Abstract:

Abstract:[Objective] Mutants of Aurantiochytrium sp. PKU#SW7 were screened for high docosahexanoic acid (DHA) yield. [Methods] UV mutagenesis and chemical stress screening were used to mutate Aurantiochytrium sp. PKU#SW7, with biomass production, lipid content and DHA yield as screening criteria to obtain mutant strains. [Results] Mutant PKU#PM003 presented stable high DHA yield in 4 generations. In lab fermentation the biomass of this strain reached 6.62 g/L, improved by 11.26% compared to that of the original wild strain; the lipid content of the mutant reached 4.01 g/L, improved by 26.1% compared to the original. The DHA content in total fatty acids also increased from 29.97% to 33.43% and the DHA yield of PKU#PM003 increased by 41.1%. [Conclusion] The mutant stain PKU# PM003 could be used as a potential strain for industrial fermentation.

Abstract:

Abstract:[Objective] To study the properties of endogenous microorganisms in Weibei Oilfield, we studied the ability of biosurfactant production, crude oil degradation and enhanced oil recovery on core displacement model of the isolated hydrocarbon oxidizing bacteria. Furthermore, we evaluated the effects of huff and puff field trial to discuss the process and feasibility for microbial enhanced oil recovery in ultra-low permeability and shallow reservoir. [Methods] We collected oil-water samples from ultra-low reservoirs and isolated surfactant producing hydrocarbon oxidizing bacteria by crude oil plate, identified the isolates by the physiological-biochemical properties and 16S rRNA gene sequence analysis, and then determined their environmental adaptation. Furthermore, we used an endogenous and exogenous microbial compound system to evaluate the abilities of crude oil degradation and enhanced oil recovery on both sand-pack and core-flooding physical model. Finally, the highly optimized microbial system was performed by the huff and puff process in a production well. [Results] A hydrocarbon oxidizing bacterium Pseudomonas aeruginosa, named WB-001, was isolated from ultra-low permeability reservoir in Weibei Oilfield. The fermentation broth surface tension and wax concentration were reduced to 29.04 mN/m and 8.48%, respectively. Sand-packing model and core displacement experiments showed that the oil displacement efficiency was 9.72% and 12.54%, respectively, performed with WB-001 and OPUS-HOB-001 compounding fermentation liquor injection compared with water flooding. The field trial showed that the daily oil production was increased from 0.42 t to 0.89 t, and total oil increase production was 44.47 t after microbial huff and puff process. In addition, the laboratory experiments on collecting samples showed that oil viscosity, freezing point and fluid surface tension was reduced by 11.70%, 9.41% and 18.93%, respectively. [Conclusion] This successful field trial proved that microbial enhanced oil recovery has its applicability in ultra-low permeability reservoirs and provides theoretical reference and a novel approach to high oil production of tight and ultra-low permeability reservoirs.

Abstract:[Objective] To study the diversity and composition of Bdellovibrio-and-like organisms (BALOs) in mariculture environment of Apostichopus japonicus. [Methods] The aquaculture water of A. japonicus was filtered to obtain the microorganisms. Then the total DNA of microorganisms was extracted. The purpose fragments of 16S rRNA gene of BALOs were amplified using two pairs of primers Per and Bac by PCR, respectively. Different patterns were determined with restriction enzymes Hae Ⅲ and Msp I by amplified ribosomal DNA restriction analysis (ARDRA). Positive clones were verified from 60 monoclonal strains. [Results] Eight and nine differences base sequences were obtained from two pairs of primers, respectively, and which were all belong to Bacteriovorax sp. and affiliated with three different clusters. Clusters Ⅰ and Ⅱ are dominant type which are known clusters; Cluster Ⅲ is a new one; The representative strains of Per1 (KP214541) and Bac44 (KP214551) are the dominant species. [Conclusion] The BALOs presented high diversity in mariculture environment of A. japonicus, including known and unknown, and the BALOs should be further isolated and cultured for the study of disease control in A. japonicus culture.

Abstract:[Objective] With the universal application of CeO2 nanoparticles (CeO2 NPs), its biological toxicity and environmental effect have been paid more and more attention. The purpose of this study was to acquire the process and influence of CeO2 NPs on activated sludge from activated microorganisms metabolites. [Methods] EPS and SMP are the two main types of microbial metabolites, the content and composition of which were both examined after dosing different concentrations of CeO2 NPs in laboratory-scale activated sludge. [Results] The content of EPS and SMP increased with the increasing dosage of CeO2 NPs during a short period. The content of TB-EPS and LB-EPS didn’t have changed observably at low concentration of CeO2 NPs. The content of TB-EPS and its compositions were not significantly affected, while the polysaccharide and protein of LB-EPS increased for resistant to the toxicity of CeO2 NPs (more than 25 mg/L). Compared to the control upon exposure to CeO2 NPs (50 mg/L), the protein and polysaccharide increased 35.18% and 46.57% respectively. The content of two types of EPS increased in the presence of CeO2 NPs, and LB-EPS increased significantly higher than the LB-EPS growth. When the concentration of CeO2 NPs was more than 25 mg/L, the content of protein of SMP appeared, polysaccharide and humic also increased significantly as well. [Conclusion] The protein generated from SMP might be combined with nanometer materials to reduce the toxicity of nanomaterials. At the low concentration of CeO2 NPs, the adsorption effect of EPS would prevent CeO2 NPs from passing into the cell. However, when the concentration was higher, CeO2 NPs would stimulate the microorganisms to produce more EPS which could form a thicker outer barrier layer to protect cells better. EPS and SMP worked together to resist the toxicity of CeO2 on microbial cell.

Abstract:[Objective] To study the effect of Cr(Ⅵ) reduction ability of Serratia sp. S2 under ammonia nitrogen (AN) and nitrate nitrogen (NN) with different concentration. [Methods] Simulated the nitrogen pollution of common environment in the laboratory and added different doses of AN or/and NN to the cultivation system in order to evaluate the effect of different types and concentrations of nitrogen, S2 was cultured in the premise of the Cr(Ⅵ) containing culture. Shake culture at constant 37 °C and measure A600, removal rate of Cr(Ⅵ), amount of AN and NN at a regular intervals. [Results] The growth inhibition of Cr(Ⅵ) to S2 was remittenced under the low and middle AN groups. The decline of S2 was accelerated under the high AN and NN groups. The removal rate of Cr(Ⅵ) and amount of AN among experience and control groups according to the independent effect of AN had no significant relationship. The removal rate of Cr(Ⅵ) of low concentration groups had a significant reduction of more than 10.0% and a increase of 7.1% in high concentration groups according to the independent effect of NN. S2 had the ability to reduce 200 mg/L NN to the level of control groups within 4 h. While AN and NN effect at the same time, AN played a leading role in low concentration groups and NN in high groups during the course of Cr(Ⅵ) reduction. [Conclusion] The presence of AN had no significant effect to the Cr(Ⅵ)-removal ability. Different concentrations of NN had different effect to Cr(Ⅵ)-removal ability. High concentration groups had an advantage effect. S2 had a significant ability of reducing NN.

Abstract:[Objective] To investigate the biodegradation of 2,4-dinitrotoluene (2,4-DNT) under various environmental conditions by Rhodobacter sphaeroides. [Methods] 2,4-DNT was anaerobically biodegraded under 30 °C in illumination incubator using photosynthetic bacterium Rhodobacter sphaeroides. The concentration of 2,4-DNT in liquid medium were detected by HPLC. [Results] Optimum conditions for the removal of 2,4-DNT were initial concentration of 40 mg/L, initial pH 7.0 and inoculation quantity of 15%. In addition, 2,4-DNT could be absorbed by the cells in lag phase, then degraded as carbon source in exponential phase. The removal rate of 2,4-DNT achieved 98.8% at 72 h. Using HPLC, two different intermediate metabolites were also observed, however, these decreased gradually within 120 h. Furthermore, the removal kinetics of 2,4-DNT corresponded with the first-order rate model. [Conclusion] The removal rate of 2,4-DNT under different conditions indicated that Rhodobacter sphaeroides is efficient in biodegrading 2,4-DNT.

Abstract:[Objective] Thermophilic filamentous fungus Thermomyces lanuginosus is capable of secreting a host of thermostable enzymes. We are committed to find a high activity and thermostable β-glucanase gene. [Mehtods] a novel exo-β-glucanase gene (glnB) was cloned from T. lanuginosus and successfully expressed in Pichia pastoris GS115. [Results] SDS-PAGE shows that GlnB has a molecular weight of about 48 kD. Levels of recombinant GlnB reached 11.5 U/mL in the flask fermentation broth of recombinant Pichia cells. GlnB exhibited optimum temperature and pH at 65 °C and 5.0, respectively, and was stable below 50 °C and over the pH range 3.0?10.0. Hydrolysis of laminarin by the recombinant GlnB produced monosaccharide and disaccharide, and extended incubation resulted in partial hydrolysis of disaccharide. [Conclusion] The recombinant enzyme GlnB from T. lanuginosus is an exo-β-glucanase and has good command of thermo stability and pH stability.

Abstract:[Objective] In order to improve the expression of Antheraea pernyi lysozyme in Pichia pastoris and determinate the lysozyme activity. [Methods] According to the Pichia pastoris codon preference, we optimized the codons of Antheraea pernyi lysozyme gene and added six histidine residues at the 3′ terminal of the gene. The optimized gene cloned into pPIC9K vector. Then we transfered it into Pichia pastoris GS115 through the electroporation method. High copy transformants were screened by using different concentration gradient of G418 and realized the secretory expression by methanol induction. We separated and purificated Antheraea pernyi lysozyme by ammonium sulfate precipitation and Ni-chelating affinity chromatography. Then we determinated the Antheraea pernyi lysozyme activity by turbidimetric method and detected antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck by the agar diffusion method. [Results] The expression conditions were optimized, and the highest expression level of Antheraea pernyi lysozyme occurred when the recombinant strain was induced with 0.75% methanol under pH 7.0 at 25 °C for 96 h. The expression quantity reached 2.4 g/L and the specific activity of Antheraea pernyi lysozyme towards Micrococcus lysodeikticus was 23 970 U/mg. [Conclusion] Codon optimized Antheraea pernyi lysozyme was successfully produced as a single major secreted protein in Pichia pastoris GS115. Antheraea pernyi lysozyme exhibited antibacterial activity against Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis, Ochrobactrum tritici, Escherichia coli and Acetobacter beijerinck.

Abstract:[Objective] To investigate the influence of complex microbial agent on the soil bacterial populations in apple replanted orchard. [Methods] After the treatment with complex microbial agent, soil specimens were sampled from new crop and replanted apple orchard then analyzed by the method of automated ribosomal intergenic spacer analysis to monitor the bacterial community. [Results] Two years after treatment with complex microbial agent, the operational taxonomic units (OTUs) and Shannon-Wiener diversity index of replanted orchard was 250 and 4.44, respectively. In comparison, corresponding values were 234 and 3.81 in new crop orchard. One-Way ANOSIM analysis further showed that the difference coefficient was 0.108 3 and 0.084 3 respectively. In addition, the new crop and replanted orchard shared 89 core OTUs, and had a moderate similarity (corresponding Jaccard community similarity coefficient was 0.47). [Conclusion] The application of complex microbial agent could improve the soil microbial diversity and benefits to the recovery of the soil microbial ecosystem in replanted orchard.

Abstract:[Objective] The preponderant strain was isolated from brick tea. Studies on its physiological and biochemical characteristics in cultivation, which provide the basis for the actual production of brick tea fermentation. [Methods] Strains were obtained from brick tea by dilution plate method in PDA. Based on common fungus fermentation method, the culture conditions was selected by the single factor method, and than culturing conditions were optimized using response surface methodology. Contents of reducing sugar, polyphenols, enzyme activity and antioxidant capacity from fermented liquid were determined. [Results] The preponderant strain was identified as Eurotium cristatum by the morphology and ITS sequence analysis. The optimization culturing conditions were: 187 r/min, 10 d, 28 °C. And then yield of biomass was 21.52 g/L. The four kinds of extracellular enzyme activity reached the maximum value at 10th days, meanwhile extracellular polyphenol (EP) 0.588 g GAE (gallic acid)/mL at 9th day. The fermentation liquid of E. cristatum showed antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical. The scavenging effects towards DPPH and hydroxyl radical were 90% and 94%, respectively. [Conclusion] Fungal strain MJAU EC021 was E. cristatum. And then the fermentation liquid had good antioxidant capacity, which is a potential antioxidant additive.

Abstract:[Objective] Via researches on phenotypic features (optimal growth temperatures, sporulation quantity, etc.) and fungicide sensibility of five strains of Corynespora cassiicola with different phenotypes isolated from cucumber in Gansu Province, to provide technical support for control of cucumber Corynespora leaf spot and stem blight. [Methods] The optimal growth temperatures were evaluated by temperature gradient method; The sporulation quantity was assessed by conidia yields per unit area on PDA plate; Fungicide sensitivity of Corynespora cassiicola strains was assessed by amendment plate method, autoclaved PDA media were amended prior to pouring with 8 different fungicides. [Results] The optimal growth temperatures of all the five strains were among 25?30 °C; Conidia amounts of the strains with scanty aerial hyphae were significantly more than the strains with luxuriant aerial hyphae on PDA plate (cultured 5 d at 25 °C); Under test concentrations, the sensitivity of the five tested strains of Corynespora cassiicola to eight fungicides successively were: mancozeb>flusilazole>tebuconazole>difenoconazole>procymidone>chlorothaloni>azoxystrobin>carbendazim. [Conclusion] Different phenotypic strains of Corynespora cassiicola isolated from cucumber were different in optimal colonial growth temperatures, growth speeds, sporulation quantity and fungicide sensitivity. At the tested concentrations, the strains showed very low sensitivity to carbendazim and azoxystrobin (inhibition rates<40%), which implied that the two kinds of fungicides had lost its effect on control of cucumber Corynespora leaf spot and stem blight in the region.

Abstract:[Objective] The aim of this study was to screen autotoxicitic compounds degradation bacteria from apple rhizosphere soil, and investigate the capacity of degradating phloridzin, phthalic acid, P-hydroxybenzoic acid and pyrogallic acid of the bacteria. [Methods] Bacteria were enriched and isolated by the sole carbon source Phthalic acid, Then，the strains were identified by the determination of physiological and biochemical characteristics, as well as 16S rRNA gene sequence analysis. Additionally, the ability of degradating phthalic acid, pyrogallic acid, P-hydroxybenzoic acid and phloridzin using the ultraviolet spectrophotometry and high-performance liquid chromatography. [Results] A total of five bacteria were isolated and named as BL1, BL2, BL3, BJ1 and BJ2, respectively. All of them degraded phthalic acid, pyrogallic acid, P-hydroxybenzoic acid and phloridzin. The five strains BL1, BL2, BL3, BJ1 and BJ2 were identified as Cupriavidus necator, Azospirillum lipoferum, Microbacterium hydrocarbonoxydans, Paenibacillus phyllosphaerae and Ochrobactrum cytisi respectively. The degration rates to phthalic acid, pyrogallic acid, P-hydroxybenzoic acid and phloridzin of BL1, BL2 and BL3 were all more than 50%. The degradation rate to phthalic acid, pyrogallic acid, P-hydroxybenzoic acid and phloridzin of BL2 was highest, and the degradation rate was 91%, 72%, 84% and 84% respectively. [Conclusion] We firstly discovered that Cupriavidus necator, Azospirillum lipoferum and Microbacterium hydrocarbonoxydans had the ability to degrade those four autotoxictic compounds, and they were expected to be applied in continuous cropping obstacle caused by autotoxicitic compounds in apple.

Abstract:[Objective] To detect a novel signaling molecule of quorum sensing, diketopiperazines (DKPs), in spoilage bacteria of aquatic products, the simple and sensitive technique of GC-MS method was developed for the quantitative analysis. [Methods] By optimizing the conditions of gas chromatography and mass spectrometry, media and extracting solvent, the quantitative measure of diketopiperazines (DKPs) was established. The characteristic ions of four kinds of DKPs standards, cyclo-(L-Pro-L-Gly), cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe), was ensured. The DKPs activity in two spoilage bacterial of aquatic product, Pseudomonas fluorescens and Shewanella baltica, were detected. [Results] Four DKPs had good linear at range of 1?200 mg/L. The limits of detection were 0.06, 0.10, 0.06 and 0.04 mg/L, respectively. The limits of quantification were 0.16, 0.18, 0.14 and 0.12 mg/L, respectively. The recovery of four DKPs in the medium was among 51.8%?88.5% with the RSDs of 1.4%?8.3%. The higher level of DKPs was detected with chloroform served as extracting solvent and LB used as medium. Two kinds of DKPs, including cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), were found in P. fluorescens and S. baltica. The level of DKPs in two spoilage bacteria increased with the bacterial growth, and arrived to the highest activity for 12 h of culture. [Conclusion] The technique of GC-MS method developed could analyze quantitatively the levels of four kinds DKPs in spoilage bacteria, with high precision and accuracy.

Abstract:[Objective] The purpose of this research was to analyze the Bacillus species, cellulose decomposition, antimicrobial activity and antibiotic resistance in giant panda intestines. [Methods] Bacillus was isolated based on the resistance of high temperature, then to construct the phylogenetic tree with 16S rRNA gene sequences. Hydrolyzed circle on the cellulose-congo red medium was measured to detect the cellulose decomposition. The oxford cup method was adopted to examine the bacteriostatic ability and then we analysed the relationship between the bacteriostatic ability and phylogenetic tree by software. The distribution regularity of Bacillus antibacterial peptides was detected by PCR. At last, susceptibility testing was put into use to detect the antibiotic resistance of Bacillus. [Results] Twenty-one strains was isolated and divided into 6 categories. All of the strains are able to decompose the cellulose on different levels by measuring the hydrolysis circle diameter. Most strains can inhibit the enteric pathogens obviously. Cluster analysis shows that the antibacterial ability is relevant to gene classification based on 16S rRNA gene in some cases. Susceptibility testing shows that most Bacillus strains are sensitive to common antibiotics, but a few strains are still resistant to the antibiotics. [Conclusion] The Bacillus strains isolated in giant panda intestines are abundant and able to decompose cellulose. The metabolic product of the antimicrobial peptide gene contained in all of 21 Bacillus strains can significantly inhibit three kinds of enteric pathogens. The Bacillus are sensitive to common antibiotics which is helpful to standardize clinical medicine.

Abstract:[Objective] To breed L-arginine high-yielding strains by atmospheric and room temperature plasma (ARTP), and to explore the optimal fermentation conditions of mutants by response surface method. [Methods] The L-homoarginine and 8-azaguanine resistant strain was obtained from the strain Corynebacterium glutamicum GUI089 (SGr, L-His?) that was treated by ARTP. Based on the results of single factor experiments, ammonium sulfate, glucose and urea were selected as independent variables to optimize fermentation conditions for L-arginine production from 7 factors by Plackett-Burman design. Subsequently, the levels of the three variables were further optimized by response surface methodology. [Results] After several rounds of screening, a strain was selected resistant to 15 g/L L-homoarginine and 0.7 g/L 8-azaguaine, and designated as C. glutamicum ARG 3-16 (L-HAr, 8-AZr, SGr, L-His?). L-arginine production of ARG 3-16 was 49.79% higher than that of the original strain. Beyond the higher production of L-arginine production, ARG 3-16 accumulated fewer by-products than the original strain, especially the accumulations of L-proline, L-glutamate and L-valine. Under the optimal conditions, the yield of L-arginine reached 39.72±0.75 g/L, 10.49% higher than before optimization. [Conclusion] We successfully bred a high-yielding L-arginine producing strain by ARTP breeding system, and optimized fermentation conditions by response surface methodology, the results suggested that ARG 3-16 was a promising L-arginine producing strain.

Abstract:[Objective] The effect of cooperative interaction between Bas1p and Bas2p on cAMP production by recombinant Saccharomyces cerevisiae strain was studied, and fermentation medium was preliminarily optimized. [Methods] Bas1p and Bas2p were over-expressed by using the co-integrated expressing strategy in the cAMP-producing strain G5. The growth and extracellular cAMP production of the resultant strain were investigated by fermentation in shaking flasks. The effects of the amount of yeast powder and peptone, and the precursor adenine added in media were studied. [Results] The cAMP concentration at 120 h produced by the G5 strain over-expressing Bas1p and Bas2p was 2 253.8 μmol/L when fermentation medium 1×YP was used, which was increased by 51.4% compared with control strain. The product concentration was further increased to 4 450.4 μmol/L when the content of yeast powder and peptone in media was doubled. By adding 0.5 g/L adenine in media, the cAMP concentration at 120 h was further increased to 5 314.3 μmol/L. [Conclusion] All those results proved that the reinforced cooperative interaction between Bas1p and Bas2p as well as medium optimization can improve cAMP production.

Abstract:[Objective] To identify the taxonomic status of a stilbaceous entomopathogenic fungus collected on the soldier ant of Ponera. [Methods] The analysis mainly based on phenotypic traits (especially from the nutritional supplies), combined of molecular phylogeny and splits network. [Results] The strain GZAC XCH-ant-710 distinguished from the other Tilachlidium species by the ant host, the size of phialide and conidia, (11.9?16.2) μm×1.1 μm, (2.2?4.3) μm×1.1 μm, respectively. [Conclusion] The strain GZAC XCH-ant-710 is a new species, supported by the phenotypic traits (especially from the nutritional supplies), species delimitation, theory of evolutionary relationship, and multipath of combined analysis, and named Tilachlidium poneraticum.

Abstract:[Objective] To biosynthesis silver nanoparticles using Trichoderma hamatum and detect antibacterial activity of silver nanoparticles. [Methods] Silver nanoparticles were biosynthesized using Trichoderma hamatum mixed with AgNO3 and were characterized by UV-vis, XRD and TEM. Thermogravimetric analysis and atomic absorption were used to detected productivity of silver nanoparticle and conversion rate of Ag+. Antibacterial activity of silver nanoparticles was detected using Escherichia coli and Bacillus subtilis. [Results] Culture solution of Trichoderma hamatum mixed with AgNO3 was reddish-brown; UV-vis spectrum showed a significant peak at 420 nm; XRD spectrum showed four peaks, which corresponded to four crystal faces of silver nanoparticles; TEM images suggested that silver nanoparticles were monodisperse with shape in spherical. Size distribution suggested that they had narrow distribution, between 1–13 nm and the average size was 6.69 nm. Atomic absorption showed conversion rate of Ag+ was 84.41%. Thermogravimetric analysis showed that the productivity of silver nanoparticles was 67.12%. MBC of silver nanoparticle for E. coli was 10 mg/L and MIC was 7 mg/L, while for Bacillus subtilis was 5 mg/L and 4 mg/L. [Conclusion] After mixed with AgNO3, Trichoderma hamatum can biosynthesis silver nanoparticles. Silver nanoparticles were uniform, cubic and pure. Lethal effect of silver nanoparticles for Bacillus subtilis was much greater than that for E. coli.

Abstract:Proteomics is one of the main contents of functional genomics era, and plays an important role in understanding gene function, molecular mechanisms, etc. Plant proteomics, which is regarded as a branch of proteomics, has been applied and investigated more and more extensively, especially in exploring how plants and pathogens recognize each other and differentiate to establish either a compatible or an incompatible relationship. This review have summarized the proteomic studies on plant-pathogen interactions from both the plant and pathogen view points in recent years, particularly focused on plant response to fungi, viruses and bacteria infection. Finally, the prospects are forecasted to provide some references and theoretical basis for further study.

Abstract:Because of the purpose, pertinence and timeliness of the case-based teaching method, it has been more and more applied in the class teaching in universities. In the Microbiology teaching, teachers can pull in abundant materials as cases according to the teaching arrangements, such as classical experiments, social hot pots, histories of science and so on. By the cases carefully selected before the class and effective guidance in the class, teachers can train the abilities of students to find, realize and solve problems and develop their active scientific thinking. Meanwhile, teachers should try to avoid the case teaching to be just a form.

Abstract:In order to improve students’ ability of autonomous learning and cooperative learning, we adopted the teaching model of autonomous and cooperative learning in a team based on task driven in Microbiology teaching. In the model, the main line was task, in which the teaching contents and ability objectives were implied, and the teacher was a leader, who guided the students (the main body) to make use of learning resources, construct knowledge and improve ability through individual autonomous learning and team cooperative learning. Three rounds of teaching practice showed that the pattern stimulated the students’ leaning enthusiasm, cultivated students’ comprehensive ability and innovation consciousness. What’s more, the pattern promoted the teacher’ teaching level.

Abstract:In view of the fact that medical laboratory specialty in higher vocational college recruit students from arts and science currently and combined with students’ weak foundation, in course teaching of microbiological examination, led by microbiological testing job tasks as well as reinforcement of skills training, the curriculum has designed four modules, basics, basic skills, technology applications and unit training namely, fusing the theoretical knowledge of microorganism to skills training programs, to build “Serial Architecture” course architecture. Part of the experiment is designed by students themselves, with various measures teaching methods and diversified assessment methods. The results show that: curriculum reform and practice has stimulated students’ interest in learning and cultivated the students’ creative ability and practical ability, with obvious teaching effects.

Abstract:[Objective] Fluorescence quantitative PCR can absolutely or relatively quantify the target genes in soil samples. However, the accuracies of absolute quantification of target genes are influenced by DNA yields during DNA extraction and inhibition caused by co-extracted humic acids. [Methods] Using prepared mutated plasmid DNA, this paper quantified the 16S rRNA gene from one paddy soil by the methods of adding such plasmid containing mutated internal gene before DNA extraction. Two-way primers amplification showed that the added internal gene inserted in this mutated plasmid DNA was specific to studied soils when used as internal standards. [Results] The results showed that DNA yields varied greatly among these paddy soil samples, leading to incorrect results from absolute quantification. Using the mutated plasmid DNA, the precision of resulted data points has been improved significantly. It showed that the coefficient of variation for absolute 16S rRNA gene copies of these samples was 17.8, which was 66.7% lower than the values calculated for soil samples without internal gene correction. It indicates that added internal gene can correct the effects of varied DNA yields. Such tested methods were further used to quantify 16S rRNA gene from 6 wetland soils with great spatial and physic-chemical differences in soil organic matter contents and water moisture. The total bacterial abundance indicated by absolute 16S rRNA gene copies was linearly correlated (R2=0.694, p<0.001) with soil microbial biomass carbon, demonstrating that after addition of internal standards, the quantified 16S rRNA gene copies can accurately reflect the absolute microbial abundance in soil samples. [Conclusion] The added internal gene can correct for DNA yields and the effects of inhibition of humic acids on PCR amplification. Using plasmid containing mutated internal gene as internal standards, this method, as a nucleic acid assay can be applied in soil ecology to quantify soil microbial biomass and absolute abundance of microbial functional genes.

Abstract:[Objective] To develop a rapid, specific and sensitive method for detecting the serum antibodies against porcine epidemic diarrhea virus (PEDV). [Methods] The major S protein antigenic region were expressed after analyzing the site antigenic of PEDV S protein by bioinformatics softwares and the recombinant protein was detected by SDS-PAGE and Western-blot. Furthermore, indirect ELISA was developed by using the recombinant protein as coating antigen and though optimizing conditions, specificity and reproducibility tests. [Results] The recombinant protein was expressed successfully, the recombinant S protein could react with PEDV positive serum specifically, and the indirect ELISA method for serum antibodies against PEDV was developed successfully. Both the intro-batch and inter-batch variation coefficient were lower than 10%. The developed indirect ELISA method compared with commercialization of PEDV antibody detection kit and Western-blot, the coincidence rate was 86.67% and 88.89% respectively. [Conclusion] The established indirect ELISA could be used for detecting PEDV antibodies.

Abstract:[Objective] Simple sequence repeat (SSR) molecule markers were used to separate and identify protoplast monokaryons, sporulated monokaryons and their hybrid progenies of two Xianggu (Lentinula edodes) strains. [Methods] SSR primers developed from the whole genome sequence of Xianggu were used to identify the genotypes of different monokaryons and hybrids, and then to separate them according different genotype information. [Results] Employing Lefp-55 SSR markers, we obtained both protoplast monokaryons of strain “L808” without matching crossing of monokaryons, and we found the segregation ratio of both protoplast monokaryons was high to 191:1. This result was confirmed by other SSR markers, random amplified polymorphismic DNA (RAPD) markers and tranditional method. In the identification of sporulated monokaryons and their hybrid progenies, cooperation of several SSR markers was able to identify numerous hybrids, and it was useful in genetic and breeding research. [Conclusion] Protoplast monokaryons separation would be fast and accurate by using SSR markers, which also can be used in identification of sporulated monokaryons and their hybrid progenies of Xianggu in relevant genetic and breeding research.