Table of Content

09 January 2015, Volume 31 Issue 1

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Research Progress of MicroRNAs in Plant Stress Responses

Wang Wei, Zhang Yujuan, Chen Jie, Liu Jubo, Xia Minxuan, Shen Fafu

2015, 31(1):  1-10.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.001

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MicroRNA（miRNAs）is a class of endogenous non-coding small RNAs, which participate in regulation of physiological activities by target mRNA cleavage or translational repression in plants. Some miRNAs play important roles in stress-related signal transduction and improving resilience against adverse environmental hazards of plant capacity by regulating gene expression under stressed conditions. This article focused on the production of miRNA, mechanism, research approaches and characteristics of resisting the biotic and abiotic stresses in plants, the development trends and future prospect of plant miRNAs research.

Research Advances in the Selective Relationship Between Ubiquitin Ligases and Substrates

Li Yang, Li Dong

2015, 31(1):  11-20.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.002

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Ubiquitin is a small-molecule protein composed of 76 amino acids. The covalent-bonding process between ubiquitin and substrates is defined as Ubiquitination. Ubiquitination modification, an efficient, ATP-dependent, and highly specific process, is mediated by a cascade regulation of ubiquitin activating enzymes, ubiquitin conjugating enzymes, ubiquitin ligase and deubiquitinating enzymes. Ubiquitination is highly relevant with biological processes like cell cycle regulation, cell apoptosis, transcription regulation, DNA damage response and so on. In the process of ubiquitination, the recognition between ubiquitin ligases and substrates is critical for the specificity. The mechanisms of such recognition are being reported moreover the high throughput methods identifying E3’s substrate are being improved and developed. With the accumulation of ubiquitination data from the deep-going research, the bioinformatics approach studying the selective relationship between E3s and substrates is becoming a hot spot. This article will discuss the recognition mechanism between E3s and substrates, the high throughput methods used for the identification of E3’s substrates, review the bioinformatics study about E3s’ substrates and highlight the future research direction.

Research Progress of the Arginine Biosynthetic Pathway in Prokaryotic Cells

Yan Hongbo, Wang Wei, Li Lingdi, An Wanchang

2015, 31(1):  21-28.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.003

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Arginine biosynthesis in prokaryotes consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase（NAGS）. After metabolisation of N-acetylglutamate, biosynthesis proceeds via three enzymatic steps which form further acetylated intermediates, until the acetyl group is removed in the fifth step of this process. The resulting ornithine is carbamoylated to citrulline. Addition of aspartate leads to N-argininosuccinate, which is finally converted to L-arginine. Here we summarize the arginine biosynthetic pathway, catalytic mechanisms of enzymes and the molecular mechanisms of feedback inhibition of ArgR protein. In addition, we briefly outline existing problems in the research of arginine metabolism and directions for future research.

Research Progress in EZH2 as a Target for Anti-tumor Immunotherapy

Li Qian, Wang Yanlin

2015, 31(1):  29-32.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.004

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EZH2（enhancer of Zeste homolog2）, the core subunit of polycomb group protein complex（PcG）, is a histone methyltransferase which involves in cell density maintenance, stem cell pluripotent, cell cycle regulation and other important physiological roles. The study has found that EZH2 is over-expressed in many tumor tissues and can be used as a carcinogen to promote tumorigenesis. Since EZH2 has been proved to be non- or low-expressed in normal tissues, it has recently been identified as a tumor-associated antigen. Multiple antigen peptides derived from the EZH2 protein have been identified and their ability in stimulating the killing activity of immune cells against the tumor cells with over-expressed EZH2 has been proved. These studies indicate that EZH2 could be a new molecular target for anti-tumor therapy and has potential value in tumor immunotherapy. This paper gives a brief review on the research progress in these study fields.

Quorum Sensing and Its Application in Preventing and Therapeutic Effect for Pathogenic Bacteria

Liang Xinyan, Ruan Haihua

2015, 31(1):  33-38.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.005

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Bacteria releases one or several chemical molecules served as signal to estimate the density of bacteria and sense the change of environment. This chemical communication, called as “quorum sensing”（QS）is defined as a density dependent mechanism by which bacteria coordinate expression of specific target genes in response to a critical concentration of signal molecules. A many of studies had showed that the construction of various QS system depends on the type of bacteria. QS system exists widely in pathogenic bacteria, which build up the capability of infection, expression of toxic genes and pathogenesis. Therefore, it is a concerned topic in medicine realm that prevents and cures the diseases caused by pathogenic bacteria by targeting the QS system. Here, this review discussed the QS and its application in preventing and therapeutic effect for pathogenic bacteria.

Research Progress on Microbial Community Structure in Solid-phase Denitrification Systems

Zhang Lanhe, Zuo Zhengyan, Wang Xuming

2015, 31(1):  39-45.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.006

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Solid-phase denitrification（SPD）is a new type of heterotrophic denitrifying process, in which solid organic matters are used simultaneously as carbon source and biofilm support. SPD process has been applied on nitrogen removal from groundwater and wastewater with the low ratio of carbon to nitrogen. Microbial community structure in SPD system determines not only degradation efficiency of solid carbon source but also denitrification rate and stable running of the SPD system. Therefore, the research on microbial community structure is significant for the optimizing SPD process and exploring its mechanism. This review summarized the present research status and progress on microbial community structure in different SPD systems. In addition, the main problems and the new perspectives in the present study were also discussed.

Research Progress in Genomic Imprinting in Mammals

Chen Xiuli, Ma Libing

2015, 31(1):  46-50.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.007

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Genomic imprinting is a genetic phenomenon because of the different parents resulted from the allelic gene expression differences. The causes and the process of genomic imprinting is a hot issue of modern genetics. Mammals many genomic imprinting characteristic makes it become a focus biology problems in the post genome era. The evolution of genomic imprinting has played a special role in mammalian reproduction and development. This paper reviewed the characteristics of genomic imprinting、imprinting mechanism of gene imprinting, gene imprinting and the development of cloned animals, the research progress of the imprinting genes and the disease.

Research Methods of Abiotic Stress-related Transcription Factors in Plants

Su Mingxing, Sun Yinghao, Shi He, Li Qiuli

2015, 31(1):  51-60.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.008

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Transcription factor is a kind of proteins which bind to the cis element region of the promoters specifically and a kind of transcription regulatory factors, and it is also one of the biggest gene families in plants. Many transcription factors can regulate the expression of downstream genes, and play important roles on plant growth and development, morphogenesis, hormone adjustment, and resistance to a variety of biological and abiotic stress. In combination with the research progress of transcription factors in recent years, we have summarized main strategies and methods of transcription factors to the abiotic stress, including the transcription factor domain structure, subcellular localization and transcriptional activation, transcription factor complex and functions of transcription factors, to provide related research theories and methods to plant transcription factors for reference.

Research Progress on Technology ofIn-gelProteinDigestion

Jiang Chao, Zhang Xuewen, Pan Yinghong

2015, 31(1):  61-66.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.009

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In-gel digestion is the important step that connects electrophoresis separation and mass spectrometry identification in proteome research, and can significantlyaffect the results of qualitative and quantitative analysis of proteins. The technology was firstly proposed in 1992, and from then on, many progresses have been made and various improvement plans have been developed. In order to more effectively use the technology of in-gel digestion, the progress on the aspects of gel decoloration, impurity removal, protein digestion and peptides extraction were reviewed from the literature.

Advance Based on Signal Amplification Technology with Aptamer Biosensor

Xue Mingyue, Qin Yingfeng, Li Jian, Ye Gaojie, Zhan Zhihua

2015, 31(1):  67-72.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.010

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Signal amplification technology has grown immensely in many fields because of its high accuracy and sensitivity at low concentrations. As a recognized molecule, aptamer has been used on many biosensors, and also has shown a good prospect in medical diagnosis, environmental monitoring and biological analysis. In recent years, biosensors with aptamer as recognized molecule has attracted more and more attention. The new research development of aptamer biosensors based on signal amplification technology in nearly three years was summarized especially.

Optimization Method for the Extraction of Total DNA from the Intestinal Microbes in Quails

Zhang Zhenhua, Bao Wangbo, Yu Ran, Wang Changyong, Liu Yan

2015, 31(1):  73-78.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.011

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Comparing the effects of total DNA extracted by different methods from intestinal microbes in quails, the study aimed at investigating the influences of DNA quality and PCR-DGGE conditions on intestinal microbial diversity of quail. In this study, the experiment used four methods to extract the DNA. The first was a conventional method for using saturated phenol/chloroform, other three commercially available DNA extraction kits（QIAGEN, SHENGGONG, TIANGEN）were assessed. The ribosomal gene sequences were amplified with V3 and V6-V8 hypervariable region universal primers at different annealing temperature and subjected to denaturing gradient gel electrophoresis（DGGE）. The QIAGEN DNA Kit generated the greatest bacterial species eveness and DNA quantity and quality in compare with other methods（A₂₆₀/A₂₈₀=1.8, 200 ng/μL）. The resulting DGGE profiles were substantially different in terms of the number and relative intensity of the amplification products. The V6-V8 region produced better DGGE profiles than V3 region. The QIAGEN DNA Kit and V6-V8 hypervariable region universal primers will be used in future studies of intestinal microbial diversity of quail.

Optimization of Ultrasonic Assisted Extraction of Lentinan by Response Surface Methodology

Wan Yue, Qi Jiying, Zeng Hong, Han Yang, Han Jing

2015, 31(1):  79-85.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.012

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Technological conditions of extraction of Lentinan with ultrasonic extraction are optimized for developing and utilizing the resource of Mushrooms by using the response surface method（RSM）. Firstly, single factor experiment was investigated, and then based on single factor experiment, a 3-factor, 3-level Box-Benhnken experiment was designed, in which ultrasonic power, ultrasonic time and the ratio of solid to liquid were chosen as influencing factors, and the yield of polysaccharide was selected as response value. The result shown that the regression model fit well with experimental data, and it can well predict the polysaccharideyield of Lentinan. The optimum conditions of ultrasonic extraction were ultrasonic power of 300 W, ultrasonic extraction time of 25 min and solid-to liquid ratio of 1:30, and the polysaccharide yield was 25.71%. Compared with the theoretical value（25.55%）, it has a smaller relative error at 0.63%. Compared with traditional method, ultrasonic extraction method is more efficient and less time consuming, so it is the ideal extraction method of Lentinan.

Physiological and Biochemical Analysis of Young Shoot Purple-related and Gene Expression of Key Enzymes in Tea Plant（Camellia sinensis）

Zhou Qiongqiong, Sun Weijiang

2015, 31(1):  86-91.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.013

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The young purple shoots of tea plant（Camellia sinensis）were compared to mature green leaves based on the biochemical and molecular level. Results showed that the content of tea polyphenols，catechins，caffeine and anthocyanins in the tender purple leaves were significantly higher than those in mature green leaves，while the content of chlorophyll and chlorophyll a，b，carotenoids were significantly lower than those mature green leaves. Q-PCR analysis indicated that the key genes of PAL、C4H、CHS、CHI、F3H、F3'H、F3'5'H、DFR and ANS in the anthocyanin biosynthesis were up-regulated in the tender purple leaves，which may contribute to the synthesis of anthocyanins and the color of the young shoots.

Establishment of Mannose Selection System in Sugarcane Transformation

Wang Wenzhi, Yang Benpeng, Cai Wenwei, Feng Cuilian, Wang Jungang, Xiong Guoru, Zhang Shuzhen

2015, 31(1):  92-97.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.014

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In this research the killing curve of mannose was tested at the subculture，differentiation and rooting stages of sugarcane cultivar ROC22. Then the expression cassette containing a pmi selection marker gene and a GFP report gene was transformed into sugarcane embryonic callus by Agrobacterium-mediated method. Resistant plants were obtained after selection by Mannose. The PCR detection result and GFP microscope observation result both show that our lab had established a high efficiency PMI/Mannose selection system in sugarcane transformation.

Determination the Content of Total Flavonoids and Hypericin in Callus from Hypericum attenuatum Choisy

Liu Xiaodan, Zhang Keqin, Liu Lian, Li Wenchang

2015, 31(1):  98-103.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.015

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With fresh leaves as test material, this study established a relatively stable system of callus culture for Hypericum attenuatum Choisy, and determined the content of total flavonoids and hypericin in callus. With MS as the basic culture medium, the effects of different hormone combinations were investigated on callus induction. The results showed that the optimum culture media for callus induction was MS + 2，4-D 4.0 mg/L + 6-BA 0.2 mg/L. The total flavonoids and hypericin of callus were extracted using ultrasonic method, respectively, using UV spectrophotometry and HPLC method for determination total flavonoids content and hypericin content in callus. The results showed that total flavonoids was 27.99 mg/g, and hypericin content was 0.515 mg/g in dry callus.

The Antioxidant Properties of Chickpea Protein Hydrolyzed Fractions

Chen Xiaofei, Sun Yufei, Feng Fei, Wang Xueyan, Zhou Fuzhong

2015, 31(1):  104-108.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.016

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It was to assay the antioxidant activities of the Chickpea protein isolates（CPI）and its peptide fractions in vitro. CPI were hydrolyzed with alcalase, and the hydrolysates were further separated into peptide fractions of <3 kD, 3-5 kD, 5-10 kD and >10 kD, respectively, using membrane ultrafiltration. Results showed that, the <3 kD peptides exhibited better ferric reducing power and radicals scavenging activities when compared to peptide fractions of higher molecular weights. CPI and its peptide fractions had significant ability to chelate metal ions compared to glutathione（GSH）, and also had some ferric reducing powers. The remarkable antioxidant properties indicate that CPI and its peptide fractions have the potential to be used in manufacturing antioxidant functional foods and healthy foods.

Studies on the Genetic Diversity of Polyscias by ISSR Marker

Zhang Guowu, Liu Xuefeng, Zhou Xinghua, Huang Tao

2015, 31(1):  109-114.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.017

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In this study, nine species Polyscias leaves as experimental material, we analyzed the genetic diversity and relationship by ISSR markers, 9 stable and high polymorphism ISSR primers screened from 100 ISSR primers by optimized the system of PCR for Polyscias, a total of 70 bands were obtained by the polymorphic primers of 9 test specimens, including 61 polymorphic bands, the polymorphic percentage was 87.14%. The genetic similarity coefficients ranged from 0.346 9 to 0.816 3, suggesting that the substantial genetic divergence between some varieties. The clustering scheme showed that Polyscias had small differences of genetypes their genetic basis was comparatively narrow.

Cloning and Analysis of Paternally Expressed Gene EB30 in Zygote of a Tobacco Interspecies Hybrid

Luo An

2015, 31(1):  115-121.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.018

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As known that EB30（EST accession number EB426694）paternally expressed in zygote of a tobacco interspecies hybrid, its CDS sequence was then cloned by using the EST database of NCBI. Also some characters of EB30 protein were analyzed by the methods of bioinformatics. Genome walking and hiTAIR-PCR were used to obtain the 5' flanking region of EB30 including the promoter and 5'UTR. The EB30-EGFP fusion protein expression vector driven by the 5' flanking region of EB30 was constructed and transformed into the tobacco. The results showed that EB30 gene encoded 211 amino-acid residues with a molecular weight 22.616 2 kD and pI5.63. This protein was hydrophilic, and predicted to locate in the mitochondrion. The phylogenetic tree showed that EB30 protein of tobacco was so closed to the tomato and potato, the amino acid homology was about 73%. There were two conserved regions near the N-terminal and C-terminal in homologous proteins from different species. Finally, the 5' flanking region of EB30 was proved to have a promoter activity in the early embryogenesis by fluorescence observation in the zygote and 2-celled proembryo from transgenic plants.

Genetic Analysis of cryIAC Gene in Transgenic Maize Plants and Its Effects on Insect-resistance

Wang Jianjun, Yang Huizhen, Liu Jiao

2015, 31(1):  122-130.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.019

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In order to analyse the genetic regularity of target gene cryIAc in transgenic and their descendant plants（lines）, and to study the effect of target gene on insect-resistant activity of transgenic plants at the same time, this research work was conducted. Firstly, cryIAc gene was transformed into maize inbred ‘Zheng 58’ and ‘Chang7-2’ separately by pollen-mediated transformation method. PCR, Southern, ELISA analysis and bioassay in the field of each generation transgenic plants were then conducted according to experimental requirements. Results showed that ⑴ A total of 24 and 41 transgenic plants were obtained through transformed cryIAc gene into maize inbred ‘Zheng 58’ and ‘Chang7-2’, respectively. ⑵ The molecular detection results of transgenic T₂, hybridization F₂ and backcross B₁ plants suggested that the segregation of target gene in these lines followed 3: 1, 3: 1 and 1: 1 segregation ratio of the Mendel laws of heredity, respectively. ⑶ The molecular detection results of T₁ to T₄ transgenic plants（lines）showed that the target gene could stably inherited and expressed effectively, the expression amount of the target gene was from 9.8 to 14.3 ng/g leaf fresh weight. ⑷ Moreover, the results of bioassay in the field indicated that in the case of high insect-susceptibility of negative control line, the transgenic lines still showed highly insect-resistant activity. ⑸ In addition, the target gene integrated into genomics of transgenic plants could inherit to the next generation plants by hybridization. ⑹ At last, 5 high insect-resistant transgenic lines, SZ003, SZ005, SC001, SC004 and SC007, was gained by screening. The pollen-mediated transformation method was a effective and shortcut tool used in plant transformation, cryIAc gene could confer and enhance insect-resistant activity of transgenic plants（lines）tansfomated with it.

Construction and Prokaryotic Expression of Trichoderma viride TvALP-linker-TvRBL Fusion Gene

Xu Yangyu, Wen Shijie, Li Haifen, Li Ling, Liang Xuanqiang

2015, 31(1):  131-137.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.020

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Fusion gene were constructed by linking the TvALP and TvRBL genes with a flexible chain（linker）. Bioinformatics analysis showed that the fusion gene contained a signal peptide. The TvRBL、TvALP and ?TvALP genes were cloned by RT-PCR from the total RNA of Trichoderma viride mycelium, which in turn were cloned into expression plasmid pET30a to construct prokaryotic expression plasmids pET30a-TvALP-linker-TvRBL and pET30a-?TvALP-linker-TvRBL, then these two expression plasmids were transformed into E.coli BL21（DE3）pLysS, and protein expression were induced by IPTG. SDS-PAGE was used to analyze the expression of the fusion protein. The result showed that expression plasmid pET30a-?TvALP-linker-TvRBL was obtained expression in E.coli BL21（DE3）pLysS, which was a specific band at about 60 kD in size and identical with the expected molecular weight of the fusion protein.

Identification and Polyphasic Taxonomy Character of a Strain of Streptomycete

Jiao Linpeijin, Huang Yunhong, Li Suzhen, Long Zhonger

2015, 31(1):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.021

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To identify a strain of actinomycetes JXNU03 through the sequences of 16S rRNA gene. The physiological characteristics were acquired through physiological and biochemical experiments. The antagonistic activity was determined by plate assay and adopting mycelium growth rate method respectively. When the actinomycetes JXNU03 was cultured on Gause 1 medium, the vegetative hyphae were well developed and abundant without fracture, and the aerial hyphae were grown well with straight or spiral spore hypha. There were some thorns on the surface of spores. The glucose was detected in the whole cell hydrolysis, while not other dominant diagnostic sugars, and the L, L-DAP was detected in cell wall. The phylogenetic analysis showed that the level of 16S rRNA gene sequence similarity between the actinomycete JXNU03 and Streptomyes gancidicus NBRC 15412. The strain JXNU03 demonstrated a board spectrum of antagonistic activity against. The Gram-positive bacteria, the Gram-negative bacteria, yeasts and molds. The actinomycetes JXNU03 was identified as Streptomyes gancidicus, and termed Streptomyes gancidicus JXNU03. As its antagonistic activity against bacteria, yeast and filamentous fungi. The actinomycetes JXNU03 possessed an important prospect for antibiotics development.

Screening and Identification of High-yield Thermostable Lipase Producing Microorganisms

Li Yang, Cai Haiying, Zhao Minjie, Zhang Hui, Feng Fengqin

2015, 31(1):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.022

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Two strains of microorganisms（J2 and J3）with a high-yield lipase producing ability had been screened from dumplings’ steamer cushions, and identified as two variants of Aureobasidium sp. according to morphological observation and 26S rRNA gene（26S rDNA）sequencing. After being incubated at 30℃, 200 r/min for 3-5 d, the lipase activities of their fermentation supernatant were 10.61 U/mL and 14.43 U/mL, respectively, measured by spectrophotometry with p-nitrophenol palmitate（p-NPP）as a substrate. The investigation of physicochemical properties of the produced lipases（Lip2 and Lip3）showed that the optimum catalyzing temperature of Lip2 is 50℃ and there is no significant activity loss after being incubated at 50℃ for 5 h, while the optimum catalyzing temperature of Lip3 is 60℃ and there is no activity loss after being incubated at 40℃ for 5 h, but only 42.19% of lipase activity remained when incubated at 50℃ for 5 h. The results indicated that the lipases produced by strain J2 and J3 have good thermal stability and high optimal reaction temperature.

Isolation and Identification of Saline-alkaline Tolerant Hydrocarbon-degrading Strains and Study on Their Saline-alkaline Tolerant Characteristics

Zhang Hairong, Tang Jingchun, Sun Kejing, Zhang Qingmin

2015, 31(1):  151-159.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.023

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10 saline-alkaline tolerant strains which can degrade hydrocarbons were isolated from a petroleum-contaminated saline soil in Dagang oilfield of China. According to their morphology, physiochemical characteristics and 16S rRNA sequence analysis, the 10 strains were identified as Ochrobactrum, Staphylococcus, Dietzia, Corynebacterium, Achromobacter, Microbacterium and Bacillus. Moreover, a series of liquid incubation experiments were conducted to investigate their saline tolerant and alkaline tolerant characteristics. The salt resistance test demonstrated that 10 strains grew well at NaCl concentrations ranging from 0.5% to 5.0% or even higher except B07 which could grow under 3% of NaCl. B02 and B05 still had high growth activity under a salinity of 11%. The alkalinity resistance test demonstrated that all the 10 strains grew well at pH 9. Besides, B01, B03, B04, B06 and B09 could endure the pH of 10, B03 and B04 still had high growth activity with the pH of 11. The results indicated that petroleum-degradation bacteria with high saline tolerant and alkaline tolerant abilities were widespread among different microbial species. Therefore, they are expected to be widely used in the remediation of oil-contaminated saline alkaline soil.

The Agar-degrading Enzymatic System of Marine Bacterium Agarivorans sp.HZ105

Lin Bokun, Lu Guoyong, Song Yan, Xie Ruiquan, Chen Honglin, Hu Zhong

2015, 31(1):  160-166.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.024

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Three agarase genes（hz1, hz2, hz3）of 2 988 bp, 1 437 bp and 1 362 bp respectively, were cloned from strain Agarivorans sp.HZ105. These three agarase genes（hz1, hz2, hz3）encoded agarase HZ1, HZ3 and HZ4 and belonged to glycoside hydrolase family GH50, GH118 and GH16, respectively. The agarase genes of strain HZ105 were linked with vector pET-32a（+）and then were transfered to Escherichia coli BL21（DE3）. These agarase genes were successfully expressed in E. coli cells and their recombinant agarases were prepared. Agarose degradations of the recombinant agarases were studied. Agarase HZ1 could degrade agarose and neoagarooligosaccharides with high degrees of polymerization（8, 10, 12 and 14）into neoagarobiose and neoagarotetraose. Agarase HZ3 digested agarose to produce neoagarooligosaccharides with high degrees of polymerization, while agarase HZ4 decomposed agarose and neoagarooligosaccharides with high degrees of polymerization to yield neoagarotetraose and neoagarohexaose. Therefore, strain HZ105 might produce firstly agarases HZ3 and HZ4 to degrade agarose into neoagarooligosaccharides with high degrees of polymerization, and the products of agarases HZ3 and HZ4 would be then digested into neoagarooligosaccharides with low degrees of polymerization by agarase HZ1 and another agarase（HZ2）from strain HZ105 reported previously by our research group. Strain HZ105 is the first reported strain that produces four agarases and this study is the first report of the agar-degrading enzymatic system in the genus Agarivorans. The results could enrich the knowledge about the bacterial agar-degrading enzymatic system and the roles of the agarases in the system. The application of agarases from strain HZ105 should also benift from this study.

Preparation of Algae Agents for S. miltiorrhiza Bunge Root Rot Biocontrol Bacillus subtilis2-1

Zhou Ying, Yuan Mengjuan, Han Jun, Chen Fang

2015, 31(1):  167-172.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.025

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With algae residue as carrier, S. miltiorrhiza bunge root rot biocontrol agents: Bacillus subtilis2-1（CGMCC No.9256）, were developed into algae agents, which features both nutritional and cell biological control function, also in line with the basic indicators of agricultural products, technical indicators microbial agents. Then appropriate ratio of seaweed broth and residue was chosen by physical means. Besides, the number of viable cells, moisture content, pH value and expiration date were measured by agricultural product quality testing methods, and field experiments was conducted to test biocontrol effect. Results showed that number of effective viable agents was 2.32×10⁹/g, moisture content was 6.75%, pH value was 6.7, and the expiration date was about 11.6 months , all of which were in line with the technical specifications of the particles. The incidence of root rot in field trials rate decreased 37.6% compared with the blank control group, the effect of biocontrol was obvious.

Isolation and Identification of A Tetracycline-degrading Bacterium and Optimizing Condition for Tetracycline Degradation

Zhang Xinyang, Cai Tingjing, Xu Xuping

2015, 31(1):  173-180.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.026

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Tetracycline antibiotics have been widely used in the livestock industry, but its complex structure, difficult to degrade, easy to accumulate in the water environment and produce a far-reaching impact on the ecological environment, antibiotic pollution as an important environmental issues has expanded the related basic research. Microbial degradation of the environment antibiotic pollution in recent years become a hotspot method. The selective media was used to isolate and screen the tetracycline-degrading bacteria. A high efficiency tetracycline-degrading bacterium（4002）was selected from sewage of a pharmaceutical factory. According to the morphological features, physiological and biochemical characteristics and the sequence analysis of 16S rDNA, the strain was identified as Advenella kashmirensi. Besides, the pH, dissolved oxygen, inorganic salts and other aspects of the strains were analyzied to study the degradation performance of tetracycline. The results showed that, the strain suitable conditions for the degradation of tetracycline are pH7.0, under aerobic condition, 150 r/min and cultured with shaking 6 d, the initial concentration of 50 μg/mL tetracycline degradation rate of 57.8%. FeSO₄ and MnSO₄ can promote degradation of tetracycline and little impact on MgSO₄, but CaSO₄ can inhibit degradation. After cultured for 8 d, the culture solution by HPLC detection showed at 6.022 min emergence of a new absorption peak, which was speculated as the degradation product.

Expression of Nitrilase Gene from Acidovorax facilis in Escherichia coli

Guan Qiwang, Ma Jiangfeng, He Aiyong, Jiang Min, Gong Changbin

2015, 31(1):  181-185.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.027

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Plasmid containing Acidovorax facilis gene, was used as a template, and length of 1 080 bp nitrilase（Nitrilase）gene fragment was obtained by PCR. Expressed genes were sequenced and compared to gene sequences found in GenBank. There are 2 bp mutation was found, which can not affect the expression and characterization of the enzyme. The recombinant plasmid was transformed into the Escherichia coli Rosetta（DE3）competent cell, which was induced IPTG for strain- expression. SDS-PAGE analysis was used and showed that the protein molecular weight was about 41.28 kD. Enzyme activity analysis showed that the specific activity of the supernatant was 3 U/mg. Induction conditions were optimized, including induction temperature, IPTG concentration, induction time and a series of conditions. Under optimal conditions and with the expansion of the culture volume, the specific activity can up to 15 U/mg, and activity increased by about five times.

Cloning，Expression，Purification and Characterization of Soluble Recombinant Endoproteainase AspN

Zheng Juan, Guo Huaizu, Li Jing, Duan Shuyan , Zhao Ziye, Zhang Dapeng, Guo Shangjing, Wang Hao

2015, 31(1):  186-192.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.028

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Endoproteinase AspN（flavastacin）is a zinc metalloendopeptidase which can selectively cleave peptide bonds N-terminal to aspartic acid residues, also it is one of most widely used proteolytic enzyme used for protein digestion prior to MS or HPLC analysis. The natural endoproteinase AspN was extract from the bacteria, Elizabethkingia meningoseptica. Some factors including low yields, difficulty in purification and high cost, greatly limit the application of the enzyme. Target gene was cloned into an pET32a expression vector and transformed into E.coli BL21（DE3）. This is the first report for the soluble expression of AspN-Trx in prokaryotic expression system and a poly-histidine tag enabled purification by Ni affinity chromatography. The activity of the recombinant endopeptidase AspN was identified by HPLC, SDS-PAGE and the fluorogenic substrate Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe（NO2）-Tyr-Asp. The results showed that the recombinant endopeptidase AspN was consistent with the standard enzyme and can be used widely.

Prokaryotic Expression of Alpha Fetoprotein and Optimization of Renaturation

Cai Lei, Jiao Liyuan, Wang Jihua, Tang Shixing

2015, 31(1):  193-197.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.029

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Plasmid pET32a-AFP was constructed to express recombinant human alpha fetoprotein, which was in the form of inclusion body, and the protein was obtained after renaturing optimization. The recombinant plasmid pET32a-AFP was transformed in E.coli, after induced by IPTG and purified by affinity column, the inclusion body was renatured under different conditions, including renature method, pH and addictives. The results showed that it had the highest renature efficiency when 1.0 mg/ml AFP was processed under one-step dialysis renature method and the pH is 8.5, with 0.5 mol/L L-Arg. This renature process made the recombinant AFP protein having the highest efficiency and easy to manipulate.

Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System

Xie Jingying, Feng Ruofei, Xu Lei, Zhang Haixia, Li Xiangrong, Hou Lanxin, Ma Zhongren

2015, 31(1):  198-202.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.030

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Bait vector pDHB1-VP1 was constructed for screening cellular proteins interacting with VP1 protein of encephalomyocarditis virus from yeast two-hybrid cDNA library of target cells in this study. VP1 gene was amplified and cloned into pMD18-T vector. After being verified by sequencing, it was directional cloning into bait vector pDHB1 of yeast two-hybrid system. Then the recombinant plasmid was identified by enzyme digestion and sequencing and transformed into yeast cells NMY51．The bait vectors’ expression and self-activation to reporter genes were tested．The results showed that the bait plasmid pDHB1-VP1 could express in yeast cells, and product size was 66 kD. It was specific binding with rabbit anti EMCV serum, which showed better immunogenicity. Bait plasmid pDHB1-VP1 was successfully constructed, could express in yeast cells and proved to be no self-activation to reporter genes．It could be used in the yeast two-hybrid system screening test．

Effect of Cell Density and Nutrition Supply on Cell-specific Virus Yields of Influenza Virus H1N1

Kong Wengang, Huang Ding, Luo Jian, Zhou Linting, Chen Ze, Liu Xuping, Tan Wensong, Deng Xiancun

2015, 31(1):  203-208.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.031

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It was to investigate the phenomenon that high cell density with low cell-specific virus yields and find out the solution. Methods：High cell density was achieved by increasing the microcarrier concentration and enhancing nutrition supply, then the effect of cell concentration at infection（CCI）and virus maintenance medium（VMM）on virus titer and cell-specific virus yield（Svy）was studied. Results：The maximum cell density was up to 1.47×10⁷cells/mL in 12.6 g/L microcarrier culture using medium exchange strategy. Under high CCI（1×10⁷cells/mL）condition, the Svy was up to 5.14×10³ virions/cell by choosing appropriate virus maintenance medium, which is nearly twice of Svy in DMEM. Conclusion：In order to achieve higher influenza virus titer, culture conditions and nutrition demands at different stages of influenza production should be taken into consideration. The results in this work provide guidance for further development of industrial-scale vaccine production processes.

Effect of Streptococcus dysgalactiae equisimilis on Total SOD Activity in Macrobrachium nipponense

Zhao Weihong, Zhang Fengying, Yu Yebing, Wang Zisheng, Qi Zhitao

2015, 31(1):  209-213.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.032

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Total superoxide dismutase（T-SOD）activities were investigated in hepatopancreas, muscle and gill of Macrobrachium nipponense at 3 h, 6 h, 9 h, 12 h, 24 h, 5 d and 10 d after being challenged by Streptococcus dysgalactiae equisimilis at levels of 0.6×10⁶, 2×10⁶, 4×10⁶ or 6×10⁶ CFU/mL in this study. Results showed that activity of T-SOD in hepatopancreas of the prawn reached the maximum at 5 d after infection. The values in muscle and gill reached to a maximum at 6 h. The activities of T-SOD in the hepatopancreas and gill of prawns infected with 6×10⁶ CFU/mL bacteria, in the muscle of prawns infected with 4 × 10⁶ or 6 × 10⁶ CFU/mL bacteria at 10 d decreased to about half of that in the control group. And the maximum in muscles was higher than those in hepatopancreas and gills. The results showed that Streptococcus dysgalactiae equisimilis could affect the activity of SOD in M. nipponense, and the response of the prawn to the bacteria was different in hepatopancreas, muscle and gill.

Effect of Xist Gene Repression on Early Development of Bovine SCNT Embryos

Liu Liming, Sun Wei, Zhang Jindun, Zhao Lixia, Li Shuyu, Wang Biao, Chen Yanglin, Hu Shuxiang, Wu Baojiang, Li Xihe

2015, 31(1):  214-219.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.033

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Xist is an X-inactivation related gene that produces a noncoding RNA, and it is one of the first imprinted genes to be expressed in the early embryo with expression beginning at zygotic genome activation. This experiment aimed at investigating Xist repression in fetal bovine fibroblast by TALE-REPRESSOR（TALER）which contains mCherry as reporter, and the impact of Xist repression on early development of cloned cattle embryos using the transfected cells as nuclei donor. The results showed TALER repressed Xist gene expression by 93.85% in fluorescence-activated sorted cells compared with wild type fibroblast, indicating the TALER vector designed in this experiment effectively suppressed expression of Xist. The mCherry positive cells were selected for somatic cell nuclear transfer（SCNT）. The 2-cell, 8-cell, morula and blastocyst rates of suppressed group vs. control group were 78.8% vs 75.1%（P>0.05）, 54.4% vs. 50.6%（P>0.05）, 12.3% vs. 27.8%（P<0.01）and 0 vs. 26.6%（P<0.01）, respectively. These results indicated that TALER vectors effectively inhibited Xist gene expression in female fetal bovine fibroblast. Suppression of Xist gene promotes the 2-8 cell embryonic development of cloned embryo, however, development of morula/blastocyst decreased significantly. Therefore, the mechanism by which Xist gene expression regulates early embryonic development of cloned embryos needs further investigation.

Expression and Bioinformatics Analysis of the msp4 Protein of Anaplasma Phagocytophilum

Zhao Qingliang, Yang Sufang, Liang Tian, Zhang Dianqing, Yang Xia, Sheng Jinliang

2015, 31(1):  220-224.  doi:10.13560/j.cnki.biotech.bull.1985.2015.01.034

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The purpose of this study was to analysis the antigenicity of msp4 protein of anaplamsa phagocytophilum. The informatics analysis was used to predict and analyze the important parameters of the msp4 protein, such as the physical and chemical properties, protein secondary structure, and its hydrophilicity, hydrophicity, bepipred linear epitope. According to the advantage epitope, we selected the high hydrophilic region of protein membrane outside domains, and cloned to pET32a prokaryotic expression vector. The bioinformatics analysis revealed that the msp4 protein was composed of 283 amino acids, and that its isoelectric point was 6.52. The msp4 protein was an stable hydropathilic protein（the instablility coefficient was 32.79, the grand average of hydropathicity was 0.063）. The secondary structure of the msp4 protein was mainly composed of random coils, extended strands, and alpha helixes. B cell epitope predicted that the msp4 protein has 14 linear epitopes. From the result of bioinformatics analysis, we selected the the 28-158 aa of the high hydrophilic region of protein membrane outside domains, and cloned to pET32a prokaryotic expression vector, the msp4 protein was successfully expressed and purified. The expressed and purified the recombinant msp4 protein lays a material foundation for the establishment of serological methods for anaplasma phaagocytophilum detection.