Abstract:[Objective] Micromonospora carbonacea JXNU-1 is an actinomycete with broad-spectrum antimicrobial activity. This study aims to reveal its genomic sequence information. [Methods] The genomic DNA from M. carbonacea JXNU-1 was sequenced by Sanger technology using Illumina MiSeq and Illumina HiSeq 2500 sequencers. The genome was assembled by using SOAPdenovo software; multiplex PCR was used to close the gaps, and the genome sequence information was analyzed by bioinformatics methods. [Results] The whole-genomic DNA of M. carbonacea JXNU-1 was sequenced and annotated, and a fine genome map of M. carbonacea JXNU-1 was completed. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JXSX00000000. [Conclusion] This study provides the basis to explore biosynthesis of antimicrobials from M. carbonacea JXNU-1.

Abstract:[Objective] The gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase were investigated from fecal microbiome of Nycticebus pygmaeus. [Methods] Degenerate primers were used to amplify phenol hydroxylase and catechol 1,2-dioxygenase gene fragments from metagenomic DNA. Phenol hydroxylase and catechol 1,2-dioxygenase gene clone libraries were constructed, and some of clones were sequenced separately. [Results] The BLAST analysis of phenol hydroxylase and catechol 1,2-dioxygenase sequences showed 92%?100% and 87%?100% identities to the known phenol hydroxylase and catechol 1,2-dioxygenase sequences in GenBank. Phylogenetic analysis showed that phenol hydroxylase sequences in gene clone libraries had high similarity with phenol hydroxylase sequences from Neisseria, Burkholderia, Alcaligenes, Acinetobacter. And catechol 1,2-dioxygenase sequences in gene clone libraries had high similarity with catechol 1,2-dioxygenase sequences from Acinetobacter. Sequence alignment showed two DEXRH motifs of LmPH sequences were detected in phenol hydroxylase sequences, and the conserved cysteine was detected in catechol 1,2-dioxygenase sequences which was inhibited by Ag+ and Hg2+. [Conclusion] The phenol hydroxylase from fecal microbiome of Nycticebus pygmaeus was multicomponent phenol hydroxylase, and catechol that middle production of phenol degradation can be cleaved by catechol 1,2-dioxygenase through ortho-pathway.

Abstract:

Abstract:[Objective] Specific growth rate of Escherichia coli could be significantly decreased by L-alanine, which would result in reduction in L-alanine volumetric productivity. Therefore, the λpR-pL promoter was used as a thermo-controllable genetic switch to coordinate the processes of cell growth and L-alanine production in E. coli. [Methods] Synthetic routes for acetate, formate, ethanol, succinate and lactate as well as for L-alanine recemization were inactivated by deleting the corresponding genes (ackA-pta, pflB, adhE, frdA, ldhA, dadX) in the wild-type E. coli B0016 to generate B0016-060B. Subsequently, alanine dehydrogenase derived from Geobacillus stearothermophilus was cloned downstream of the pL promoter and expressed in B0016-060B to produce B0016-060B/pPL-alaD. Shake-flask and bioreactor experiments were conducted to investigate the properties of cell growth and L-alanine fermentation. [Results] Deletions of the competing pathways significantly decreased by-products accumulation with formation of low levels of acetate, succinate and ethanol. Strain B0016-060B/pPL-alaD hardly produced L-alanine during cell growth phase at 28 °C, which facilitated high growth rate. Meanwhile, efficient L-alanine production was obtained when cultured at 42 °C under oxygen-limited conditions. In bioreactor experiment, strain B0016-060B/pPL-alaD produced 67.2 g/L L-alanine, with a productivity of 2.06 g/(L·h). [Conclusion] Efficient cell growth and L-alanine production were realized simply by switching the cultivation temperature.

Abstract:

Abstract:[Objective] To screen microsatellite markers associated with acetic acid tolerance of Saccharomyces cerevisiae. [Methods] One hundred and sixty segregants were obtained by crossing two yeasts (high acetate tolerant YHA and low acetate tolerant YLA) with different phenotypes. The alleles of 15 microsatellite markers were amplified from 40 offspring yeasts and SPSS 11.5 software was used to analysis the relationship between acetic acid tolerance and microsatellite markers. [Results] Three microsatellite loci were identified. Among them marker 14P2 had significant positive correlation with acid tolerance (P<0.01), marker 15P2 and marker 15P3 had significant negative correlation with acid tolerance (P<0.01 and P<0.05). Furthermore, 70.6% of high tolerant individuals contained the marker 14P2 (344 bp) derived from YHA, the high tolerant strain; 91.3% of low tolerant individuals contained the marker 14P2 (331 bp) derived from YLA, the low tolerant strain. [Conclusion] This study indicated that the microsatellite marker 14P2 has the obvious tendency of heredity correlative with genes for acetic acid tolerance, it can be used in molecular marker assisted selection.

Abstract:[Objective] This study aimed to purify and characterize protease from Aspergillus oryzae, and to apply in casein phosphopeptides (CPPs) preparation. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography and Butyl-Sepharose HP hydrophobic chromatography were performed to purify the enzyme. The molecular weight and purity were determined by SDS-PAGE, and cleavage sites were detected by MALDI-TOF-MS. [Results] A protease named PE with the weight about 58 kD was obtained from Aspergillus oryzae. Protease PE shows maximal activity at pH 8.0 and 55 °C. It was inhibited by Fe3+, and activitied by Mn2+. With casein as the substrate, Km and Vm of the protease were 0.36 g/L and 18.18 mg/(L?min), respectively. Protease PE has cleavage ability in -Leu-Cys-, -Val-Glu-, -Tyr-Leu- and -Arg-Gly- residues of bovine insulin chain B, showing a wide range of residue specificity. CPPs were obtained after proteolysis of casein using PE by barium-ethanol precipitation. Yield and r (N/P) of the CPPs were 15.87% and 6.17, respectively, and delayed calcium deposit for 35 min. [Conclusion] The research provided a reference for Aspergillus oryzae protease using in the functional food industry.

Abstract:[Objective] In order to enhance the production of 1,3-propanediol by Klebsiella pneumoniae, the regeneration of reducing power was strengthened. [Methods] The xylose isomerase gene (xylA) from Escherichia coli was cloned and expressed in Klebsiella pneumoniae. The relevant metabolites and NADH concentrations of the recombinant strain were analyzed when it was cultured with xylose and glycerol as co-substrates. [Results] The intracellular reducing equivalent of the recombinant Klebsiella pneumoniae was increased by 0.1?0.3 fold. The titer of 1,3-propanediol of the recombinant strain reached 23.31 g/L, which was 20% higher than that of the parent strain. The conversion rate of 1,3-propanediol of the genetic engineered Klebsiella pneumoniae was improved from 0.60 mol/mol to 0.73 mol/mol. [Conclusion] The xylose metabolic pathway and intracellular reducing power is enhanced with the expression of xylA gene, resulted in the improved 1,3-propanediol concentration.

Abstract:[Objective] Objectives of this study were to investigate the impacts of two major lactic acid bacteria (L. homohiochii XJ-L1 and L. buchneri XJ-L2) on the microbial communities and flavor quality of Chinese Maotai-flavor liquor. [Methods] Bacteriostatic tests in vitro and mixed culture fermentation were carried out to investigate the interactions between main lactic acid bacteria and the other major microorganisms during fermentation. [Results] L. buchneri XJ-L2 could inhibit the growth of three Bacillus strains (B. amyloliquefaciens XJ-B1, B. subtilis XJ-B2, B. licheniformis XJ-B3), five mold strains (A. oryzae XJ-M1, A. niger XJ-M2, A. flavus XJ-M3, A. albicans XJ-M4, R. oryzae XJ-M5) and two yeasts (Sc. pombe XJ-Y4, G. candidum XJ-Y5). L. homohiochii XJ-L1 and L. buchneri XJ-L2 could promote the growth of three main yeasts (S. cerevisiae XJ-Y1, Z. bailii XJ-Y2, P. galeiformis XJ-Y3), and benefited for the production of acids, alcohols, esters etc. [Conclusion] The two main lactic acid bacteria, L. homohiochii XJ-L1 and L. buchneri XJ-L2, could maintain the ecological status of the main yeasts in the fermentation process through promoting the growth of three main yeasts and inhibiting bacillus, mold and few yeasts (L. buchneri XJ-L2 was more obvious). In addition, lactic acid bacteria affected the formation of acids, alcohols, esters and other flavor compounds and benefited for Maotai-flavor liquor fermentation. The appropriate proportion of lactic acid bacteria could enhance the balance of fermentation microflora. It is significant for the aroma production of Chinese Maotai-flavor liquor.

Abstract:[Objective] Saccharomyces cerevisiae is the predominant microbe in Maotai-flavor liquor producing which has several stressors. Learning its physiological characteristic would provide an insight in improving the yield and quality of liquor. [Methods] One S. cerevisiae strain with excellent performance was selected from the fermented grains of Maotai-flavor liquor making. Then the physiological characteristics of this strain were compared with other S. cerevisiae strains. [Results] One strain named as S. cerevisiae MT1 with excellent performance was selected from the fermented grains in Maotai-flavor liquor making. MT1 could tolerate high temperature (42 °C), high concentration of ethanol (16%, V/V) and strong acid environment (pH 2.0). The maximum specific growth rate and the maximum ethanol production rate of MT1 were as high as 125% and 114% of those of S288c, respectively. Its ethanol conversion rate was also higher than other strains. Several volatile compounds only can be detected in fermentation liquor of MT1. MT1 could also yield more phenethyl alcohol, farnesol, nerolidol, beta damascene and acetoin. In addition, MT1 could utilize and ferment a variety of carbon source to produce ethanol. [Conclusion] Compared with the model strain S288c and other strains, MT1 has the higher environmental stress tolerance, more efficient fermentation performance and wider range of carbon source utilization.

Abstract:[Objective] To study the function of Mbp1 gene, including the effects of Mbp1 gene mutation on ethanol fermentation of Saccharomyces cerevisiae. [Methods] The Loxp-KanMX-Loxp was constructed and transformed into haploid Saccharomyces cerevisiae MATa and MATα. After haploid mating test, the diploid mutant was selected. Ethanol fermentation and physiological studies of the mutant were performed and compared with those of the wild type strain. [Results] The growth rate of the mutant was similar to that of the wild strain; the budding rate of mutant strain is lower than that of the parent strain during the fermentation before 48 h; the mutant is approximately 19.2% bigger than the parent strain in cell volume and was more sensitive to nutrient starvation; in addition, pseudomycelium was also observed in the mutant. The ethanol production and sugar utilization ability of the mutant is slightly lower than that of the parent strain. The ethanol production level of mutant strain is lower than that of the wild strain in static culture, while there is no different between the mutant strain and the wild strain in agitated culture at 130 r/min. [Conclusion] Mbp1 is involved in fermentability and morphogenesis of Saccharomyces cerevisia.

Abstract:[Objective] To investigate the bacterial community structure in the rhizosphere of Eichhornia crassipes. [Methods] Terminal restriction fragment length polymorphism (T-RFLP) technique was used to analyze the characteristics and diversity of bacterial community in the rhizosphere of E. crassipes and the water samples near and far from it. Clone library technique and culturing method were also used to identify bacteria in the rhizosphere of E. crassipes. [Results] The bacterial community diversity (Shannon-Weiner diversity index H′ or Simpson diversity index D) in the rhizosphere of E. crassipes was the most abundant. The second and the third were that near and far from the rhizosphere of E. crassipes respectively. The bacterial community diversity in October was higher than that in May. According to the clone library of the bacteria in rhizosphere of E. crassipes, the dominant bacteria group was Proteobacteria (65.1%), including Bacteriovorax sp., Dechloromonas sp., Leptothrix sp., Rhodospirillaceae, Rhodoferax sp. and Rhodocyclaceae, etc. T-RFLP profiles revealed that the dominant bacteria was 159 bp, and the second dominant bacteria was 247 bp. 247 bp was identified as γ-Proteobacteria and 159 bp was belonged to Proteobacteria according to the result of the clone library and culture method. [Conclusion] This research indicates that the bacterial community diversity in the rhizosphere of E. crassipes was abundant, and its abundancy changed slightly in different period. The dominant bacterial community in the rhizosphere of E. crassipes was Proteobacteria.

Abstract:[Objective] This study aims to clone, express and purify an organic solvent-tolerant β-fructofuranosidase (β-FFase). [Methods] The recombination plasmid pET22b-pelB-bff by fusing the signal peptide gene pelB with bff was expressed in Escherichia coli BL21(DE3), encoding β-FFase from Arthrobacter arilaitensis NJEM01. Ammonium sulfate precipitation and DEAE anion exchange chromatography were used to purify β-FFase. Crystallization condition was determined using the sitting drop diffusion technique. [Results] Plasmid pET22b-pelB-bff was constructed. Under the optimal induction condition as 0.1 mmol/L IPTG at 30 °C for 10 h, β-FFase with the specific activity of 108 U/mg was expressed in the culture medium, which was further purified to the crystalline purity. The single crystal of β-FFase could be diffracted up to 2.1 ? resolution at the optimal culture condition consisting of 0.15 mol/L calcium chloride dehydrate, 0.1 mol/L HEPES (pH 6.7) and 26% (V/V) polyethylene glycol 400. [Conclusion] β-FFase has been successfully expressed, purified and crystallized with an efficient transfructosylation activity, which lays a foundation for the investigation on the structure-function relationship and improvement of β-FFase catalytic efficiency by directed evolution of gene manipulation in the future.

Abstract:[Objective] The novel gene encoding agarase in a strain of Vibrio shilonii screened from marine was cloned and expressed in E. coli BL21. [Methods] Isolate an agarase producing strain BY, 16S rRNA gene sequence analysis and construct phylogenetic tree. The degenerate primer was designed according to the known agarase gene sequence, touch-down PCR and chromosome walking techniques were used to obtain agarase full-length gene sequence. After being assembled, the full-length of agarase gene was inserted into expression plasmid pET22a(+), then transformed into E. coli BL21(DE3). Activity of recombinant enzyme was determined utilizing DNS method, study the enzymatic properties of the recombinant enzyme. [Results] A new gene Vibrio sp. BY was cloned from strain BY genomic. The full length of Vibrio sp. BY consists of a 2 232 bp, encoding 744 amino acids, with a putative molecular mass of 85 kD, the amino acids sequence of agarase Vibrio sp. BY shared 86% identity with the agarase of Vibrio sp. EJY3. The activity of agarase was 71.73 U/mL, which indicated that Vibrio sp. BY was an agarase gene. The optimum temperature and pH for the recombinant activity was at 50 °C and 7.0, have a high agarase stability. [Conclusion] A new agarase gene was cloned by chromosome walking techniques, realized recombinant in E. coli BL21(DE3), which lays a good foundation for the research on application of agarase.

Abstract:[Objective] The β-mannanase gene (man) from Bacillus subtilis BE-91 which is an efficient strain for hemi-cellulose degradation was cloned and expressed in Escherichia coli BL21(DE3) , then the enzymatic properties of the β-mannanase (Man) were studied. [Methods] The man was amplified from B. subtilis BE-91 by PCR, respectively linked to pEASY-E1 and pET28a, and finally expressed in E. coli BL21(DE3). After comparing the extracellular and intracellular β-mannanase activities from the gene engineering strains, the β-mannanase with highest activity was selected to study the enzymatic properties fully. [Results] The manA (GenBank: KP277209) was 960 bp in length, including a termination codon and encoded 319 amino acids. The highest activity of the extracellular β-mannanase from pEASY-man/BL strain was 229.1 IU/mL. The optimal temperature and pH of the β-mannanase were 65 °C and 6.0. The catalytic activity of the enzyme was stable at no more than 65 °C and pH 4.5?7.0 after being incubated for 30 min. It was promoted by Cu2+, Mn2+, Zn2+and Ca2+, while inhibited seriously by Ba2+ and Pb2+ at concentration of 1 mmol/L. [Conclusion] A precious β-mannanase gene has been excavated from B. subtilis BE-91, and its expression product with properties of thermostability and weak acidity may be available for feed additive.

Abstract:[Objective] To screen infection cushion-deficient mutants in Botrytis cinerea strain RoseBC-3 transformed with Agrobacterium tumefaciens. [Methods] Mycelial plugs of B. cinerea were inoculated on onion epidermal strips, which were then incubated for 12 to 120 h, stained with Medan dye and finally examined with a light microscope. Colony morphology, formation of infection cushions, pathogenicity, and production of pectinase and toxic metabolites by the selected mutants were determined using routine methods. [Results] One hundred and sixty-eight transformed mutants of B. cinerea were divided into three types: the rapid formation type (158 mutants), the slow formation type (9 mutants), and the defective formation type (only one isolate, namely AT19). On potato dextrose agar, isolate AT19 grew slowly, produced conidia and formed colonies with the normal appearance. However, it could hardly infect leaves of tobacco, strawberry, broadbean and pea, although it was detected to be able to produce pectinase and some toxic metabolites. [Conclusion] Formation of infection cushions appears important for B. cinerea to infect plant tissues.

Abstract:[Objective] To further explore the effect of Trichoderma viride TV41 on spatial distribution of Fusarium oxysporum FW0 around watermelon plant and fusarium wilt control of watermelon. [Methods] The number of F. oxysporum in plant rhizosphere, root surface, and internal of the plant was counted. The proportion of lateral roots infected by F. oxysporum and the infecting process of F. oxysporum in the plant were investigated. The pot experiment was carried out for three times and the incidence rate was recorded. [Results] The results showed that the number of TV41 was always significantly higher than that of F. oxysporum in watermelon rhizosphere when the inoculation concentration of TV41 and FW0 was same with 5×105 spores/g substrate. The number of F. oxysporum in plant rhizosphere, root surface of TV41+FW0 treatment was 103 CFU/g substrate, which was decreased significantly compared with the control of 104 CFU/g substrate. The Trichoderma viride TV41 not only retarded the infecting process of F. oxysporum in plant, but also reduced the number of F. oxysporum in plant root and stem. The disease incidence was reduced greatly when TV41 was applied. [Conclusion] The T. viride TV41 can decrease disease incidence rate of Fusarium wilt of watermelon by 60% through impacting the spatial distribution of F. oxysporum around watermelon plant.

Abstract:[Objective] The whole genome sequencing of Lactobacillus kefiranofaciens ZW3 is already known, it carries a 14.4 kb EPS (exopolysaccharides) gene cluster (WANG_1283 to WANG_1299) containing 17 EPS-related genes. In this study, we analyzed the expression quantity of 17 EPS genes in Lactobacillus kefiranofaciens ZW3 growth, and explored one expression quantity changed gene how to influence EPS yield. [Methods] Expression quantity of 17 EPS genes was analyzed by semi-quantitative RT-PCR technique; By constructing a recombinant strain of Lactococcus lactis containing an expression quantity changed gene, the EPS yield was compared between mutant strain and the wild strain. [Results] WANG_1284, WANG_1286, WANG_1287, WANG_1288, WANG_1290, WANG_1291, WANG_1292, WANG_1294, WANG_1296, WANG_1297, WANG_1298, WANG_1299 have the highest expression level at 50 h and 60 h when EPS was highly synthesized, suggesting that these genes play a role in the polymerization of polysaccharides. WANG_1291 was picked out from EPS genes to be further studied. This gene was inserted into plasmid pMG36e for the recombinant expression. The recombinant expression vectors were electroporated into Lactococcus lactis WH-C1. Assessing growth curves showed that the mutant strain and the wild strain have some differences. EPS yield of the recombinant was 2.1 times higher than that of the wild strain as analyzed by phenol-sulfuric acid method. [Conclusion] We provided evidence to show that WANG_1921 is a key gene to regulate Lactobacillus kefiranofaciens ZW3 EPS yield.

Abstract:[Objective] Thirty four strains isolated from the root nodules of Acacia melanoxylon R. Br. in Fujian and Guangdong were studied about the taxonomic position in order to abundant germplasm resources of rhizobia. [Methods] 16S rRNA genes and housekeeping genes (atpD and glnII) of all 34 strains were sequenced, 14 type strains of which were used to perform phylogenetic analysis. Furthermore, some representative strains were chosen to perform nodule experiment. [Results] The results of phylogenetic analysis of 16S rRNA genes and housekeeping genes (atpD and glnII) were mostly in agreement with which of 16S rRNA PCR-RFLP. And 14 type strains were divided into 10 groups, 2 of which belonged to Mesorhizobium and the others were Bradyrhizobium. In addition, nodule experiment suggested that the tested strains have a large rang of hosts because they could nodulate with A. melanoxylon, Leucaena leucocephala, Albizia falcataria and Acacia aneura. Moreover, the growth promoting effect of these stains was significant when inoculated on A. melanoxylon and L. Leucocephala. [Conclusion] This study illustrated genetic and symbiotic diversity of rhizobia isolated from the root of A. melanoxylon.

Abstract:[Objective] To understand the diversity of culturable bacteria isolated from Periplaneta americana adult gut. [Methods] Bacteria were isolated from the sample by using conventional culture-dependent method and then investigated by using numerical taxonomy and phylogenetic analysis based on 16S rRNA gene sequences. [Results] Totally 54 bacteria strains were selected on nutrient agar plate. On the basis of morphological, physiological and biochemical characteristics, we selected 32 strains to perform a phylogenetic analysis based on 16S rRNA gene sequences. The results showed that the strains can be divided into 12 apparent groups on the 82% similarity level by numerical taxonomy; 32 isolates represented 20 species, belonging to 15 genera of 10 families in four phylogenetic groups (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria). The most abundant and diverse isolates were within the Proteobacteria (15 strains, 46.9%) and the Bacteroidetes (10 strains, 31.3%). [Conclusion] Overall, the study results presented above demonstrated that there are abundant bacteria diversity in the Periplaneta americana adult gut.

Abstract:Integrative and conjugative element (ICE) is a kind of novel mobile genetic elements found in bacteria, which is a chromosomal mobile element and can transfer between bacteria through conjugation. The horizontal gene transfer contributes to the adaptation of microorganisms under special environmental conditions. Because of many ICEs associated with antibiotic resistant genes, the transfer of these genetic elements would speed up the dissemination of antibiotic resistance genes within and between microbial genera. It results in the growing problems of drug resistance, even multi-drug resistance and makes the mechanism of resistance become exceedingly complex. In addition, ICEs are closely related with genomic islands, and intensive research leading to deeper understanding is imperative.

Abstract:Inorganic polyphosphate (Poly P), is a linear chain of tens or many hundreds of phosphate residuals linked by high-energy phosphoanhydride bonds, and widely exist in prokaryotes and eukaryotes. Many researches demonstrated that Poly P can affect the virulence of bacteria, and help bacteria to survive in nutrient-deficient environment. Poly P has been identified to be related with the RNA transcription in nucleolus of eukaryotes. Poly P can also promote cell differentiation and modulate inflammatory response. Additionally, Poly P is related to mineralization and demineralization vertebrate bones. Poly P is the activator of mitochondrial permeability transition pore. This review is aim to summarize the impacts of Poly P to microorganism or mammal, and get more attention for the importance of Poly P.

Abstract:DNA cloning and assembly techniques are essential tools for molecular biology research. With the recent advances in synthetic biology, efficient and fast assembly of large DNA including a number of genes is becoming more and more important. Meanwhile, a variety of DNA assembly methods are also developed very quickly. In this paper, various DNA assembly methods based on atypical enzyme digestion and ligation, PCR, homologous recombination, single strand annealing and splicing are summarized for providing effective technique tools for the further development of synthetic biology.

Abstract:Shewanella oneidensis MR-1 is a model strain of electricigens Shewanella, which are widely distributed in natural water environments. As a facultative anaerobic bacterium, Shewanella is capable of using a multitude of electron acceptors to anaerobic respiration. Through the complex electron transport network composed by diverse cytochromes, Shewanella can not only reduce soluble electron acceptors permeating the periplasm, but also utilize insoluble electron acceptors extracellularly via transmembrane electron conduit. Here, this paper provides an overview of recent progress in the study about anaerobic metabolic pathways of Shewanella and is helpful for exploring the importance of electronic delivery network to the respiratory diversity and environmental adaptability of Shewanella.

Abstract:Microbiology is a professional basic course in agricultural faculties and universities. Currently, in agricultural faculties and universities, the specialty groups are complex, the students’ individual differences are obvious, and the social competitiveness of the students is weak. Aimed at the above problems, based on the specialty features and educational objectives, hierarchical teaching method was applied in construction of the teaching programs, teaching contents, teaching objectives and examination methods, and the students were taught according to their personal aptitude, so as to fully develop the students’ initiative, improve the teaching effect and enhance the talent-development quality.

Abstract:Problem based learning (PBL) is a new problem-based and student-centered teaching method. In order to improve the teaching quality, we adopted this method in the reform of Fermentation Engineering teaching. The teaching effects were analyzed by final examination and self-evaluation investigation questionnaire. The results showed that PBL teaching method did have a large impact on stimulating students’ learning interesting, and improving the ability of self-study, the ability of research and creative talents, which brings good teaching effects. PBL teaching method could effectively improve the teaching quality, and had a certain value to popularize.

Abstract:Current examination system should be further reformed to improve the teaching quality and to train high-quality talents in colleges and universities. In present paper, reform attempts were discussed. The characteristics of General Plant Pathology, and the main problems existing in the examination system of this course were analysed. A multiple comprehensive examination mode and a platform were built. The new examination system integrated theory with practice and was carried out through the whole teaching process, which can greatly improve students’ abilities and qualities.

Abstract:[Objective] Riemerella anatipestifer (RA) is an important poultry pathogen. Considering the twenty-one serotypes, it still lacked a method for detecting multi-serotypes antibodies. Previous studies showed that the Outer membrane protein A (OmpA), widely existing in multi-serotypes of RA strains, is a protein with immunogenicity. Its encoding gene is highly conserved among the genomes of RA of different serotypes, which indicates that OmpA can be used as a target molecule for serum antibody detection of RA infection. An indirect ELISA method to detect the antibody caused by RA infection was developed with the recombinant OmpA as the coating antigen. [Methods] The recombinant OmpA was purified after optimizing the prokaryotic expression conditions, and its immuno reactivity was tested by Western-blot with sera obtained from ducks infected by different serotypes of RA. Furthermore, the optimum testing conditions of ELISA were determined by phalanx test. Reproducibility, specificity and sensitivity tests were performed to estimate the practicability. [Results] The recombinant protein would be more soluble by adding 1% of ethanol in the medium. As analysed by Western-blot, the purified protein is immunogenic among 1, 2, 6, 10, 11, 13, 14, 17 serotypes of RA. Phalanx test results showed the optimal concentration of coated antigen is 8 mg/L, and the optimal dilution degree of serum is 1:160. Also, the developed assay was proved to be quite repetitive, specific and sensitive. [Conclusion] An indirect ELISA method for detecting the infection of multi-serotypes of Riemerella anatipestifer was established in this study and could be used for evaluating the immune effects of RA vaccine and for diagnosing RA infections with different serotypes of RA.