Abstract:

Abstract:[Objective] To further investigate molecular mechanism of conidiospore formation, infection and pathogenicity of Botrytis cinerea, the gene related to conidiospore formation was cloned and characterized. [Methods] A mutant with no conidiospore, named BCt78, was found by screening T-DNA insertional mutant library of B. cinerea and it was testified by PCR and Southern Blotting techniques. The flanking sequence of T-DNA insertion site was acquired by using TAIL-PCR. The sequence of the T-DNA inserted gene was obtained by scanning the B. cinerea gene bank. The mutant was further identified by PCR and RT-PCR respectively. The function of the gene was studied by analysing the colony morphology, growth rate, cell wall degrading enzyme activity, biological activity of crude toxin and pathogenicity of the mutant strain on tomato leaves as well as the expression level of genes related to pathogenicity. [Results] T-DNA insertion site was defined in the initiation codon of BC1G_12707.1 gene, and the mutant gene was identified as BC1G_12707.1 by RT-PCR technology. The full-length DNA sequence of BC1G_12707.1 was 135 bp, and encoded a 44 amino acids hypothetical protein. Compared to the wild type strain, the mutant strain colony was white, growed slowly, did not produce conidium and sclerotia on PDA medium, but showed stronger pathogenicity on tomato leaves and cell wall degrading enzyme activity, higher expression level of genes related to pathogenicity, such as cell wall degrading enzyme gene cutA and Bcpg, genes (PKA1, PKA2, Bmp3 and Bac) involved in signal pathway, gene (BcBOT2) encoding sesquiterpene synthase, gene (Lac1) encoding melanin and transmembrane protein gene Btp1. [Conclusion] The BC1G_12707.1 gene was involved in conidiation, sclerotia formation and pathogenicity in B. cinerea.

Abstract:

Abstract:[Objective] The study aims to heterologously express Aspergillus fumigatus prolyl endopeptidase cDNA and to characterize the recombinant enzyme. [Methods] The cDNA from A. fumigatus CICIM F0044 was obtained by reverse transcription using the total RNA as the template. The PEP gene that encodes the mature prolyl endopeptidase was amplified using polymerase chain reaction with the cDNA as the template. The recombinant expression vector pPIC9K-PEP was constructed by inserting the PEP gene into pPIC9K, which was then transformed into Pichia pastoris GS115 by electroporation. The resulting recombinant enzyme was purified and characterized. [Results] A maximum yield of 647.3 U/L enzyme activity was obtained from the recombinant yeast. The molecular weight of the purified recombinant enzyme was approximately 63 kD. The optimal reaction temperature of the recombinant enzyme was 65 °C. The enzyme is highly thermostable, retaining 90% of enzyme activity after 8 h of exposure to temperatures 55 °C. The recombinant enzyme exhibited an optimal reaction pH of 5.5 and its activity was highly stable from pH 3.0 to 9.0. No decrease in enzyme activity was observed after 10 days of exposure to pH ranging from 6.0 to 8.0 at 37 °C. [Conclusion] The A. fumigatus prolyl endopeptidase cDNA was expressed in P. pastoris. The activity of the recombinant enzyme was stable, which indicates that the recombinant yeast has potential value in industrial applications.

Abstract:[Objective] A chelate carrier with agarose containing Cu2+ iminodiacetic acid (IDA) for hydrolysis saccharification enzyme was prepared and the fixed the glucoamylase process conditions with the chelate carrier was optimized. [Methods] Based on the technology of protein separation with immobilized mental ion affinity chromatography, the method for immobilization of glucoamylase has been selected, and the enzyme activity of the immobilized glucoamylase was determined using the UV spectrophotometric for research the process influencing factors. [Results] The optimum immobilization conditions of enzyme were as flows: enzyme load of 80 mg/g carrier, Cu2+ of 1.0×10?2 mol/g carriers, immobilized time of 4 h, pH value of 4.6, and the activity of immoblized glucoamylase was 252.1 U/g. The activity of immoblized glucoamylase on the regenerated matrix keep 65.1% effective of the new enzyme activity after used 5 times. [Conclusion] The Cu2+-IDA-agarose magnetic metal chelate can be used excellent immobilization carriers for starch hydrolysis saccharification enzyme.

Abstract:[Objective] The aim of this study is to improve the yield of Rhizopus oryzae lipase in Pichia pastoris by co-expression of chaperones. [Methods] By high-density fermentation in 7 L bioreactor, the expression of lipase in strain BH128 co-expressed with two chaperones Ero1p and PDI was compared with that of H238 co-expressed without any chaperone. [Results] The results showed that the highest lipase activity, the specific growth rate, the specific production rate and the specific consumption rate in BH128 reached 2 388.7 U/mL, 0.02 h?1, 944.5 U/(gDCW·h) and 0.15 gmethanol/(gDCW·h), which is 1.7, 0.5, 4.1 and 1.3 fold higher than those in H238, respectively. Moreover, the fermentation period in BH128 was 20 hours shorter than in H238. [Conclusion] The high-level expression of Rhizopus oryzae lipase could be achieved by co-expression of chaperones Ero1p and PDI, which provided the potential application of Rhizopus oryzae lipase in industry.

Abstract:[Objective] Human adenoviruses (40/41) have been related to acute gastroenteritis, and used as an index of human viral pollution in bathing waters. Traditionally, fecal coliform (FC) was used as an bacterial indicator by using cultivation techniques to estimate risks posed by pathogen in environmental samples. The spatial-temporal detection of waterborne pathogens is of great importance to public health and the prevention of illness. [Methods] In this study, a total of 30 bathing water samples were collected from ten representative bathing beaches of China from May to October, 2008. The quantification of human adenoviruses and FC were analyzed. [Results] The concentrations of adenoviruses ranged from 1.7×106 to 1.1×108 genomic copies/L detected by real-time PCR assay. Occurrence of adenoviruses was determined by real-time PCR and compared to that of common PCR, the positive rate was 30% and 26.7%, respectively. The FC values in seven sampling beaches were higher than 2?000?CFU/L. The temporal distribution trend of adenoviruses presented from August to October were much more than that of other months (p<0.05). Under this experimental conditions, when the sample areas changed in spatial scale, including not only among these ten bathing beaches, but also among different sites of each beach, adenovirus distribution had no obvious difference (p>0.05). Results also showed that spatial and temporal variation of FC were not significant (p>0.05). While there was a correlation between the concentration of FC and the distance from the seashore (P<0.05). The result further confirmed that bacterial and viral indicators were not correlated with each other in the chosen beaches. [Conclusion] In order to prevent a major outbreak of gastroenteritis disease in the swimming season, the monitoring of viral and bacterial indicators as well as sanitation management must be strengthened.

Abstract:[Objective] A microbial consortium Cf3, which was obtained and enriched from an e-waste contaminated river sediment, was applied to study the characteristics for decabromodiphenyl ether (BDE-209) degradation in order to pave a way for the bioremediation of PBDEs contaminant in sediment. And the composition of the microbial consortium was also studied. [Methods] Congeners of BDE-209 after the biodegradation were analyzed by GC-MS and the degradation rates were calculated. the composition of the microbial consortium was analyzed by DGGE. [Results] High BDE-209 degradation rates were obtained by consortium Cf3. After 120 days incubation, 80.03% BDE-209 was transformed by consortium Cf3 when the initial amount of BDE-209 was 2.6 μmol. Meanwhile, the biomass was obtained and the OD600 value increased from 0.01 to 0.21. The pH also changed from 6.93 to 8.50 during the degradation process. Ten cultivable strains were isolated from this consortium, six of which associated with Citrobacter spp. and four with Alcaligenes spp. based on the 16S rRNA gene sequences. Denaturing gradient gel electrophoresis (DGGE) results showed that other four major genera Wolinella spp., Acidaminococcus spp., Acetobacterium spp. and Desulfovibrio spp. were presented besides Citrobacter spp. and Alcaligenes spp.. However, the bands for Acetobacterium spp. and Desulfovibrio spp. disappeared with prolonging the incubation time. [Conclusion] A microbial consortium with high PBDEs degradation rate was obtained. The data obtained in the study about the characteristics of the degradation of BDE-209 by the microbial consortium and the composition of the consortium could provides some useful information and precious strains resources for the anaerobic bioremediation of polybrominated diphenyl ethers (PBDEs) in the persistent organic polluted environment.

Abstract:[Objective] The aim of this study was to screen laccase-producing bacterial strains and to investigate the enzymatic properties as well as decolorization ability of the laccase. [Methods] Enrichment medium supplemented with copper ions was used to isolate bacterial strains exhibiting laccase activity. The isolated strain was identified by morphology observation, physiological and biochemical tests and 16S rDNA sequence analysis. The enzymatic properties of laccase were investigated with syringaldazine as substrate. Dye decolorization ability of the laccase was tested by determining the change at maximum wavelength of synthetic dyes. [Results] A bacterial strain LS05 with high laccase activity was isolated from forest soil, and was identified as Bacillus amyloliquefaciens. The spore laccase of strain LS05 demonstrated optimum pH and temperature at pH 6.6 and 70 °C, respectively. It also showed high stability, retaining its activity after incubation at 70 °C for 10 h or at pH 9.0 for 10 d. Resistance towards SDS and EDTA was found for the spore laccase. The enzyme could efficiently decolorize different synthetic dyes at alkaline conditions. More than 93% of remazol brilliant blue R, reactive black 5 and indigo carmine were decolorized within 1 h. [Conclusion] The spore laccase of Bacillus amyloliquefaciens LS05 was highly stable at high temperature and alkaline pH, which was more advantageous in industrial application than fungal laccase. It showed high potential in treatment of industrial dye effluents.

Abstract:[Objective] In order to investigate microbial diversity of the Qinling-Daba region, collect microbial culture and metabolites aimed to screen for active compounds, and establish the collection database as a platform for sharing inform. [Methods] The microbial strains from different habitats were isolated by the different methods and identified by physiological, biochemical and morphological characteristics and 16S rRNA gene sequence analysation, while the microbial metabolites were prepared for screening for active compound by fermentation in shaker flasks, extraction with methanol. [Results] An actinomycete, named strain Str-4331, was isolated from a soil sample in the Qinling-Daba region. It produced grayish-white aerial mycelium and solvable-violet-blue pigment on Gause’s synthetic agar, microscopic observation revealed that it produced helically spore hypha, the spores were elliptic to oblong. It was primarily identified as genus Streptomyces according to its morphological, physiological and biochemical characteristics. The 16S rRNA sequence of strain Str-4331 was analyzed by ClustalX, the phylogenetic tree was derived with NJ and analyzed with bootstrapping. The results showed that the 16S rRNA gene sequence of the strain Str-4331 shared the highest identity (99%) with that of S. lateritius strain: LMG 19372. The antibacterial activity of the extracted metabolites was determined and the results showed that the metabolites had the activity against the Gram-positive bacteria. The aqueous solution containing the metabolites showed different colors under different pH conditions. Liquid chromatography-mass spectroscopy analysation showed that the metabolites produced by the strain Str-4331 composed of three main compounds with the molecular weight separately 557.68 kD, 588.43?kD, 485.18 kD, while the molecular weight of granaticin B was 558 kD according to the documents. [Conclusion] The strain Str-4331 was identified as S. lateritiu from its morphological and cultural characteristics and the 16S rRNA gene sequence. This is the first report that S. lateritius strain was isolated in the Qinling-Daba region, and this study provided basis for the further exploration of the strain.

Abstract:[Objective] We cloned the cDNA of β-carotene converting enzyme gene (asy) from Phaffia rhodozyma 7B12, a high astaxanthin-producing strain, and expressed the recombinant pET32-asy in E. coli BL21(DE3). Our work could lead to an important use for the study of Asy properties and further applications in vitro. [Methods] Using RACE method, we cloned the asy cDNA from Phaffia rhodozyma 7B12, and constructed recombinant plasmid pET32-asy. After optimizing the temperature and IPTG concentration, the soluble expression of Asy was achieved in E. coli BL21(DE3). pET32-asy and pACCAR16Δcrtx which carried the genes chain on the synthesis of beta-carotene by acetyl CoA were co-transformed into E.?coli BL21(DE3), and the changes of carotenoid species were analyzed by HPLC to detect the activity of Asy. [Results] The homology between new cloned cDNA sequence of asy gene (accession No. HM204708.1) and the only reported asy mRNA sequence (accession No. DQ002007.1) was 97%. The obtained cDNA was 1 971 bp in length, the longest open reading frame was 1 614 bp encoding 538 amino acids, and therefore the fusion protein expressed in E.?coli BL21(DE3) was about 70 kD. At the optimizing condition (induced by 0.5 mmol/L IPTG, at 26 °C, 5 h), 85% fusion protein expressed by recombinant pET32-asy was soluble. Compared the components of pigment in the E. coli strain only transformed with pACCAR16Δcrtx with the strain co-transformed with pACCAR16Δcrtx and pET32-asy, we found some changes of carotenoid components. The peak presented α-carotene was disappeared and three new peaks were shown, suggested that β-cryptoxanthin which is one of themetabolic intermediates between β-carotene and astaxanthin were produced because of Asy expression. [Conclusion] Astaxanthin synthase cDNA was cloned from Phaffia rhodozyma 7B12 and the soluble expression of astaxanthin synthase in E. coli BL21(DE3) was obtained. The fusion protein had a certain activity to transform β-carotene.

Abstract:[Objective] NRPS genes in 14 Eurotium spp. strains obtained from dark tea samples were detected. The potential of secondary metabolite biosynthesis mediated by some NRPS genes were predicted. Distributive characteristics of NRPS genes may be useful for understanding the genetic diversity of Eurotium spp.. [Methods] Fourteen Eurotium strains were obtained from dark tea samples. Fungal genomic DNA samples were extracted by IQS DNA extraction method. Distribution of 11 NRPS genes were detected by specific DNA primers for these strains by PCR and analyzed by DNA sequencing and sequence analyses. UPGMA was conducted to discuss distribution pattern of NRPS genes. [Results] Numbers of NRPS genes involved in Eurotium strains tested were ranged from 4 to 10. NRPS7 and CPS1 genes were detected from all the Eurotium strains, while NRPS2 and NRPS6 genes were only detected in few strains, and other NRPS genes diverse. Fw-30813-1, Fw-30813-4, Fw-30813-7 and Fw-30925-5 from the same tea brick were different each other in detection of NRPS1, NRPS2, NRPS3, NRPS4 and NRPS8 genes, demonstrating an extremely high genetic diversity in Eurotium spp. from dark teas. NRPS5 and NRPS8 genes were not detected from strain Fa-20719-3 from Fuzhuan samples made in Baishaxi, Hunan, while detected from all of 7 strains obtained from Fuzhuan samples made in Yiyang, Hunan. Distinct genetic differences were demonstrated among Eurotium strains obtained from separate manufactures and sites. [Conclusion] Detection and distribution and genetic diversity of NRPS genes were first reported from Eurotium strains obtained from Chinese dark tea. Detection of NRPS genes could be effective as an easy method to evaluate genetic diversity of Eurotium spp..

Abstract:[Objective] The objective of this study is to define the species and distribution of the fungi in mangrove wetlands, and to lay the foundation for the development and utilization of marine fungi. [Methods] Five hundred fifty soil samples were collected from different soil types of trees (Avicennia maria, Aegiceras corniculatum, Bruguiera gymnorrhiza, Rhizophora stylosa) and tidal zone (low, middle, high). The fungal isolate was isolated by dilution plate technique from the soil samples. Fungal diversity and species identification was carried out by based on the fungal morphology and rDNA ITS analysis. [Results] A total of 274 fungal isolates were isolated from the soil samples. Among the 274 isolates, 39 fungal species in 19 genera were found. The dominant fungal genera within Zhangjiang mangrove tidal-flat area were Aspergillus, Penicillium and Trichoderma. In addition, the fungal species of Talaromyces helicus was a new record specie in China. [Conclusion] There is a wide variety of fungal species in mangrove wetlands of Zhanjiang, and with potential prospects off development and utilization.

Abstract:[Objective] Genetic and phenotypic diversity of β-rhizobia associated with Mimasa spp. in Dehong district of Yunnan province were performed to revealed the diversity of rhizobial resources in China. [Methods] 60 isolates were analyzed by 16S rDNA PCR-RFLP, total cell protein SDS-PAGE analysis and 16S rDNA sequencing. [Results] All the isolates were clustered two genetic groups and two phena groups respectively, by characterizing 16S rDNA PCR-RFLP and SDS-PAGE anlaysis. The strains in each group are in coinciding with between two methods. All strains clustered with type strain of Cupriavidus, and Burkholderia. From the phylogenetic tree of 16S rDNA sequence, the test strains were belonged to the species of Cupriavidus taiwanensis, Burkholderia mimosarum and Burkholderia phymatum. [Conclusion] At present study, we find that β-rhizobium from Dehong district were belonged to Cupriavidus and Burkholderia mainly. The Cupriavidus spp. were predominated in these strains. These records showed the diversity of Rhizobium from Mimosa spp. and enriching resources of β-rhizobium in China.

Abstract:The survival rate and low temperature stability of lactic acid bacterial starter obtained by vacuum freeze-drying are governed by several factors. In this paper, the influence of the technology of high cell density cultivation and vacuum freeze-drying on cryotolerance of lactic acid bacteria for use as starters was analyzed. During fermentation, the following factors had a significant effect on the cryosurvival of lactic acid bacteria: culture medium, temperature control, pH stat, the neutralizer used, the harvesting stage of the cell crop, and post-fermentation handling of the concentrated cells. Factors affecting cell viability subjected to lyophilization include the following: cryoprotectants used, conditions used in initial freezing of the cell concentrate, and during vacuum freeze-drying. A good understanding of these factors will provide a reliable technology for preserving high cell density starter. The use of starter bacteria with high cryotolerance and viability can improve the quality of fermented milk products and boost economic benefits to the dairy industry.

Abstract:The widely spread bacteria of Pseudomonas genus could produce siderophores with different chemical structures and pigments with special colors. These siderophores and pigments confer the Pseudomonas bacteria great practical application potency in diseases controlling and medical researches. In some content, siderophores could be used as a tool for Pseudomonas taxonomy. Pigments produced by Pseudomonas have diverse dues, chemical structures and functions, in addition, some of them also function as siderophores. It is relatively simple to isolate and purify the Pseudomonas’s siderophores and pigments, while the biosynthesis and transport regulation mechanism are complicated very much.

Abstract:s an important part of the microbiology experiment, the microbial morphology experiment is really an eye-opener for students majoring in biology-related specialties to perceive the microscopic world. With the reference of the existing situations of the microbial morphology experiment teaching, the microscope digital mutual system was introduced into experiment teaching, which not only effectively improved the quality of experiment teaching, but also strengthened both the students’ practical and innovational abilities. Meanwhile, the method of experiment examination was perfected as well.

Abstract:This paper summarizes the practical experiences of constructing excellent course of Food Microbiology, including the development of the constructions of network resource and teaching materials, the modifications of course content and teaching methods, etc. These reform measures are helpful to improve the teaching quality.

Abstract:According to the reality of Science and Arts for vocational enrollment and students’ learning , in Cell Biology and Medical Genetics teaching, students’ sensory perception will be enriched through case teaching, and their abilities of active learning will be trained in problems teaching. Also, students’ abilities to obtain information and analyse problems will be improved through using internet. Interesting and open experiments will be created to cultivate their practical ability. All these ways are to help students’ understanding of knowledge so that their skills and teaching efficiency will be improved.