/CHN/2010 and the influence of S-fragment on FMDV characteristics.

Abstract:[Objective] To screen actinomycetes strains with calcineurin inhibitory activity. [Methods] Actinomycetes strains with calcineurin inhibitory activity were screened by a yeast report gene (CDRE::LacZ) based high throughput screening model and a calcineurin phosphatase assay kit. The isolated actinomycetes strains were indentified by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. And the fermentation conditions were optimized by single factor and orthogonal experiment. [Results] strain CZW-15 with high inhibitory activity on calcineurin was isolated from soil, and it belongs to the Streptomyces spp.. Yeast report gene assay showed that the fermentation supernatant of CZW-15 inhibited yeast calcineurin-dependent gene expression with IC50=7.4 mL/L, and the IC50 of FK506 was 65 μg/L. The optimum fermentation conditions of CZW-15 were as follows: 2% corn flour, 0.1% yeast extracts, 0.04% K2HPO4, 0.04% MgSO4, 0.05% NaCl, 0.001% FeSO4, at initial pH of 7.5, 28 °C, 180 r/min, liquid volume 50 mL in 250 mL flask, inoculation size of 4% for 4 d. At the optimum conditions, the calcineurin inhibitory activity of 1.5 μL fermentation supernatant of CZW-15 reached 117%, increased 4.5 times. [Conclusion] CZW-15 belongs to Streptomyces spp., and produces highly active substances with calcineurin inhibitory activity.

Abstract:[Objective] Isolation of a bacterium strain named TZ-1 synthesized magnetic nano-particles from Dongchang Lake in Liaocheng City. [Methods] Proceed the morphological study and 16S rRNA gene identify to the strain TZ-1, extract and depurate the magnetic nano-particles form TZ-1 culture. Observe and analyze TZ-1 cells and the magnetic nano-particles through transmission electron microscope (TEM), scanning electron microscope (SEM) and X-ray diffraction (XRD). [Results] Concluded that TZ-1 belongs to Burkholderia sp.. The cells of TZ-1 is rod under the TEM, possess flagellum and capsule easy to gather. The approximate polygon in like electron dense granules arounded biomembrane in the cells include two types in size for 60 nm approximate located at the cytomembrane and 180 nm approximate located inner the middle of cells. The cells are rod under SEM, and the sizes are the same as the TEM. EDAX spectrum for the magnetic nano-particles on SEM shows that the main elements are Fe, P, O. According to the result of TEM, SEM and XRD that TZ-1 can compose magnetic nano-particles. [Conclusion] The bacterial strain TZ-1 we isolated synthesized magnetic nano-particles, XRD shows the ingredient of the magnetic nano-particles are Fe3(PO4)2·8H2O and Fe3O4, and the crystal structure of the magnetic nano-particles is monoclinic crystal.

Abstract:[Objective] The interaction between the conserved amino acid in Magnaporthe oryzae CYP51 F helix and Diniconazole was studied, aiming to help for designation of new specific and effective demethylase inhibitors for M. oryzae. [Methods] Six mutants (P222C, P222H, I223A, I223W, N224A, N224S) of M. oryzae sterol 14α-demethylase (MGCYP51) F helix were constructed with truncation of N-terminal 36 residues and heterologously expressed in Escherichia coli BL21(DE3) Rosetta. The binding ability of the recombination proteins to the diniconazole was detected by using the binding spectrum method. [Results] All of the recombination proteins had the activity to binding to the diniconazole and presented type II spectrum. Compared with the wild type protein, the Kd values of the mutations I223W and I223A binding to diniconazole were essentially unchanged, and the Kd values of N224S, N224A, P222C increased slightly with no significant difference (P>0.05), while the Kd of P222H increased significantly (P<0.05), indicating that the ability of the mutation P222H binding to the diniconazole significantly reduced. [Conclusion] The hydrophobic of the site P222 could play a major role in the binding of MGCYP51 to the diniconazole.

Abstract:Bacillus subtilis has been a model organism for gram-positive bacteria and has played important roles in metabolic engineering and industrial microbiology. Genetic manipulation technologies in Bacillus subtilis are critical for investigation of gene functions and modulation of bacteria physiologies in post-genomic era. In this review, we firstly summarize commonly used counter-selection markers for marker-free genetic manipulation. Then, we describe and compare different marker-free genetic manipulation strategies. Finally, we discuss challenges and future trends for developing more efficient tools for genetic manipulation of Bacillus subtilis.

Abstract:The process of nitrate-dependent anaerobic ferrous/iron oxidation (NAFO) is performed by specific microorganism, called nitrate-dependent anaerobic ferrous/iron oxidation microorganism (NAFOM), with nitrate/nitrite as electron acceptor and ferrous/iron as electron donor in anaerobic conditions. The discovery of NAFO is of great significance for the exploration of new microbial resources, the development of new denitrification techniques, and the new insight into Fe and N cycle in geoscience. In this paper, literatures about NAFO in recent decades were reviewed to analysis phylogeny, distribution, nutrition and metabolism of NAFOM. All the analysis aim at providing basis for discovering new NAFOM species, researching Fe and N cycle and optimizing NAFO process.

Abstract:The roles of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), as drivers of ammonia oxidation, in nitrogen cycle have been one of the most attractive topic in the field of microbial ecology. The relative importance of AOA and AOB in nitrogen cycle currently is still under debate since significant variations in the relative abundance, community structure and activity between them under different environmental conditions were observed. This discrepancy could be attributed to differences in the physiological ecology of AOA and AOB as evidenced by kinetics and genomes of cultures and environmental samples studies. Metabolic pathway and environmental factors like ammonia concentration, pH, dissolved oxygen and temperature were potential factors causing niche differentiation between AOA and AOB. Here, current knowledge of differences in the physiology, phylogeny, environmental responses and metabolic pathway between AOA and AOB were summarized, in order to gain deep insight into their relative contribution to nitrogen cycling under different environments. Finally, the future perspectives of AOA and AOB were proposed.

Abstract:Due to the difficulty in doing the productive practice for students majoring in bio-pharmaceutical in higher vocational education because of the particularity of industry, on-campus simulating-enterprise model for productive practice was constructed to solve the problem. Through this model, the consistency of training and production was achieved; students’ vocational skills and occupational quality were improved, as well as the remarkable results have been obtained. There are mainly five contents in the model. The first is to form a teaching team with rich production management and teaching experience, the second is to develop project named “Polymyxin E Productive Practice” based on antibiotics production process followed by development of the productive practice textbooks, the third is to establish a higher simulating and open training base used for scientific research and social service, the fourth is to build the mode of enterprise-style production organization and management, and the last is to create a grading evaluation and procedural assessment system.

Abstract:Massive open online course (MOOCs) and micro-lecture were products of the information age, and also were the developing direction of higher education in the future. In this paper, MOOCs and micro-lecture were applied to the reform and exploration of fermentation engineering course at the localized application-oriented universities and to supplement and extend the classroom teaching, they were new models for both taking into account the classroom teaching and the personalized learning, and will bring out the best in students’ personalized learning. For more than a year of teaching reform and exploration, it indicated that the teaching reform of MOOCs and micro-lecture had been approved by the students and the teachers and it indeed conducive to improving the students’ learning performance. In particular, the promotion of practice results achieve to a significant level (P<0.05), it was consistent with the purpose of training applied talents, and it was worth learning for localized application-oriented universities learning.

Abstract:[Objective] The quality and efficiency of genomic DNA extraction play a key role in molecular biology research. The cell wall of Gram positive bacterium is thick and hard to be destroyed, which makes it difficult for genomic DNA extraction. The objective of this study was to find an efficient and repeatable method to extract the genomic DNA from Gram positive bacterium. [Methods] The genomic DNA of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum were extracted using six extraction methods. The DNA concentration and DNA purity were compared for these methods. [Results] The results showed that the concentration of DNA was highest (about 400 mg/L) and the consistency of DNA concentration and DNA purity were best when improved SDS alkaline lysis method was used. [Conclusion] This research provided a reference for the extraction of genomic DNA from Gram positive bacterium.

Abstract:[Objective] A rapid, simple and sensitive visual reverse transcription loop-mediated isothermal amplification method was established to detect group A rotavirus. [Methods] The method employed a set of four specially designed primers that recognize six distinct sequences of the VP6 gene for amplification of nucleic acid under isothermal conditions at 64 °C for one hour. The amplification process of RT-LAMP was monitored by the addition of Calcein dye prior to amplification. The specificity and sensitivity of the RT-LAMP assay was assessed and the assay was further evaluated with 90 clinical specimens of diarrhea patients with RT-LAMP and RT-PCR detection. [Results] The results showed that the RT-LAMP was able to achieve a sensitivity of 103 copies/μl with a high specificity. The detection limit of RT-LAMP was 100-fold higher than that of RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of RT-PCR as well. [Conclusion] The RT-LAMP assay has been proven to be a rapid, sensitive, specific and visual method for detection of the group A rotavirus, and that the RT-LAMP assay is potentially useful for the field detection and rapid detection on spot.