Abstract:[Objective] To obtain the high-yield κ-carrageenase by the recombinant strain BL21-HTa-cgkZ. [Methods] The optimal fermentation conditions of the recombinant strain BL21-HTa-cgkZ were determined by using single factor design and response surface analysis in the flask. [Results] The optimal medium compositions were composed of lactose 7 g/L, tryptone 10 g/L, yeast extract powder 5 g/L, NaCl 10 g/L and CaCl2 0.666 g/L. The optimal induction condition was adding IPTG 0.89 mmol/L after inoculation for 1.55 h and maintaining the fermentation at 23.03 °C for 24 h. The distribution of κ-carrageenase expressed by recombinant strain BL21-HTa-cgkZ was also affected by the additive such as lactose and triton X-100 in the medium. [Conclusion] The results provide the clues for the mass production of κ-carrageenase by the recombinant strain BL21-HTa-cgkZ.

Abstract:

Abstract:[Objective] The application of chemical fertilizers and straw returning are important agricultural strategies to improve the fertility of lime concretion black soil. However, there is little information about the impact of fertilization on microbial community in lime concretion black soil. The objective of this study was to investigate the effect of long-term application of chemical fertilizers and wheat straw returning on bacterial community in lime concretion black soil. [Methods] Quantitative PCR (qPCR) and 454 pyrosequencing-based analysis of the V4-V5 16S rRNA gene region were used to determine bacterial abundance, community structure and diversity in soils under different fertilization strategies. [Results] The most dominant bacterial phyla in lime concretion black soil were Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi and Bacteroidetes. Long-term application of chemical fertilizer greatly increased soil fertility, but resulted in significant bacterial community shift and diversity loss. Although wheat straw returning improved soil fertility, it could not mitigate the negative impact of chemical fertilizers on soil bacterial community. Statistical analysis revealed that changes in microbial community composition and diversity were strongly correlated with changes in pH induced by the fertilization regime. [Conclusion] Long-term application of chemical fertilizers and straw returning improved the fertility of lime concretion black soil, but soil acidification caused by fertilization had negative impact on soil bacterial community. Our results emphasized the importance of soil pH in addition to soil fertility for agricultural sustainability in lime concretion black soil region, and suggest that straw returning has little help for the stability of microbial community in lime concretion black soil, and comprehensive means must be taken to develop environmental friendly agriculture.

Abstract:

Abstract:[Objective] To improve thermal tolerance of Saccharomyces cerevisiae strains for efficient ethanol fermentation under high temperature. [Methods] Vacuolar proteinase B encoding gene PRB1 was integrated into the genome of S. cerevisiae, and cell growth and ethanol fermentation of the recombinant strain were compared with those of the control strain carrying the empty plasmid at 41 °C. [Results] PRB1 overexpressing strain consumed glucose completely in the fermentation broth within 31 h, whereas high residual glucose was detected in the fermentation broth of the control strain (35 g/L, about 40% of the total sugar amount) at 23 h and the glucose level was not changed until 31 h. [Conclusion] Overexpression of vacuolar protease B encoding gene improved yeast thermal tolerance and ethanol production under high temperature. The robust strain developed in this study has provided basis to further improve thermal tolerance and efficiency of high-temperature ethanol fermentation of the industrial strains and reduce the production cost of fuel ethanol.

Abstract:[Objective] The aim of this study is to screen culturable phosphate-solubilizing bacteria from water samples of the Indian Ocean, and tackle their mechanism of regenerating phosphate from organics and their phylogenetic diversity in the region. [Methods] Phosphate-solubilizing bacteria were selected by inoculating bacteria isolated from the Indian Ocean waters onto solid lecithin medium, and the phosphate-solubilizing bacteria were identified according to their 16S rRNA genes. Three strains of high phosphate-solubilizing activities were studied for their alkaline phosphatase production and phosphate-solubilizing in fermentation liquid. [Results] A total of 99 phosphate-solubilizing strains from 16 genera were selected from the 916 available bacteria strains. The dissolved inorganic phosphate (DIP) concentration presented N-type curves in cultures of all the three active phosphate-solubilizing stains India-BSP-1 (Cobetia sp.), India-BSP-21 (Pelagibaca sp.), India-BSP-23 (Pelagibacterium sp.), and the alkaline phosphatase was detected after DIP production. [Conclusion] Phosphate-solubilizing bacteria of the Indian Ocean waters presented rich species diversity and three candidate new species. The characteristics of bacterial regeneration of phosphate from organics were influenced by both DIP and alkaline phosphatase.

Abstract:[Objective] To realize in vitro biosynthesis and activity detection of quorum sensing signal molecule autoinducer 2 (AI-2) of Aeromonas hydrophila. [Methods] LuxS, MtnN-1, MtnN-2 proteins were analyzed for amino acid sequences, expressed and purified. AI-2 was synthesized from S-adenosylhomocysteine (SAH) with purified LuxS and MtnN-1, or purified LuxS and MtnN-2 separately. The activity of AI-2 was detected by the reporter strain Vibrio harveyi BB170. [Results] The activity of AI-2 in A. hydrophila culture supernatant was 16.96-fold of the control at 8 h. As analysis of amino acid sequence alignment indicated, the identity of LuxS of A. hydrophila with that of V. harveyi and Edwardsiella tarda is above 76%. The identity of MtnN-1 with MtnN-2 is 26.37%. MtnN-2 has relatively high homologies with representative Pfs amino acid sequences. The identity of MtnN-2 with that of V. harveyi and E. tarda is above 53%. LuxS, MtnN-1 and MtnN-2 proteins were successfully expressed and purified. The activity of AI-2 produced with purified LuxS and MtnN-1 in vitro was 45.04-fold of the control; the activity of AI-2 produced with purified LuxS and MtnN-2 in vitro was 63.62-fold of the control. [Conclusion] A. hydrophila could secrete AI-2. MtnN-1 and MtnN-2 could both cooperate with LuxS and realize in vitro biosynthesis of AI-2, though the amino acid sequences between the two showed relatively big differences.

Abstract:[Objective] Naphthalene is the simplest of polycyclic aromatic hydrocarbons and is considered as an environmental pollutant that has gained a lot of public concern owing to its carcinogenicity and persistent organic properties. Biodegradation is generally recognized as the mass balance-wise most important route of naphthalene degradation. A previous study has revealed that Pseudomonas stutzeri YC-YH1 could degrade naphthalene effectively. This study aimed to characterize degradation, optimize the degradation rate of Pseudomonas stutzeri YC-YH1 for naphthalene and investigate naphthalene degradation pathways. [Methods] Firstly, effects of pH, temperature, inoculum amount and initial concentration of naphthalene for the naphthalene-degrading rate of Pseudomonas stutzeri YC-YH1 were determined by single factor experiments. Then based on the results of the single factor experiments, the software Design Expert 8.0.5 and Box-Behnken design was used to analyze and optimize the model of naphthalene degradation rate by three key factors (pH, temperature, and inoculums amount) through response surface methodology. LC-MS was used to analyze the degradation pathway and products. [Results] An optimization model was then constructed, and its validity was verified. The model was significant (P<0.001) and fitting well. In confirming experiments, under the optimization condition of temperature 32.4 °C, pH 7.10, inoculation 5.74% (V/V), the degradation rate of 100 mg/L naphthalene was 100% after incubation for 3 days. LC-MS data indicate that it is possible to find three major metabolites of naphthalene, which include 1,2- dihydroxynaphthalene, salicylic acid and catechol. [Conclusion] The results suggested that three key factors (pH, temperature, and inoculums amount) could affect the degradation rate of naphthalene, and optimization of the response surface methodology could improve the biodegradation efficiency of naphthalene by Pseudomonas stutzeri YC-YH1. The possible degradation pathway of strain YC-YH1 was catechol pathway. Naphthalene was first turned into1,2-dihydroxynaphthalene, and then salicylic acid and pyrocatechol, finally entered the tricarboxylic acid cycle.

Abstract:[Objective] At present, studies on terpenoid synthase mainly focus on plants and fungi. The systematic study of bacterial terpenoid synthase is still rare. Based on a large number of bacterial genomes, we identified bacterial terpenoid synthase in genome-wide and predicted their function by bioinformatics. [Methods] Local bacterial proteome database was first built based on the bacterial protein sequences, and hidden markov model (HMM) of PF03936 in Pfam was used to predict bacterial terpenoid synthase. Then multiple sequence alignment for candidate terpenoid synthase was aligned by the tool of MAFFT 7.130b, and phylogenetic tree was constructed by MEGA 6.0. Finally, analysis of motifs was conducted by MEME and point mutation of TPS by PredictProtein. [Results] In total 1 423 terpenoid synthases were identified; they are distributed in 8 phylum, including: Actinobacteria, Proteobacteria, Cyanobacteria, Bacteroidetes, Firmicutes, Chloroflexi, Acidobacteria and Chlamydiae. Phylogenetic analysis revealed that bacterial terpenoid synthases can be divided into 4 groups. Motifs analysis of each group by MEME showed that in addition to conserved motifs among groups, there were some specific motifs within each group, which implies functional differentiation among different groups. Point mutations analysis showed that mutations of amino acid sites in different positions of terpenoid synthase could have different effect on TPS function. [Conclusion] Bacterial terpenoid synthases are mainly distributed in 8 phylum, of which 2 phylum have not been reported before; they are Firmicutes and Acidobacteria. Phylogenetic analysis showed that the difference among 4 groups of terpenoid synthase is mainly caused by group-specific motifs. In addition, mutation analysis of amino acids in different positions of terpenoid synthase provides a fundament for further verification of bacterial terpenoid synthase function.

Abstract:[Objective] To isolate a chlorogenic acid (CGA)-producing endophyte, and to study the function of a key gene of the CGA synthetic pathway from the endophyte. [Methods] Endophytes were isolated from Flos Lonicerae japonicae by surface sterilization. The endophytic bacterium producing CGA was screened and confirmed by HPLC and LC-MS. Histidine ammonia lyase (HAL) gene of the endophytic bacterium was cloned and expressed in Escherichia. coli and enzyme activity of the recombinant HAL was determined. [Results] An endophytic bacterium RP1 isolated from the root of Flos Lonicerae japonicae was confirmed to produce CGA. Moreover, cinnamate, an intermediate of CGA synthetic pathway, was detected from RP1 fermentation broth. RP1 was identified as Bacillus subtilis through 16S rRNA sequence analysis. The recombination HAL protein had the dual function of HAL and PAL (phenylalanine ammonia lyase). Cinnamate produced by PAL activity of recombination protein agreed with the result of LC-MS. [Conclusion] Presumably endophytic RP1 laded cinnamate, produced from phenylalanine by the PAL activity of HAL, to the phenylpropanoid metabolic pathway to produce CGA.

Abstract:[Objective] The mesotrione-resistant strain was isolated from soil, and the resistant gene 4-Hydroxyphenylpyruvate dioxygenase was cloned and characterized. [Methods] Using tyrosine as the sole carbon source, the strain was isolated by gradient enrichment culture. The isolated strains were identified based on 16S rRNA gene sequence. The resistant gene 4-Hydroxyphenylpyruvate dioxygenase was cloned by PCR amplification using the genomic DNA as a template. The expression plasmid pETH4 was constructed and expressed in Escherichia coli BL21(DE3). The mesotrione-resistant property of E. coli BL21(DE3)-pETH4 was tested by measuring the absorbance at 440 nm. [Results] Seven bacterial strains, which could grew on MSM medium containing 10 mmol/L mesotrione and 1 g/L tyrosine, were isolated. They were identified as Acinetobacter sp., Achromobacter sp. and Pseudomonas sp.. PCR fragment of 1 056 bp was obtained from Pseudomonas sp. AM-H4, which exhibited the best resistance to mesotrione. The cloned HPPD gene shared 99% sequence identity with that from Acinetobacter baumannii, replacing aspartate-341 with alanine. The protein of HPPD was expressed in E. coli BL21(DE3) with the molecular mass of 40 kD. The absorbance of brown pigment produced by E. coli BL21(DE3)-pETH4 in tyrosine-LB culture was significantly reduced in the presence of 40 μmol/L mesotrione. In addition, the brown pigment was still visible when the concentration of mesotrione was greater than 200 μmol/L. [Conclusion] The HPPD gene with high mesotrione-resistance was obtained in this study, which maybe a novel gene source for the application in breeding of herbicide-resistant crops.

Abstract:[Objective] The aim of this study is to improve furfural tolerance of Saccharomyces cerevisiae by constructing its recombinant strain with fine-tune expression of MSN2 under a self-regulated promoter control. [Methods] The ADH7 promoter, coding region of MSN2, and CYC1 terminator sequences from genomic DNA of the S. cerevisiae strain BY4742 were obtained by PCR amplification, and then were ligated into the pUG6 plasmid resulting in a recombinant plasmid pUG6-AM containing the ADH7p-MSN2-CYC1t cassette. The recombinant plasmid was transformed into the S. cerevisiae BY4742 strain by the LiAc/SS-DNA/PEG transformation method after linearization, and then positive transformants were screened. Tolerability of a selected recombinant yeast strain to furfural was compared to its control, and transcription responses of MSN2 and its representative regulons between this recombinant strain and its control under both furfural stress and normal conditions were further comparatively studied. [Results] A recombinant S. cerevisiae strain (named as AM01) with fine-tune expression of MSN2 under the ADH7 promoter control was successfully constructed. This recombinant strain showed significantly improved tolerance to furfural, and MSN2 displayed fine-tune transcription response to furfural stress and then positively affected transcription responses of its regulons. [Conclusion] Fine-tune expression of MSN2 controlled by a self-regulated promoter from a gene induced by furfural not only improved furfural tolerance of the genetically engineered S. cerevisiae strain, but also avoided side-effects due to its constitutive overexpression under the strong promoter control.

Abstract:[Objective] Strain YBT2532 was screened from 400 Bacillus thuringiensis strains against Xanthomonas oryzae pv. oryzae. Its antimicrobial substance was isolated. [Methods] Physical and biochemistry characteristics of the antimicrobial active substance produced by the B. thuringiensis strain YBT-2532 were determined. [Results] It is not sensitive to temperature, protease and pH. After incubation at 70 °C for 1 h, still 75% activity retained. Proteinase K, trypsin and pepsin had no effect on its activity. The active substance was stable in the pH range between 2.0 and 12.0. It can be dissolved in water and methanol, weakly dissolved in ethanol, insoluble in acetone, chloroform and dichloromethane. Purification of the active substance was achieved using gel chromatography, ion exchange chromatography, solid phase extraction and HPLC. The purified active substance was analyzed by HPLC-IT-MS. It is a polar water soluble small molecule of 797.8 Da. [Conclusion] A new antibacterial substance was obtained from an isolate of Bacillus thuringiensis.

Abstract:[Objective] To study bacterial communities and diversity in shrimp (Exopalaemon carinicauda Holehuis) intestine. [Methods] 16S rRNA gene was amplified and a library was constructed by genomic DNA that was extracted from the bacteria in shrimp intestine. [Results] Different RFLP patterns of the clones were obtained from analysis with Hae Ⅲ and Msp I. By comparing the obtained sequence with the published sequence in the GenBank database, 114 clones were obtained in the 16S rRNA clone library, and 11 different RFLP patterns of the clone were affirmed by Hae Ⅲ and Msp I. There were 8 clone sequences in high identity with known bacteria sequence, respectively belonging to Pseudomonas (17.5%), Enterobater (21.1%), Frigoribacterium (8.8%), Phaeobacter (5.3%), Vibrio (10.5%), Erythrobacter (9.6%), Aeromona (4.4%), and Staphylococcus (2.6%). In addition, uncultured bacteria accounted for 19.3%. [Conclusion] This study discovered the bacterial communities in the intestine of Exopalaemon carinicauda Holehuis, and the results would enhance our understanding about the bacterial community composition in Exopalaemon carinicauda Holehuis intestine and provide valuable data for the development of disease prevention mechanisms for shrimp cultivation.

Abstract:[Objective] To study recycled nutritional composition in by-products from edible mushroom deep processing. [Methods] The main nutritional composition included total sugar, protein, crude fiber ect. in ten kinds of edible mushroom by-products were measured, and the glucan configuration and amino acid composition were also quantitatively analyzed. [Results] The edible mushroom by-products still contained abundant protein and polysaccharide. The polysaccharide in by-products mainly consisted of bioactive β-glucan. All the by-products contained 17 kinds of amino acids and 7 kinds of essential amino acids. Among them, the total sugar and β-glucan contents in Poria cocos by-product were 66.04% and 59.46%, the protein content in Ganoderma lucidum spore by-product reached to 36.17%, the essential amino acids content in Cordyceps militaris by-product was 10.43%. [Conclusion] Edible mushroom by-products contain high contents of nutritional composition, which have large reused value, this study establishes a basis for the sustainable development of edible mushroom industry.

Abstract:[Objective] The study aims to optimize solid-state fermentation conditions of citrus processing by-products with Aspergillus aculeatus for naringinase production. [Methods] The naringinase activity was detected by a high performance liquid chromatography method. The effects of solid water ratio, loaded sample, inoculation quantity and culture temperature were investigated on producing naringinase by single factor experiments and the fermentation conditions were further optimized with 3 factors at 3 levels in an orthogonal experimental design. [Results] Single factor experiments showed that solid water ratio, loaded sample and culture temperature had significant effects on the yield of naringinase, while inoculation quantity has no significant effect. The optimized fermentation conditions for A. aculeatus are: solid water ratio of 1:1 (W/V), loaded sample of 5 g/250 mL triangular flask, inoculation quantity of 1 mL at 30 °C for 8 days. Under optimal culture conditions, the activity of naringinase reached 8.19 IU/g dry substrate, which was 7.38-fold higher than that achieved before optimization. [Conclusion] There is significant improvement in naringinase production in pomelo peel solid-state fermentation by A. aculeatus after optimization. This study established a high-efficiency fermentation process for naringinase production.

Abstract:[Objective] To eliminate virus contamination in Flammulina velutipes, we assessed five detoxification methods. [Methods] F. velutipes F4889 was used to isolate 2.0 kb dsRNA, and the virus was identified as F. velutipes browning virus (FvBV) by RT-PCR. Five virus-eliminating methods, i.e. hyphal tips isolation virus elimination (HTIVE), primordium tissue isolation virus elimination (PTIVE), protoplast monokaryotization virus elimination (PMVE), sexual reproduction virus elimination (SRVE) and nuclei migration virus elimination (NMVE) were applied to prepare virus-free strains. The effect of virus-elimination was detected and identified through dsRNA technique and RT-PCR. [Results] Only one virus-free strain was obtained by HTIVE method. Strains of PTIVE method remained the virus. PMVE method achieved three monokaryon strains and two protoplast monokaryon hybridization virus-free strains. Twenty-three sporulated monocaryotic strains and 8 single spore hybridization strains were virus-free after using SRVE method. NMVE method acquired 5 virus-free strains. Hyphal growth rate, biomass and laccase activity of virus-free strains by PMVE, SRVE and NMVE methods were obviously superior to the original strain and strains by HTIVE and PTIVE methods. [Conclusion] The detoxification effect of PMVE, SRVE and NMVE methods was apparently better than others. These three methods completely eliminated FvBV and had higher virus-free rate. Moreover, mycelia growth rate, biomass and laccase activity of virus-free strains significantly improved.

Abstract:[Objective] To research the alternative resources of Rhodiola crenulata, a tyrosol-producing strain was isolated and identified from the root of Rhodiola crenulata. The production features of the strain were preliminary studied. [Methods] Endophytic bacteria were isolated from Rhodiola crenulata with NA medium. The best potential strain to produce tyrosol was selected through thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), followed by identification by colony morphology, gram staining analysis and 16S rRNA gene sequence profiles. Single factor experiments based on initial pH, culture temperature, incubation time and inoculum size were performed to optimize the conditions for tyrosol-production. [Results] In this study, a total of 14 strains of endophytic bacteria were isolated from the root of Rhodiola crenulata. The best potential strain B3 was selected from 8 strains which can produce tyrosol. B3 strain was preliminary identification to Rahnella aquatilis by colony morphology, gram staining analysis and 16S rRNA gene sequence analysis. Researching its fermentation conditions, we found out that the optimum pH was 6.0, optimum temperature was 32 °C, the best fermentation time was 42 h, the best inoculation amount was 15%, and at the best fermentation conditions, the yield of tyrosol can reach 15.68 mg/L with improved NA culture medium. [Conclusion] B3 strain is able to produce tyrosol, and the yield can reach to 15.68 mg/L at the optimal fermentation condition. The tyrosol-producing strain is expected to be explored its marketing values.

Abstract:[Objective] To study the transcriptional regulation of vopT by AphA in Vibrio parahemolyticus. [Methods] Total RNAs were extracted from the wide-type (WT) strain and the aphA mutant (ΔaphA). Primer extension assay was employed to detect the transcription start site and the promoter activity of vopT in WT, and that in ΔaphA. Quantitative RT-PCR was also applied to calculate the transcriptional variation of target genes between WT and ΔaphA. The entire promoter region of vopT was cloned into the pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and ΔaphA, respectively, to measure the promoter activity (the β-Galactosidase activity) of vopT in WT and ΔaphA by using the β-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), and the entire promoter region of the target genes was amplified by PCR. Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to target promoter regions in vitro. [Results] Primer extension assay detected only one transcriptional start site located at 86 bp upstream of vopT, whose transcript was negative regulated by AphA in an indirectly manner. Moreover, RT-PCR and EMSA results showed that the transcription of vtrA was also indirectly controlled by AphA. [Conclusion] AphA repressed the transcription of vopT indirectly, and this indirect inhibition was not dependent on VtrA.

Abstract:[Objective] To clone, express and purify envelope protein sVP53B of WSSV (white spot syndrome virus) for polyclonal serum preparation. [Methods] Based on the GenBank sequence of WSSV vp53B, primers were designed, and PCR was done to amplify functional sequence of Svp53B. Svp53B was inserted into prokaryotic expression vector, pET-16b. Recombinant plasmid was transformed into Escherichia coli Rosetta 2. The expression of sVP53B was detected by SDS-PAGE and Western blotting. The protein was purified with Ni-NTA agarose beads, then applied to immune rabbits for polyclonal serum detecting it’s titer by indirect ELISA (enzyme-linked immuno sorbent assay). [Results] The sVP53B was expressed most by 1 m mol/L IPTG (isopropyl-β-D-thiogalactoside) inducing and mainly expressed as inclusion bodies. Polyclonal serum, prepared by immunizing rabbit, revealed a titer as high as 1:150 000. [Conclusion] Recombinant protein sVP53B was highly expressed in E. coli and showed high purity after purification. The prepared polyclonal serum had high affinity and specificity against sVP53B. The results pave the way for functional study of VP53B during WSSV oral infection.

Abstract:[Objective] To investigate and discover resources and diversity of plant pathogenic fungi in China. [Methods] Classical morphological features and ITS rDNA sequences were used for fungal identification. [Results] Gonatobotryum apiculatum was isolated from the fallen forest leaves in Pingshui Town, Shaoxing City, Zhejiang Province. The morphological characteristics of G. apiculatum were observed and described in detail. In addition, the ITS rDNA of G. apiculatum was amplified and sequenced. [Conclusion] G. apiculatum causing leaf spot of Hamamelis sp. was recorded for the first time from China.

Abstract:Many clinical trials showed that chronic alcohol leads to imbalance of intestinal flora which is characterized by the decrease of Bifidobacterium and Lactobacillus, with a proportional increase of gram-negative bacteria. Alcohol causes the destruction of intestinal barrier function, increases intestinal permeability, releases bacteria endotoxins, and causes liver damage. This paper reviews the research progress in using probiotics to repair alcoholic liver injury by adjusting the intestinal flora and improving intestinal barrier function.

Abstract:Folate are presented in all kinds of foods, but folate deficiency in human body still exist in every country due to the difference of dietary habit and unstability of folate. Folate are the important material that participate in synthesis of nucleic acid and the cell division and differentiation. Folate deficiency lead to the disorders of body functions, thus lead to a serise disease such as cancer. Even though most lactic acid bacteria (LAB) are folate-defective strains, more and more studies indicated that a lot of strains among LAB have the capability to synthesize folate. This review summarized the progress on species of LAB with the capacity of synthesizing folate, their folate synthesis mechanism, and folate gene engineering carried by LAB.

Abstract:The Na+/H+ antiporters play a primary role in the maintenance of intracellular pH homeostasis and dynamic balance of cellular Na+, and in the regulation of cell volume. Currently, many Na+/H+ antiporter genes involved in the resistance to high salt and alkaline pH stresses have been identified and functionally characterized in various bacteria. The prospection for novel Na+/H+ antiporters from unexplored habitats, in conjunction with our understanding of the mechanisms responsible for Na+/H+ antiport activity, will provide new insights into industrial and agricultural biotechnology. In this review, we chose four physiologically distinct bacteria as examples, and summarized the classification, structure and function of bacterial Na+/H+ antiporters.

Abstract:Small RNAs in bacteria is a kind of 40?400 nt non-coding RNAs which play an important role in regulation while the environmental conditions (such as temperature, nutrition, outer membrane protein, pH and iron) change. Small RNAs generally transport environmental stress signals and stress responses through the two component signal system and regulatory proteins, and play functions via antisense base pairing with target mRNAs to regulate translation and degradation of target mRNA, or via directly binding proteins and then modulating their activities. This paper reviewed the regulation roles and the response mechanism of sRNAs in bacteria in different stress.

Abstract:The plant-derived terpenoids are widely used in the industries of food, medicine, cosmetics as well as agriculture, and it plays an increasingly vital role in the people’s life. However, terpenoids of natural sources are unavailable in sufficient amounts to meet the requirement of people’s life, because of the slow growth, complexity of extraction techniques and high cost of production, which make its application has been greatly limited. S. cerevisiae, as one kind of simple eukaryotes has the advantages of clear genetic background, fast growth and easy manipulation by genetic engineering. Furthermore, natural biosynthetic pathway of isoprenoids is also found in yeast cells. Therefore, yeast can be take as engineering host to reconstruct the isoprenoid pathway to fit the biosynthesis of plant terpenoids after fermentation. In this article, the biosynthesis process of terpenoids, along with the achievements and problems of engineering yeast to produce terpenoids will be reviewed.

Abstract:The cultivation aim of Bio-engineering experiment teaching is to construct “bead-chain” mode. This paper shows some approaches in reforming experimental teaching. By optimization of experiment teaching contents, improving experiment teaching method and examination way, increasing comprehensive and designing experiments of project, constructing of the open laboratory and so on, good effect was achieved by the formation of an orderly “chain” of knowledge system of Bio-engineering. An innovative experimental teaching system of “bead-chain” is constructed which based on the microorganism producing amylase as a starting point and the experiment project for “bead” in the mode of genetic engineering, enzyme engineering, fermentation engineering. The experimental teaching system enables the students to have the ability of application, innovation and competitiveness of employment.

Abstract:[Objective] Prepare anti-aflatoxin B1 monoclonal antibodies to develop an indirect competitive ELISA (ic-ELISA) applied to detect AFB1 in samples. [Methods] The aflatoxin B1 (AFB1) was conjugated with bovine serum albumin by the carbodiimide method and used to immunize Balb/c mice. Hybridoma cell strains could be obtained by clonal screening and monoclonal antibodies (mAb) could be obtained by inducing ascites in vivo. The subclass and titer of mAb were identificatied by indirect ELISA. After optimizing experimental conditions, a stable ic-ELISA would be developed and applied to detect AFB1 in feed samples. [Results] Four hybridoma cell strains stably secreting specific antibodies to AFB1 were obtained. One of the cell strains 3B9 was chosen to prepare mAb, The mAb belongs to IgG1 with a titer of 1: 204 800. Its cross-reactivity with aflatoxins B2, G1, G2 and M1 was 2.2%, 33.9%, 1.8% and 4.1% respectively. It did not react with ochratoxin A, fumonisin and zearalenone. An indirect-competitive ELISA (ic-ELISA) coated with the mAb had a detection range of 1.04?25.00 μg/L with the detection limit at 1.04 μg/L and the 50% inhibitory concentration (IC50) at 6.03 μg/L. The average recovery rate ranged from 85% to 120% with the variation coefficient less than 10%. [Conclusion] The ic-ELISA has good consistency compared with a commercial kit. We suppose that the ic-ELISA could be used to detect AFB1 from food or feed samples.

Abstract:[Objective] Oolong tea extract (OTE) was tested for its potential as an electron staining reagent to substitute for uranyl acetate (UA) in electron microscopy of bacterial cells. [Methods] Three electron microscopic staining methods (standard, 0.05% OTE, and 0.1% OTE) were compared for electron microscopic observation of ultrathin sections with or without lead citrate (Pb). First floating the sections on the compared staining solutions for 10?15 min, then if need to post-stained with lead citrate, after rinsing with deionized water three times, the sections were re-floated on lead citrate for 8?10 min. Electron microscopy was performed on sections from LR-White-embedded cells of a Gram-negative bacterium, Escherichia coli, and a Gram-positive bacterium, Staphylococcus aureus. [Results] Both concentrations of the OTE preparations showed the staining results were similar to that of standard method with UA and Pb in E. coli and S. aureus cells. [Conclusion] This study demonstrated that OTE could potentially be used as an alternative to UA in electron microscopy staining for certain structures.