Abstract:[Objective] [Methods] The fermentation parameters on the β-1,3-1,4-glucanase production by Bacillus subtilis D-6 in a batch fermentor (5 L) were optimized by statistical analysis using response surface methodology (RSM) based on Box-Behnken design (BBD) analysis of variance (ANOVA). [Results] The results showed that under the optimized conditions, with agitation, aeration, and pH at 500 r/min, 1.05 vvm and 5.08, respectively, the highest β-1,3-1,4-glucanase activity of 2 294.4 U/mL was obtained after 22 h of cultivation. [Con-clusion] This study proved that RSM could efficiently be applied for modeling of β-1,3-1,4-glucanase production in submerged fermentation.

Abstract:[Objective] To identify and characterize a crude oil degrading bacteria 2-9 isolated from a crude oil contaminated marine sponge from Dalian Bay, China. [Methods] According to the 16S rRNA genes sequences analysis, physiological and biochemical characterizations, DNA G+C content assaying, determination of cellular fatty acids and testing of carbon sources. Its capability of degrading crude oil was further determined. [Results] The strain 2-9 was identified as Nitratireductor basaltis. Cells are Gram-negative with catalase-positive and oxidase-positive. The similarity between its 16S rRNA gene and that of its most closely related type strain in GenBank Nitratireductor basaltis J3T was 99%. Growth of strain 2-9 occurred with 0?8% (W/V) NaCl (optimum 2%); strain 2-9 grew at 15 °C?42 °C (optimal 30 °C) and at pH 6.0?10.0 (optimum pH 8.0). It metabolized many carbohydrates and organic acids; the G+C content of its genomic DNA was 57.29 mol%. The major fatty acids were C18:1ω7c (63.61%), C19:0 cycloω8c (16.97%), C18:0 (4.28%) and C16:0 (3.39%). When the initial concentration of crude oil was 1 g/L, the strain 2-9 could consume 63.5% of the crude oil in 14 days. [Conclusion] The strain 2-9 was a crude oil degrading bacteria, holding the potential of being applied in the bioremediation of oil spill.

Abstract:[Objective] study on yeast and yeast-like collected from the Rumex hastatus in five regions (Jinning, Xiangyun, Chenghai, Luguhu and Erhai) of Yunnan province. [Methods] The yeasts and yeast-like were isolated by spreading plate and identified by using large-subunit 26S rDNA gene D1/D2 domain sequence analysis and morphological. The ability of the yeast strains to produced extracellular enzyme and lipid were tested by screening media and Sudan black-B staining method. [Results] 82 yeasts and 99 yeast-like strains were isolated from the Rumex hastatus in five regions. The yeast strains were identified as belonging to 6 genera and 16 species and 1 suspected new species. And the yeast-like strains were identified as 1 species and 3 variety. The dominant genera in Rumex hastatus flowers were yeast-like Aureobasidium, followed by Rhodotorula and Cryptococcus. It has been obtained that 134 strains produced extracellular enzyme and 83 strains produced lipid. [Conclusion] The diversity of yeasts and yeast-like species from Rumex hastatus flowers in five regions are more abundant. With characteristics of producing amylase, protease, cellulase, lipase and lipid, these strains will have a potential value of application.

Abstract:[Objective] The objective of this study was to examine taxonomic position of two Trichoderma strains which were isolated from soil in southern China. [Methods] PDAm was used as a selective medium for isolating Trichoderma, then two strain was indetified by analysis of internal transcribed spacer regions of the rRNA gene cluster (ITS), partial sequences of transcription extensions factor 1-alpha (tef1-α) and observation of morphological characters. For phylogenetic analyses in MEGA 4.0, both ITS and tef1-α gene sequences were analyzed using the maximum parsimony approach of close-neighbor-interchange algorithm with search level 3, in which the ini-tial trees were obtained with the random addition of sequences (1 000 replicates). [Results] The ITS gene sequences of the strain CM01 were naturally clustered with ITS gene sequences of Trichoderma intricatum strain GJS 02-78 in GenBank with 99% of homology, and had the closest phylogenetic relationship with T. intricatum GJS 02-78, Trichoderma atroviride DAOM 179514 in phylogenetic tree which based on ITS sequences of strains; the tef1-α gene sequence of the strain CM01 were naturally clustered with Hypocrea intricate strain GJS 02-78 are the closest phyloge-netic relatives with 99% of homology, and had the closest phylogenetic relationship with H. intri-cata GJS 02-78 in phylogenetic tree which based on tef1-α sequences of strains; its morphological description accorded with type strain’s. Meantime, the ITS gene sequences of the strain SCGA5003 were naturally clustered with ITS gene sequences of Trichoderma stromaticum strain GJS 97-181 in GenBank with 99% of homology, and had the closest phylogenetic relationship with T. stromaticum GJS 97-179, GJS 97-180, GJS 97-181, GJS 97-182, GJS 97-183 in phyloge-netic tree which based on ITS sequences of strains; the tef1-α gene sequence of the strain SCGA5003 were naturally clustered with T. stromaticum strain GJS 97-183 in GenBank with 94% of homology, and had closest relationship with T. stromaticum CQSQ1032, GXNN7006 in phy-logenetic tree which based on tef1-α sequences of strains; its morphological description were con-sistent with type strain’s. [Conclusion] Utilizing a comprehensive approaches of morphological characters and molecular identification, we concluded that we found two new Chinese record spe-cies of the genus Trichoderma, strain CM01 was identified as Trichoderma intricatum Samuels & Dodd/Hypocrea intricata Samuels et Dodd, strain SCGA5003 was identified as Trichoderma stromaticum Samuels & Pado-Schulth/Hypocrea stromatica Bezerra, Costa & Bastos.

Abstract:[Objective] To design and synthesis encoding hydrophobic ELP genes and construct ELP genes library. [Methods] The highest hydrophobic Ile(I) amino acid (hydrophobic parameters: 4.5) was chosen to replace the guest residues X of ELP pentapeptide repeat serial units building block (VPGXG)10 and the encoding block fragment was synthesized, flanking with a Dra Ⅲ and BglⅠ sites at upstream and downstream respectively. The Dra Ⅲ and Bgl Ⅰ were designed as a pair of isocaudamer for recursive directional clone to construct the ELP genes library. To confirm that the ELP library was constructed properly, the ELP[I]50 gene was randomly chosen to be expressed. The Tt of purified ELP[I]50 was detected via turbidity changing at OD350. [Results] The ELP genes library containing the ELP[I]n (n=10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120) was successfully constructed. The library was confirmed by expression of ELP[I]50 gene, protein purification by inverse transition cycling. The Tt of ELP[I]50 was 24.3 °C. [Conclusion] In order to increase the number of hydrophobic amino acids in ELP, the Ile(I) was chosen as a guest amino acid, the foundation for further screening ELP tag was builded up to get proper recombinant tag with smaller molecular weight, high levels expression, and low phase transition temperature.

Abstract:[Objective] The objective of this study was to improve the production of coenzyme Q10 of Rhodobacter sphaeroides by replacing crtB (encoding phytoene synthase) with ubiC and ubiA (encoding chorismate pyruvate-lyase and 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, respectively) from Escherochia coli DH5α. [Methods] The plasmid for gene replacement was constructed with a suicide pSUP202 as vector, and its insertions, including the up and down streams of crtB in R. sphaerodies 2.4.1, spectinomycin resistance gene, ubiC and ubiA from E. coli DH5α were obtained by PCR. RT-PCR was used to detect the transcription of genes. HPLC was used to quantify the production of coenzyme Q10. [Results] The gene replacement mutant of R. sphaerodies was constructed, in which the crtB was replaced by ubiC and ubiA. The transcription of heterologous genes was confirmed by RT-PCR. The productivity of gene engineered strain was 1.6 fold of wide type. [Conclusion] The strain of R. sphaerodies with improved production of coenzyme Q10 was obtained successfully, and the ubiC and ubiA from E. coli could transcript with its native promoter in R. sphaerodies.

Abstract:[Objective] Identification of bacterial disease of tomato in Liaoning. [Methods] According to result of the pathogenicity, physiological and biochemical characterizations, 16S rDNA sequence and specific amplification of hrpZpst gene. [Results] The isolates were identified as Pseudomonas syringae pv. tomato (Okabe). [Conclusion] hrpZpst gene can be used to identify Pseudomonas syringae pv. tomato (Okabe).

Abstract:[Objective] To isolate and identify a novel Pseudomonas aeruginosa phage named PaP4 via its biological properties. [Methods] We used the double-layer agar culture method to make a single plaque. Phage particles of PaP4 were purified with CsCl2 gradient centrifugation and the ultrastructure of PaP4 was examined by electron microscopy. We extracted the genome of PaP4 and identified the type of nucleic acid with restriction enzyme analysis. The host bacteria were infected with PaP4 at six different MOI. Three hours and thirty minutes later, the phage titers were determined, respectively. For one step growth experiments, phages and its host with an MOI of 10 were used with a 15 min adsorption. Following centrifugation, the pellet was washed twice and resuspended with LB broth and incubated. Samples were taken at 15 min-intervals (up to 120 min) and immediately tittered by the double layer agar plate method. [Results] The plaques of PaP4 were transparent and their sizes were about 3 mm?5 mm in diameter. PaP4 had an isometric head approximately 50 nm in diameter and a long tail with 30 nm in length. 0.001 MOI-infected host bacteria gave the highest phage offsprings. One-step growth curve was determined according to the results of one-step growth experiments. [Conclusion] PaP4 is a virulent phage and belongs to Podoviridae. The whole genome can be digested by the restriction enzyme, indicating that PaP4 is a dsDNA virus. The optimal MOI of PaP4 is 0.001 and one-step growth experiment shows latent and burst periods are 25 min and 20 min, respectively, burst size is 150.

Abstract:[Objective] The separation of a herbicidal active compound from Pestalotiopsis microspora PM-1 was to be accomplished by high-speed counter-current chromatography (HSCCC) for the first time. [Methods] The compound was separated by HSCCC from the ethyl acetate crude extract of Pestalotiopsis microspora PM-1 with the two-phase solvent system consisting of n-hexane:ethyl acetate:methanol:water:solution (4:5:4:5, V/V/V/V). The upper phase (aqueous phase) was chosen as the stationary phase and the lower phase (organic phase) as the mobile phase. [Results] Under the HSCCC conditions of a flow rate of 2.0 mL/min, the apparatus rotated at 900 r/min and the effluent detected at 254 nm, four fractions were obtained. FactionⅡ had the most intensive herbicidal activity on Digitaria sanguinalis, which was further analyzed by high performance liquid chromatography (HPLC) on C18 column with acetonitrile:water=75:25 (V/V) as the mobile phase under isocratic elution. Fraction A obtained at the retention time of 7.954 min showed an effective herbicidal activity on seed germination of Digitaria sanguinalis. The spectral analysis by diode array detection (DAD) for fraction A showed that fraction A is a pure component. [Conclusion] The results showed that the separation methods of high-speed countercurrent chromatography combined with high performance liquid chromatography was effective to obtain the herbicidal compound-fraction A from the metabolites of Pestalotiopsis microspora PM-1.

Abstract:[Objective] Identification of two Trichoderma isolates were isolated from the soil in vegetable greenhouses and the pileus of Asafoetida mushroom. [Methods] By combination of morphological charaters and application of internal transcribed spacer (ITS). [Results] Two Trichoderma isolates were identified as Trichoderma pleuroticola S.H.Yu & Park sp. nov. and Trichoderma pleurotum S.H.Yu & Park. The morphological charaters of T. pleuroticola is similar with T. harzianum, but its conidiospore is obviously more than T. harzianum, secretes dark brown pigment, and forms yellow crystal on PDA medium. The typical characteristics of Trichoderma pleurotum is that its conidiophores are mostly solitary and more or less prostrate, branches scattered, arising separately and bearing crowded whorls of appressed phialides at the apex rsembling the conidiophore in Gliocladium. [Conclusion] Two Trichoderma isolates are T. pleuroticola and T. pleurotum respectively, which are two new record species in China.

Abstract:[Objective] Adopted a method of mixed induction, which can selected traits stable and growing well strains at low temperature, in order to get the low temperature-resistant strain with industrial value. [Methods] Mutagenized isolated Q1 by using ultraviolet irradiation and nitrosoguanidine treatment methods, put the strains Q1-4-6 and Q1 which already selected under the 15 °C, 20 °C, 37 °C, compare their growth rate, acid production and the amount of sugar degradation. [Results] Q1 was extracted in homemade yogurt by Maqu pastoralists, the strain was classified into Enterococcus raffinosus according to its morphology, the physiological and biochemical features and 16S rDNA sequence. After several rounds of mutation breeding, select a strain Q1-4-6 that can grow well at low temperatures and genetic traits stable. Compared growth, the amount of acid and sugar degradation between the original strains and mutant strains, we found mutants were better than the original strains. At 37 °C natural conditions, there are rarely differences in growth rate between the original strains and mutants strains, but both of them are a little bit higher than the original strains. The acid amount and growth rate of Q1-4-6 are better than CL04 and CL05 under 15 °C. [Conclusion]?Through complex mutagenesis of UV+NTG, eventually bred a strain Q1-4-6 that growing well at low temperatures with stable genetic traits.

Abstract:The SSR (simple sequence repeats) marker has become one of the most popular molecular markers recently. There are three strategies to develop SSR marker in macrofungi, traditional library method, enriched library method and GenBank screening. Among them, enriched library method has been used more frequently due to its higher efficiency. The applications of SSR marker in macrofungi are reviewed, with the conclusion that genetic identification and genetic diversity analysis are studied commonly while the genetic map construction and assistant breeding are still to be improved. In addition, the existing problems and developing prospects of SSR marker in macrofungi are discussed.

Abstract:The study on marine microbial diversity is highly valuable and promising, and will greatly improve the exploitation and utilization of microbial resources. Since the vast majority of marine microbe are unculturable, the molecular biology becomes the main way in the study of marine microbial diversity. Recently, the research strategy and technology developes fast, and this paper summarized the researches of marine microbial diversity based on molecular biology and compared different research methods. Besides, it envisioned the future of marine microbial diversity study based on the progress of this field and molecular ecology technology. This paper is valuable for the further study on marine microbial diversity as well as the ex-ploitation and utilization of marine microbial resource.

Abstract:Refractory organic substances are widely released into environment, which causes adverse impact on human health and environment due to their bioaccumulation, persistence and biotoxicant. Recently, many microbial agents have been applied to treat the refractory organic contaminants. In this paper, we reviewed the developments of microbial agent in domestic and overseas, and described the microbial cell immobilizing technologies and the immobilized carries in the preparations of microbial agents. The application of micro-bial agents to the treatment of phenols, polycyclic aromatic hydrocarbons and polychlorinated biphenyls were summarized. In addition, the main problems, as well as the new perspectives, in this field were also discussed.

Abstract:Biofilm is a kind of special microbial aggregates, and exists widely in various natural environments. The paper introduced the basic principle of biofilm formation, and reviewed the effects of carrier property, key components of extracellular polymeric substances (EPS) on the formation and stability of biofilms. Finally, the cross-disciplinary research prospect on the biofilm was provided.

Abstract:Cadherin is one of the most important receptors for crystal toxins of Bacillus thuringiensis in insects. The type, structure and distribution of cadherin in insects are sketched. The binding sites of both crystal toxin and cadherin are discussed in depth. The mechanism of interaction of cadherin with crystal toxin of Bacillus thuringiensis is expounded in detail, and the relations betweeen cadherin and insect resisitance are also presented.

Abstract:The present situation and development potential of talent requirement of Bioengineering specialty are analyzed. The project research and ability training combined innovation practice teaching mode with social requirement as the direction, practice curriculum system as the carrier, capability training as the principal thread, and students’ science and technology innovation activities and teachers’ scientific research as the drive, was successfully con-structed. The practices showed that innovation practice teaching mode can greatly improve the students’ learning interest and the teachers’ working enthusiasm.

Abstract:In this paper, reform and practice were summarized in the teaching of microbiology experiment. According to the specialized characteristic, the basic experiments were set up to strengthen the training of basic experiment skills, and comprehensive and designing experi-ments based on specialized characteristic were set up to cultivate the students’ comprehensive practical ability, and innovative experiments were set up to enhance students’ creative ability, and open experiments were established depended on the information platform of network ex-periment teaching and prefect lab management institution, which established new microbi-ological experiment teaching system of the specialized characteristic. It was proved that the characteristic reform and practice could adequately improve the efficiency and teaching qual-ity of microbiology experiment.

Abstract:[Objective] Isolating chromosome DNA of wheat blue dwarf (WBD) phytoplasma and establishing an effective protocol of purification for chromosome DNA. [Methods] Dif-ferential centrifugation and plus-filed gel electrophoresis (PFGE) were used to purify the chromosome DNA of WBD phytoplasma. The band observed in PFGE was confirmed by PCR and Southern blot. The effect of each step was detected by real-time quantitative PCR analysis. [Results] Using differential centrifugation and PFGE, a band about 650 kb was observed, which was confirmed as the chromosome DNA of WBD phytoplasma by Southern blot hy-bridization and PCR. Meanwhile, real-time PCR analysis showed that the relative copies of WBD phytoplasma chromosome DNA derived from differential centrifugation and PFGE was 436.5 times than that in the total DNA of infected periwinkle. [Conclusion] The size of WBD phytoplasma chromosome DNA is about 650 kb, purified WBD phytoplasma chromosome DNA can be obtained effectively using differential centrifugation and PFGE.

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Abstract:[Objective] This study is aimed to obtain effective cellulose-degrading bacterial strains and study the characteristics of cellulase production and degradation characteristics used NH3?H2O pretreated corn stalk as substrate, and explore mechanism of cellulose enzyme so as to improve the resource utilization rate of agricultural solid wastes. [Methods] LB medium was used to obtain eleven bacterial strains (NH1?11) from earthworm farm. CMC-Na was used in preliminary medium and congo red staining method to screening strains. Influence of pretreatment to cellulose production ability of NH11 and degradation rate of substrates was studied. Morphological characteristics of NH11 was observed by electron microscope and identified by 16S rRNA and Biolog method. [Results] Bacterial strain NH11 was isolated and identified as Bacillus subtilis. The maximum degradation rate of untreated and pretreated corn stalk was 14.24% and 24.73% when culture temperature was 30 °C after five days. CMC cel-lulose activity of NH11 reached to 153.84 U/mL and FPA cellulose activity to 197.24 U/mL in treatment group, 11.45% and 10.59% higher than untreated group. [Conclusion] NH11 has a high cellulase productivity, and NH3?H2O pretreatment could enhance the degradation rate of corn stalk. NH11 has a high value in straw compost, mushroom culture medium and ruminant feed production.

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