Biotechnology Bulletin

Discussions on Main Areas and Supporting Strategies in Transgenic Crops R&D in China

Liu Rongrong

2014, 0(9): 1-6.

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Crop transgenic engineering, one of the most important and active research areas in agricultural biotechnology, is an essential stratigic pathway to develop modern seed industry in China. In this article, the weaknesses and abilities of great-leap-forward development in transgenic crops R&D in China were analyzed, meanwhile main study areas that need more investment were summarized, in order to discuss policy and advice to improve input-output ratio in transgenic crops R&D in the future.

Advances in the Study of Endophytes to Control Plant Parasitic Nematodes

Jiang Xuwen,Li Heqin

2014, 0(9): 7-12.

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Endophyte as a biocontrol agent of natural resources, has attracted more and more interest of the researchers. In this paper, the types and distribution, methods of isolation, culture and screening, bioactive substances, mechanism of nematicidal endophyte were reviewed, and the problem and possible future direction of nematicidal endophyte were discussed. To provide theoretical reference for correlative research on nematicidal endophyte.

Mannose Selection System and Its Commercial Application in Transgenic Corn

Liu Jianfeng, Wang Yanli,Li Xianggan

2014, 0(9): 13-21.

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Mannose can be converted into mannose 6-phosphate in cells and further converted into fructose 6-phosphate in transgenic cells if phosphomannose isomerase（PMI）gene（manA）is introduced as a selection marker. The transformed cells can grow normally on the media with mannose as main carbon source while the non-transformed cells are inhibited to grow. As a positive selection, Mannose / PMI system has been applied to important food crops and important economic crops. The plants selected by mannose can be analyzed through a variety of methods, including simple CPR test and convenient strip test. Mannose selection system is a safe and efficient platform suitable for commercial applications. In this review, advances in mannose selection system with its mode of action, selection characteristics, methods of transgenic plants analysis, advantages and disadvantages in transformation, as well as its applications in transgenic corn production and breeding stacks were summarized. Successful examples to use Mannose / PMI selection system in corn to generate multiple single trait events for insect resistances and high temperature resistant amylase, which have been breeding-stacked with other corn traits selected by different selection systems to provide broad spectrum of herbicide resistance and insect resistance for higher commercial value were presented.

Advances in Studies on Propagation Technology of Cardiocrinum（Endl.）Lindl.

Wang Xiaofei,Zhou Zhiguang,Wang Yuyi

2014, 0(9): 22-27.

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The Cardiocrinum are wild ornamental plants with important application value. The research on its propagation techniques system will establish the foundation for application promotion. This article reviewed the advances of its propagation techniques in the aspects of introduction, seed physiology, tissue culture etc. It is possible, the author thinks, to multiply largely by using seeds and tissue culture technique, and the cross-breeding still needs further research. The study should also be made on modern biotechnology.

Progress in Livestock Induced Pluripotent Stem Cells

Li Jianing,Liu Pengxia

2014, 0(9): 28-33.

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To date, producing functional germline-competent embryonic stem cells（ESCs）has proven to be challenging in most livestocks, like pig, sheep, cattle et al. Induced pluripotent stem cell（iPSC）technique provides a feasible approach to establish pluripotent stem cells for a species in which ESCs have previously proven to be difficult to establish from the early embryo. Livestock iPSCs can offer a route to promote developmental biology research, obtain organs for xenotransplantation, establish animal models for human genetic disease, generate high quality and disease-resistant new breeds et al. The fundamental researches and address the perspectives of livestock iPSCs were reviewed.

The Role of miR-143 in the Adipocyte Differentiation and Lipid Metabolism

Li Jing, Huang Ying, Huang Limei, Yang Minghua, Li Qihua, Jia Junjing, Zhao Sumei

2014, 0(9): 34-38.

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miR-143, discovered in recent years, is a non-coding RNA derived within a class, which plays an important role in many biological processes including cell differentiation, growth, proliferation, fat metabolism, disease and so on. miR-143 is closely related to the adipocyte differentiation and lipid metabolism, which could regulate the differentiation of adipocytes, synthetics of triglycerides primarily by the interaction with the target gene. The synthesis of miR-143, the interaction between miR-143 and the target genes to regulate adipocyte differentiation and lipid metabolism, in order to provide new ideas to the diagnosis and treatment for the obesity disease were reviewed.

Research Progress on Biological Characteristic and Applications of Laccase from Edible Fungi

Zhang Peng,Wang Yanfeng, Pan Chunlei, Sheng Chunge, Wang Jinhe, Shi Lei

2014, 0(9): 39-44.

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Laccase is a kind of polyphenol-oxidase which contains copper and has a strong capacity of decomposing lignin. It can be secreted by edible fungi during the growth and development. At present, there’s few reports on the mushroom laccase. The article summarized the biological characteristics and method of analyzing enzyme activities and the advances of the research application on the laccase.

Comparison on Optimization of Preparation Methods of Taxol Containing Liposomes

Qiao Guangjun,Zhang Baokui, Yang Rong, Jiao Yi, Ji Dalin

2014, 0(9): 45-50.

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To optimize the docetaxel-containing liposome preparation technology, some liposome preparation methods were researched, such as reverse evaporation technique, high shear, film dispersion method, injection and multiple emulsion method. The single factor experiment was adopted to inspect the reverse evaporation technique and prescription preparation. The ideal combination of preparation and formulation were as follows：taxol-phospholipids=1∶25, phospholipid content 2.5%, cholesterol-phospholipids=1∶9. The reverse evaporation technique and the optimized preparation can be used to prepare paclitaxel liposome successfully.

A Rapid and Simple Method for DNA Extraction from Plant Leaves

Chi Jing,Geng Lili, Gao Jiguo, Shu Changlong,Zhang Jie

2014, 0(9): 51-57.

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In order to resolve the needs of PCR analysis of a large number of plants in study on molecular biology, we established a convenient and quick method for extraction of genomic DNA from plant leaves without grounding or organic reagents. The genomic DNA was isolated using TE buffer containing 1% beta-mercaptoethanol and crushed by a mini-beadbeater tissue homogenizer. Through the comparison of concentration and purity of genomic DNA and the results of PCR analysis, it indicated that the supernatant collected after quick frozen was suitable for PCR analysis. The whole protocol only takes 10-12 minutes, and it is a simple, efficient and economic method which needs less amount of samples. The genomic DNA of tobacco, rice, soybean, maize, rape and peanut extracted by this methord is suitable for PCR amplicafition. A 3 244 bp gene can be amplified and 4 new actin genes of peanuts are obtained using genomic DNA isolated by this method.

The Research of an Improved Method for Protein Staining in Electrophoresis with Coomassie Brilliant Blue

Rao Weiqiao, Xiao Weimin, Zhang Jiyuan, Rao Yuan, Zi Jin

2014, 0(9): 58-64.

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In order to establish a convenient, sensitive, economic and Mass Spectrometry compatible staining method for protein polyacrylamide gel electrophoresis, herein, we established optimized staining methods based on Blue Silver Staining, by optimizing the gel immobilization time and buffer content. Comparison result indicated the method 1 keep the similar high sensitivity and well Mass Spectrometry compatible feature as Blue Silver Staining, what’ s more, is more easily conducted, less organic solvent consuming and have better correlation between protein amount and staining density.

A Novel Plant miRNA Knockdown System in Vivo

Jiang Feng, Xu Shuo, Hu Zheng,Jiang Qiyan, Zhang Hui

2014, 0(9): 65-71.

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MiRNA is a kind of endogenous small RNA about 21-25 nuleotides in length that plays important roles in plant morphology and development. The present study was performed to establish a novel system for plant miRNA knockdown in vivo, supply a enegetic tool for miRNA function analysis. Unlike the original target mimicry vector method, we obtained multi-copyed target mimicry sequences using self-ligation method with designed specific DNA fragment based on miRNA motif, construct expression vector and transform to Arabidopsis thaliana. Starting with the conserved miRNA in Arabidopsis, we obtained six transgene lines of miRNA knockdown. We detected the relative expression of related miRNA targets in transgene plants, resulting obviously up-regulated compared with the relative expression in wild type. Based on target mimicry vector construction, we obtained MIM stable lines in Arabidopsis, successfully established a general and high effective miRNA knockdown system in plant species.

Expression Analysis of TCTP Gene in Susceptible Wheat Jing411 Induced by Powdery Mildew

Zheng Shuyang, Wang Yanhong, Xiao Ying, Liu Xiaoying, Wang Zhenying

2014, 0(9): 72-77.

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TCTP plays an important role in biological process, which widely exist in plants, animals, and microorganisms. The expression patterns analysis indicated that TCTP was rapidly and strongly induced by Bgt inoculation in susceptible wheat Jing411. After TCTP gene was silenced by VIGS method, we found that the percentage of succeed Bgt inoculation declined, the proportion of resistant structure increased, such as papilla, and the accumulating level of H2 inhanced rapidly. These results indicated that TCTP gene play an important role in wheat-Bgt interaction.

EMS Mutagenesis，Mutant Screening and Identification of Sorghum

Wang Chunyu,Zhu Zhenxing, Li Dan, Cong ling,Zhang Lixia

2014, 0(9): 78-83.

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Mutation rate and mutation type are important bases for phenotype screening and constructing saturation mutant library. Different treating time and different concentrations of Ethylmethylsulfone（EMS）for BTx623 seeds have been used in this study, mutation rate and mutation type are quite different. Mutation types are very rich, including leaf color, leaf shape, spike type, fertility, and growth period. According to the emergence rate, seed rate and mutation rate, 20 h+EMS 0.2% is considered the optimum treating time and concentration. However, if we want to build a saturated mutant library, different treating-time and various concentrations should be used.

Prokaryotic Expreesion and RNA Interference Vector Construction for Geranyl Diphosphate Synthase of Mentha haplocalyx Briq.

Yu Xu,Liang Chengyuan, Liu Yan, Li Weilin

2014, 0(9): 84-88.

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Geranyl diphosphate synthase（GPPS）gene total ORF（1 131 bp）was amplified with specific primers and linked to expression vector pET-28a. Verified by double restriction enzyme digestion and sequencing, recombined vector pET-28a-GPPS was transferred to Rosetta（DE3）and the recombined protein was overexpressed after 6 h induced by 1 mmol/L IPTG. Using RNA interference method to research gene function is matured gradually. In our research, the expression vector pBI121-RNAi-GPPS targeting to GPPS gene was structured successfully.

Establishment and Optimization of an ISSR-PCR Reaction System for Chieh-qua Using Othogonal Design

Zhao Qin, Xie Dasen, Luo Shaobo, Peng Qingwu, Li Mingzhu, Li Haida

2014, 0(9): 89-96.

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It was to identify and classify chieh-qua germplasm resources. The main factors affected ISSR reaction was tested to optimize ISSR amplification system. In this research, the orthogonal design and single-factor gradient method were used to establish and screen chieh-qua ISSR-PCR system for 4 levels of 5 factors（DNA template, dNTPs, Mg2+ concentration, primer, and Taq DNA polymerase）respectively, and then annealing temperatures were proposed by gradient PCR based on the optimal reaction system established. The results showed that the optimal ISSR-PCR system mixture（20 μL）including 70 ng template DNA, 0.2 mmol/L dNTPs, 1.2 mmol/L Mg2+ concentration, 0.96 μmol/L primer, 0.8 U Taq DNA polymerase and 2.0 μL 10×buffer. The suitable annealing temperature of primer IS807 was 53℃. Four primers were applied to four chieh-qua materials for testing and verifying stability of the established reaction system, which showed that the ISSR reaction system was proved to be stable, credible and of clear bands and rich polymorphism.

Screening SSR Core Primers for Pepper Germplasm Hybrid Purity Identification

Guan Junjiao, Zhang Jianhua, Zhang Hui, Ma Furong, Yang Xiaohong, Wang Jiangmin

2014, 0(9): 97-101.

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In order to determine a set of SSR core primers fitting for pepper hybrids’ purity identification, 100 pepper（Capsicum）hybrids were analyzed by 17 SSR primers. According to their polymorpism and heterogosite rate, three primers Hpms1-214, Es395 and Hpms1-5 were determined as preferred core primers for pepper hybrid purity identification. Using three preferred core primers, 97 hybrids（accounting for 97%）have heterozygosis band type in at least one primer. Five primers Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for pepper hybrid purity identification. 14 varieties had specific primers within the 100 varieties. Primers with complementary band types were further screened for pepper purity identification.

Isolation，Screening and Identification of Antagonistic Actinomycetes of Panax ginseng Pathogenic Fungus

Shi Ce,Han Mei,Zhang Yiming, Guo Shuangshuang, Yang Limin

2014, 0(9): 102-108.

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The antimicrobial activity of four actinomycetes isolated from ginseng rhizosphere was studied with plate-confrontation method and growth rate method. The four strains were identified through their morphological features, physiological and biochemical characteristics and 16S rDNA sequence alignment analysis. The result showed that the four actinomycetes strains could inhibit Panax ginseng pathogenic fungus obviously. In the antagonistic examination in vitro, the inhibition rate of FX-10 against Cylindrocarpon destructans, Botrytis cinerea and the inhibition rate of FX-61 against Sclerotinia ginseng were 91.28%、91.8%、90.07%, respectively. FX-10、FX-61、FX-137 could inhibit 5 kinds of ginseng pathogenic fungus, while FX-139 could inhibit 7 kinds of ginseng pathogenic fungus. The inhibition activity of both was stable. Based on the identifications of morphological, physiological and biochemical characteristics and molecular biology, FX-139 was ascertained as Streptomyces vinaceus-drappus, FX-10 as Streptomyces nigrogriseolus, FX-137 as Streptomyces vinaceus and FX-61 as griseofuscus group of streptomces.

Screening of a Fungus with Insecticidal Activity and Measure of the Stability of Its Bioactive Compound

He Haibo,Zhong Juan,Yang Jie,Zhou Jinyan,Tan Hong

2014, 0(9): 109-113.

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Using Brine Shrimp as primary screening assay and Aphid as second screening assay, the YLZ42 strain with insecticidal activity were isolated and screened from the soil in Yulin, Guangxi Province. According to the culture features, morphological characteristics, 18S rDNA and ITS sequence analysis, YLZ42 was identified as Bionectria ochroleuca. The ethyl acetate extract of YLZ42 fermentation broth have insecticidal activity to Plutella xylostella, Helicoverpa armigera and Laphygma exiguat. Results of bioassay showed that the corrected mortalities rate were more than 78.9%. The fermentation broth have high insecticidal activity under 100℃ for 60 min or pH1-11.

The Research on Mutagenesis Breeding of Phaeodactylum tricornutum and the Condition of EPA Producing

Liu Hongquan, Lin Xiaoyuan, Li Jieqiong, Pan Yihua

2014, 0(9): 114-119.

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In order to obtain algal strains of high EPA production, Phaeodactylum tricornutum was mutated by 0.6% EMS. The mutant strain EP1 was isolated by single cell clone from 200 mutant clones. The EPA yield of EP1 was increased by 19.81% compared with the parent strain. The most suitable foster condition includes NaNO3 75 mg/L, pH7.5, day-night temperature 17-15℃, 12% inoculation quantity and training 7 days. The EPA yield of EP1 could reach 26.77 mg/g under the suitable foster condition. The results of subculture test showed that the mutant strain possesses good hereditary stability.

Screen High-producing Laccase Strain of Bjerkandera fumosa Through UV and 60Co Composite Mutagenesis

Peng Yanchao, Cao Fuxiang,Dong Xujie, Peng Jiqing, Hu Shuangxi

2014, 0(9): 120-124.

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Mutagenic strains ZW-7 was obtained from the spore suspension of Bjerkandera fumosa（5.0172）by UV mutagenesis and screening. The laccase activity was 1.33 times than the original strain. After subcultured five generations, laccase activity was stable. ZW-7 was mutagenized to a new mutagenic strain Co-11 by treating with 60Co-γ ray radiation and its activity was 380.5 U/L, which was 1.18 times than ZW-7 and 1.58 times than the original strain（250.6 U/L）. After subcultured five generations, enzyme activities of Co-11 remains stable.

Identification，Expression and Insecticidal Activity Analysis of cry Genes from Bacillus thuringiensis V4

He Baonan, Li Haitao,Liu Rongmei,Wang Bo, Gao Jiguo

2014, 0(9): 125-130.

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In order to provide more gene resources for biological control, we used the cry1 class full-length primers and genomic DNA of 300 Bacillus thuringiensis strains which stored in our laboratory as the templates to identify cry1 gene-types by PCR. The strain V4 was identified containing a cry1Ea gene by PCR-RFLP, further study found that the strain also contained cry2Aa gene. The genes from the strain V4 were cloned, respectively. The two genes were designated as cry1Ea12 and cry2Aa16 by International Nomenclature Committee of Bacillus thuringiensis. Furthermore, the two genes were respectively transformed into Escherichia coli Rosetta（DE3）strain and the toxicity of the two genes was evaluated. The results showed that the insecticidal activity of the proteins is not very efficient, but the weights of the test insects are inhibited obviously, and the insecticidal activity of the protein of the strain V4 is much higher.

Variance Analysis of Different Mating Type Strains in Mononuclear Protoplast of Hypsizygus marmoreus

Pan Yue,Chen Hui,Feng Zhiyong,Zhao Jing

2014, 0(9): 131-135.

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Tow Hypsizygus marmoreus strains were studied by protoplast isolation and mating-type identification. Metabolic characteristics of different mating type strains were studied. Results showed that each of the 2 Hypsizygus marmoreus strains had 2 mating type. The ratio of two mating-type monokaryons of 2 strains was not 1∶1 and the proportion of mating types had different phenomenon. Different kinds of mating type had different hyphae form and there was difference between the conductivity, the pH, the content of reducing sugar of the broth and the mycelial biomass in different mating type strains, which indicated that hyphae form and metabolic characteristic had relations with mating factors in this two strains.

The Production of Phenolic Compounds of Inonotus obliquus by Fungal Elicitor Introducing and Its Study of Biochemical Mechanism

Wei Zhiwen,Sun Yong, Wang Fei

2014, 0(9): 136-141.

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In order to study the effects of fungal elicitor on secondary metabolites of Inonotus obliquus, using shaking flask method, the fungal elicitor was added to the liquid cultures of Inonotus obliquus, detected the accumulation of secondary metabolites, and the effects of fungal elicitor on Inonotus obliquus nitric oxide synthase（NOS）activity, discussed the influence of elicitors to Inonotus obliquus liquid fermentation process and the accumulation of secondary metabolites. The results showed that the addition of fungal elicitor could improve the metabolic rate of Inonotus obliquus and the accumulation of polyphenols. The intracellular and extracellular polyphenols were up to 102.15 mg/L and 311.91 mg/L, and could also significantly improve the activity of NOS, NOS activity was up to 151.93 U/mg prot, nitric oxide content in the fermentation liquid was up to 256.28 μmol/L. This showed, fungal elicitor could effectively active the biosynthesis pathway of secondary metabolites of Inonotus obliquus.

Cultivation of Chlorella protothecoides Using Pichia pastoris Fermentation Waste Liquor

Deng Wei, Gui Xiaohua,Wang Guilin, Yao Jie,Yan Yunjun

2014, 0(9): 142-148.

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The aim of this article was to examine the feasibility to cultivate Chlorella protothecoides using waste liquor of Pichia pastoris fermentation and then further optimize the cultivation conditions. The results showed that the P. pastoris fermentation waste liquor had more positive effect on the growth of C. protothecoides than SE culture medium. The highest biomass of 0.65 g/L was achieved after 7 days cultivation when using the fermentation waste liquor diluted to 10 times, which was 2.28 fold of that of SE medium. However, it could not significantly improve the lipid content of C. protothecoides. Then, the culture medium was optimized via single factorial experiments and orthogonal experiments. The obtained optimal culture conditions for the maximum biomass were：0.05 mol/L glucose, 0.01 mol/L NaNO3, 0.003 mol/L KH2PO4, and 300 μL/L seaweed liquid fertilizer. Under the optimal culture conditions, 6.56 g/L dry-weight of C. protothecoides biomass and 33.68% lipid content were achieved. The fatty acid profiles of the lipid were C16：0（25.12%）, C18∶0（4.69%）, C18∶1（50.46%）, C18∶2（6.78%）and C18∶3（8.58%）, which were suitable for biodiesel feedstock.

Introducing gshF into Lactobacillus paracasei L14 to Influence Its Stress Risistance Ability

Yu Rui,Zuo Fanglei, Chen Xiling, Wei Yanjie,Chen Shangwu

2014, 0(9): 149-156.

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As one of the important groups of food industry microorganisms, lactic acid bacteria will confront to various abiotic stresses when applied in industry. Previous studies suggested that some lactic acid bacteria can synthesize or absorb glutathione from the medium and protect themselves from diverse stress. In this study, glutathione synthetase gshF from Enterococcus faecalis, which can catalyze the synthesis of glutathione, was cloned and transformed into Lactobacillus paracasei L14 by Lactobacillus recombinant expression vector. The stresses resistance ability of the gshF recombinant strain was evaluated under stress conditions of hydrogen peroxide, acid, freeze-dry dehydration and osmosis. The results suggested that the survival rate of the gshF recombinant strain was greatly improved compare with the control strain.

Cloning，Expression and Characterization of Gluconate 5-dehydrogenase from Gluconobacter oxydans

Li Ling, Xu Lin,Wei Miao, Li Yan, Yan Ming

2014, 0(9): 157-163.

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Guconate 5-dehydrogenase gene from Gluconobacter oxydans H24 was amplified with primes designed based on NCBI database published sequence information. Ga5DH was cloned and expressed in Escherichia coli Rosetta strain. Results showed that the molecular mass of the enzyme was 26.5 kD by SDS-PAGE gel analysis. After purification, the enzyme activity could reach 7.83 U/mg. It was suggested that the recombinant protein had a maximum activity at 40℃, pH 11. The characterization of purified enzyme indicated that Ga5DH was highly resistant to organic solvents.

Screening，Identification of a Cellulase-producing Bacterium Strain and Its Enzyme-producing Conditions

Chai Xiujuan, Li Caolong, Kong Dezhen, Cui Zhenglong, Wang Aiying

2014, 0(9): 164-170.

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One cellulose producing strains C-8 were isolated from Xinjiang turpan area soil samples. According to morphology, biochemical and physiological characterization, and 16S rRNA sequence analysis, C-8 was identified as Bacillus sp. The celluloses produced by C-8 could hydrolyze cellulose, the optimum pH and temperature were 4.0 and 40℃, also it has good thermal stability and pH stability. In order to improve the ability of cellulose producing strain C-8, the fermentation conditions were optimized using response surface method. It was to screen out the effects of the main effect of enzyme production condition factors using Plackett-Burman Design, the steepest ascent experiment approximation maximum response area, and optimize fermentation conditions by Response Surface Box-Behnken. The results showed that the initial pH, CMC-Na% and culture tempinduedrature was the main influencing factor. The response of three factors was, obtained, and the optimal conditions for the initiation of pH8.0, CMC-Na%2.5%, culture temperature 28℃. Under the optimum condition, the activity of cellulose was up to 193.89 U/mL, and the enzyme activity was increased by 2.35 times compare to previous optimization.

Deletion of sopB Gene of Salmonella typhimurium LT2 by λRed Recombination System

Li Ye,Zhang Xixuan,Guo Mengzheng, Wang Suying, Zhang Kunsheng,Ruan Haihua

2014, 0(9): 171-177.

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In order to investigate the role of SopB effector secreted by Salmonella typhimurium LT2, the sopB gene deletion mutant with λ Red recombination system was constructed. To amplify the homologous fragment for sopB deletion, a pair of knockout primer was designed according to the full-length sequence of sopB gene of Salmonella typhimurium LT2. With pKD4 plasmid as template, the homologous fragments including homologous regions which are similar to sopB gene upstream sequence and downstream sequence respectively and kanamycin cassette with two FRT sites were amplified. Then, the homologous fragments were electrotransformed into the Red-induced S.Typhimurium LT2 competent cells. Under the pressure of antibiotic and with the work of λ Red system, homologous recombination occurred between the fragments and genome of host strain, and the recombinants were selected on kanamycin agar plate. After the resultant recombinants were verified with PCR, the positive recombinants were cultured at 43℃overnight to eliminate pKD46. To remove the kanamycin resistant relevant DNA fragment, FLP recombinase-encoding plasmid pCP20 was introduced into the recombinants, resulting in a single FRT site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR. The removal of sopB was further verified by the secretion of SopB protein and the induction of pAkt activation in HeLa cells upon S. typhimurium LT2 infection. Considering the results including genome PCR, SopB secretion and pAkt activation in HeLa cells upon infection, it was confirmed that the sopB gene was successfully knocked out from S. typhimurium LT2 genome with λ Red recombination system. In summary, it is concluded that the sopB gene of S. typhimurium LT2 could be successfully knocked out. Moreover, this paper provides a powerful tool for the functional study of SopB effectors during the interaction between Salmonella and host. Also, it supplies the clue for the gene knock-out of other types of bacteria.

The Comparison of Expression Efficiency of Human CD137L in Different Expression System

He Dongyang,Ma Chao,Gao Zhenyue,Wang Shuzhen,Chen Yijun

2014, 0(9): 178-184.

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It was to construct a suitable expression system for CD137L by comparison of expression level of CD137L in Pichia yeast, Bacillus subtilis, and Escherichia coli and determine the effect of CD137L on T cell proliferation. CD137L was amplified by PCR with designed primers from yeast expression plasmid pPIC9K-CD137L and subcloned into vector pP43 or pET11a. Then recombinant plasmid pPIC9K-CD137L, pP43-CD137L and pET11a-CD137L were transformed into GS115, WB800 and BL21, respectively. Positive clones were screened and expression of CD137L was detected by SDS-PAGE. Then inclusion body of CD137L from Escherichia coli was refolded by dilute refolding and CD137L was purified by ion exchange chromatography. After that, bioactivity of CD137L was determined by T cell proliferation assay. CD137L was expressed by all the three expression system and further confirmed by Western blot assay. But expression level of CD137L in GS115 and WB800 was too weak for further study. In contrast, CD137L was efficiently expressed in BL21 as inclusion body. It was about 0.8 g inclusion body per 1 L cultured BL21 and 200 mg denatured protein was obtained. Then inclusion body was dissolved in 8 mol/L urea at 50℃or 60℃, and active protein CD137L was obtained after dilute refolding. Then refolded CD137L was purified by ion exchange chromatography, and purified CD137L demonstrated significantly costimulatory effect on T cell proliferation in dose-dependent manner. It was demonstrated that CD137L was efficiently expressed in Escherichia coli expression system compared with Pichia yeast and Bacillus subtilis, and purified CD137L kept costimulatory activity on T cell proliferation.

Cloning and Bioinformatics Analysis of TLR3 Gene in Maiwa Yak

Chen Yabing,Lan Daoliang,Lin Baoshan,Huang Cai,Huang Yong, Li Jian

2014, 0(9): 184-188.

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The coding sequence of Maiwa yak（Bos grunnien）TLR3 was cloned in this research. Some characteristics of the TLR3 gene and encoded protein sequences were predicted and analyzed by the methods of bioinformatics in the following aspects, including the general physical and chemical properties, secondary structure and protein domain. Results showed that the coding region sequence of Maiwa yak TLR3 contains a complete open reading frame（2 715 bp）encoding 904 amino acids. The secondary structures of the protein are mainly composed of a-helix and random coli. The phylogenetic tree shows that the gene of Maiwa yak was so close to cattle, sheep and horse. Sequence alignment of TLR3 gene also showed highly homology in yak with other species.

Analysis of the Relationship Between Polymorphism of Leptin and Seasonal Estrous in Sheep

Li Hui,Lu Shouliang,Wan Pengcheng,Dai Rong,Li Liangyuan,Meng Haohao,Shi Guoqing

2014, 0(9): 189-194.

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According to the known sequence of leptin gene from GenBank, two primers were designed. The single nucleotide polymorphisms（SNPs）of the nonseasonal estrus Hu sheep and seasonal estrus Altay sheep were detected using PCR-SSCP assay. Then, the screening SNPs was subjected to the relative analysis of genotype and seasonal estrus. The results showed that three consecutive bases TTG inserted and C / T mutated intron 1 of leptin in Altay sheep, and G / T mutation in exon 3 resulted in the replacement of valine by leucine in contrast with the Hu sheep. Three genotypes AA, AB, BB were detected on the amplified fragment of the exon 2, the genotype BB was dominant in Altay. The genotype distributions were significantly different（P<0.001）between two groups, especially demonstrated that genotype BB was benefit for seasonal estrus. In conclusion, the sequence difference of Leptin in different sheep breeds may be one possible reason leading to nonseasonal or seasonal estrus, which can be used as an assistant marker for nonseasonal estrus breeding.

Preliminary Investigation on Antimicrobial of the Epidermal Mucus Secretion of Roughskin Sculpin（Trachidermus fasciatus）

Qiu Jin,Pan Liande,Zhang Shuai

2014, 0(9): 195-200.

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The epidermal mucus samples from roughskin sculpin（Trachidermus fasciatus）were extracted with aqueous, acidic and organic solvents to identify potential antimicrobial acitivity by the methods of Kirby-Bauer and two fold dilution. The crude acidic extracts only showed antibacterial activity against all six tested pathogens and the antibacterial ability of descending order is Aeromonas veronii, Staph epidermidis, Staph soparophytics, Aeromonas hydrophila, Citrobacter freundii, Staph warneri. The protein profiles of the acidic extracts mucus on tricine SDS-PAGE gel showed proteins was less than 95 kD, and the highest levels was between 34 kD to 55 kD. Preliminary studies indicated that the epidermal mucus samples from roughskin sculpin（Trachidermus fasciatus）extracted by acidic played an major role to defense against these pathogens, and to determine the antimicrobial protein remains to be further analyzed.

Detection of Pathogenic Aeromonas hydrophila in Andrias davidianus by Quadruple PCR

Ling Kong,Ding Shihua, Jin Juan,Wu Xingzhen

2014, 0(9): 201-207.

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In order to develop a rapid PCR method to detect pathogenic Aeromonas hydrophila in Andrias davidianus, four pairs of primers were designed based on the conservative sequences of 16S rDNA, outer membrane protein（omp）, hemolysin（hly）and serine protease（ahp）genes. By optimizing of PCR conditions, a quadruple PCR assay was established. The assay had a highly specific detection on pathogenic strains of Aeromonas hydrophila without other irrelative bacteria. This assay had a high sensitivity with the detection limit as low as 100 fg DNA template. From quadruple PCR assay, there were 18 positive results in Aeromonas hydrophila（n = 20）. After artificial infection of Andrias davidianus and the extracellular products assay, the positive strains with more species of virulence genes showed a highly pathogenic activity. This method also was applied in other artificial samples（n = 33）isolated from Andrias davidianus and suspicious samples（n = 34）, resulting in 100% and 21/34 positive results, respectively. In conclusion, the quadruple PCR assay, which has a highly specific and sensitive characteristic, could be applied in rapid diagnosis for Aeromonas hydrophila in Andrias davidianus.

Evaluation of Genetic Diversity in the Onchidium struma from Different Geographical Populations in China by Inter Simple Sequence Repeat（ISSR）

Wang Chengnuan, Shen Heding, Zheng Pei

2014, 0(9): 208-212.

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The genetic diversity of Onchidium struma collected from Yancheng（YC）, Shanghai（SH）, Wenzhou（WZ）, Xiamen（XM）, Shenzhen（SZ）, Hainan（HN）in China, was studied using the inter simple sequence repeat（ISRR）technique. The results indicated that the values of percentage of polymorphic band, Nei’s gene diversity and Shannon’s information index were 54.20%-70.99%, 0.1564-0.2191 and 0.2421-0.3324. According to AMOVA analysis, there were significant（P < 0.001）genetic differences among the six populations, 47.25%was attributed to among populations and the rest（52.75%）to differences within populations. The pairwise Fst =0.4725, gene differentiation coefficient（GST=0.3705）among populations and gene flow（Nm=0.8497）indicated that greater genetic differentiation occurred among the populations. Genetic drift was one of the main factors that affecting the population differentiation.

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