Table of Content

28 October 2015, Volume 31 Issue 10

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Review

Present Situation and Prospects on Application of Plant Bioreactor in Pharmaceutical

Chen Jinmei, Jiang Luyi, Hong Zhi

2015, 31(10):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.005

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With recent advances on the development and techniques in the life sciences, particularly in genetic engineering and molecular biology, a diverse range of organisms as “factories” can be utilized to produce the products to cure human disease or for preventative measures, i.e., biopharmaceuticals. Currently, bacterial, yeast, invertebrate and mammalian cells as well as plant expression system are already extensively utilized as bioreactors, whilst among them, plant bioreactors have shown the greatest potential, attracting world-wide attentions, due to their easy operation and cost efficiency. In this review, the applications of plant bioreactors, including existing problems and feasible modifying measures are summarized, followed by the prospects on the application and promotion of plant bioreactors based on the biopharmaceutical development trend.

Research Advances on Plant Inducible Promoters and Related Cis-acting Elements

Li Zhuoxue, Chen Xinbo

2015, 31(10):  8-15.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.006

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Promoter is an important element in the expression vectors for genetic expression and regulation. Accurate regulation of gene expression by promotors requires fine coordination between the core promoter and its up- or downstream cis-acting elements in the promoter. Inducible promoters can reduce the accumulation of heterologous proteins and the waste of plant energy during the expression of foreign genes. In this paper, we review current research advances on inducible promoters, including promoter structure, function, classification, application and related cis-acting elements. The existing problems and future prospects are also discussed.

Advances on the Research of Non-cell-autonomous Small RNAs in Plants

Deng Shuai, Zhang Tingting, Wang Ruru, Liu Yu, Zhang Yuanhu

2015, 31(10):  16-23.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.007

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One of the most fascinating features of RNA interference（RNAi）is its ability to transmit and spread from cell to cell. Such non-cell-autonomous silencing effects can also occur between tissues and organisms, in which the mobile small RNAs play a key role. However, the nature of non-cell-autonomous small RNAs is somewhat elusive. Recent studies have implied that small RNAs, including siRNAs and miRNAs, can transmit intercellular messages as transcriptional factors, peptide ligands and plant hormones do, and specifically are involved in a variety of biological processes of regulating developmental patterns, responding environmental stress, enhancing antiviral defense, and maintaining the silence of transposon. In this article we review the recent major research advances on the non-cell-autonomous RNAi, mainly focusing on the varied small RNAs transmitting the silence signals via the pathways of phloem and plasmodesma as well as their biological roles, also their molecular properties and regulation of mobility. Further potential problems and prospects of future researches are discussed .

Research Advances on Steroidal Glycoalkaloids

Xu Huijinlan, Wang Cuicui, Fu Daqi

2015, 31(10):  24-30.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.008

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Steroidal glycoalkaloid is a substance wildly existing in solanaceae plants, i.e., in plant’s leaves, roots, flowers, fruits and tubers, and it possesses broad biological activities, such as anticancer, antifungal, antiviral, antioxidant, and anti-inflammatory functions etc. Researches on the distribution, content, biological activities and synthesis pathways of steroidal glycoalkaloids have always been hot topics in this field. The research on the molecular mechanisms of steroidal glycoalkaloids’ biosynthesis will provide theoretical basis for breeding fine varieties. In this paper, the structure, biological activities and biosynthetic pathways of steroidal glycoalkaloids are reviewed.

Research Progress on the Role and Regulation Mechanism of Gibberellin Signal in Response to Abiotic Stress

Niu Yali, Zhao Qian, Zhang Xiaohan, Ai Qiushi, Song Shuishan

2015, 31(10):  31-37.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.009

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Gibberellin（GA）could promote seed germination and plant growth. Recent studies show that gibberellin plays an equally important role in the response of plant to abiotic stress. Plants increase stress tolerance by regulating GA biosynthesis, signal transduction and biological activity. In this paper we review the role of GA in response to common types of abiotic stress. The mechanisms of the synthesis, signal transduction and regulation of GA as well as the relationship with other hormones in response to abiotic stress are analyzed.

Research Progress on Application of Ferritin Nanoparticles in the Field of Biomedicine

Li Zhipeng, Liu Qingyou, Shi Deshun

2015, 31(10):  38-47.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.010

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Biological systems have generated wide varieties of nanoparticles during the process of long evolution. As one of naturally occurring nanoparticles, ferritin widely exists in all organisms, and is a key functional protein in life activities. Recently, the specific physicochemical properties of self-assembling ferritin nanoparticles allow it possessing the enormous advantages and application prospect in biomedical field. Current applications of ferritin nanocages include clinical measurements of trace amounts released into serum, nutritional sources of concentrated iron, nano biomaterial templates, and biological delivery of nanomaterials. This review presents an overview of ferritin nanoparticles in the diseases diagnose and treatment, delivery of medicines and development of vaccine, and outlines the potential prospects of applying ferritin nanoparticles on biomedicine.

Research Progress on Bacterial Quorum Quenching Enzymes

Xing Qifan, Liu Pengfu, Shi Jiping, Sun Yumei

2015, 31(10):  48-55.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.011

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Quorum sensing（QS）is a phenomenon of intercellular communication of bacteria via signal molecules. Bacteria secrete the specific signal molecules and respond to them, and bacterial cells enable the expression of specific genes, especially disease-causing genes while the signal molecules accumulate to a threshold concentration. This provides a new thought to prevent plants and animals from bacterial pathogenicity. Quorum quenching（QQ）based on QS system is a schema to decompose the signal molecules beyond the threshold concentration, and therefore represses the expression of specific virulence gene, so finally the prevention and control of diseases are achieved. The enzymes of QQ have been explored the most and also proved to be the most effective ways of quenching. To date, many QQ enzymes have been isolated successfully. Here we review progress on QQ enzymes with aspects of their type, property, catalytic mechanism, and physiologic function.

Research Progress on Cell Autophagy Induced by Cadmium

Wang Qiwen, Song Derong, Liu Qichang, Zhou Darong, Peng Hua, Zhang Qiongdi, Luo Yao

2015, 31(10):  56-61.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.012

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Cadmium, a highly toxic heavy metal, has been proven to be hazard for both human health and environment. Growing evidences have confirmed that cadmium caused damages to multiple organs and systems, and even led to cancer and tumors. Autophagy is an evolutionarily conserved lysosomal pathway of degrading cytoplasmic proteins and organelles. On the one hand, autophagy represents a cell survival mechanism to clear damaged organelles for preventing the cells from the further damages induced by cadmium; on the other hand, it may lead to cell death as cell death mechanism while the damage to the cell is irreversible. It is found that the role of autophagy in cadmium-induced cytotoxicity is still controversial, which might be caused from the variations on concentration and exposure time of cadmium. Current investigations on the mechanism of autophagy are mainly focusing on signal molecules of mTOR, Ca²⁺ and Beclin-1. Studies of the molecular mechanism between cadmium and autophagy can provide new ideas for the treatment and prevention of cadmium poisoning. This review summarizes the role of autophagy in cytotoxicity and signaling pathways in the autophagy induced by cadmium.

Research Progress on Sex Differentiation Related Genes on Sturgeon

Dong Tian, Liu Fengjiao, Hu Hongxia, Zhu Hua

2015, 31(10):  62-70.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.013

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Sturgeon is one of the oldest fishes in the world. Due to the delicious and nutritious flesh and caviar, sturgeon has extremely high economic value. Because of the price of caviar kept very high, breeding female sturgeon should get higher economic prospect than male. It seems impossible to identify the sex according to the exterior condition because of the similarity of sex characteristic on sturgeon. Currently, sex of immature sturgeon is usually identified through the ultrasonic instrument or with the help of endoscope. Even so, the accuracy of the identification cannot be ensured. Using molecular techniques to investigate sex determination and differentiation of sturgeon is supposed to conducively develop the technology of identifying sex and achieve the full female breeding. In this paper, the research status of genes related to sex determination and differentiation of fish is summarized, then the research progress on genes related to sex determination and differentiation of sturgeon is reviewed, and the feasible future research direction is also predicted.

Technique

Advanced in Proteomics Researches on Heterosis of Maize

Feng Wanjun, Dou Chen, Niu Xulong, Xing Guofang

2015, 31(10):  71-76.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.014

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Heterosis results in the phenotypic superiority of a hybrid over its parents with respect to traits such as growth rate, reproductive success and yield. Heterosis has immense agronomic value and for decades has been utilized in maize breeding to produce superior high-yielding hybrids. However, the molecular mechanisms underlying this event are still not completely understood. One of the approaches to study heterosis at the molecular level is to compare the gene expression of hybrids and their parental inbred lines in order to identify heterosis-associated genes and gain insight into the processes governing this phenomenon. In this review, we outlined the heterosis behaviors in maize and proteomics researches on maize heterosis.

Research Advances on Molecular Techniques for Improving Microbial Tolerance to Organic Solvents

Wang Yanxia, Liu Xiangsheng, Wang Min, Luo Jianmei

2015, 31(10):  77-88.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.015

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Organic-solvent-tolerant microorganisms can survive and grow in the higher concentrations of organic solvents, which exhibit significant application advantages in the field of the non-aqueous phase biocatalysis. Compared to natural screening, long-term domestication and traditional mutagenesis methods, molecular technique is a more rational and efficient way to obtain organic-solvent-tolerant strains. This paper mainly summarizes the research advances on the improvement of microbial organic-solvent tolerance by the reformation of functional genes related to the resistance to organic solvents and transcriptional factors using molecular biology techniques, also prospects the application of organic-solvent-tolerant strain.

Transgenic Technology: Establishment of Animal Models and Treatment of Diabetes Mellitus

Wang Hefei, Liu Dongjun

2015, 31(10):  89-98.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.016

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Diabetes mellitus endangers human health seriously. In recent years, with the development of transgenic theory and technology, many researchers are devoted to treat diabetes by applying transgenic technology. Therefore, some remarkable achievements have been achieved. This review describes the current achievements and challenges in the establishment of animal models and treatment of D. mellitus by transgenic technology. In future the breakthroughs from following aspects may accelerate the gene therapy of D. mellitus in clinical practice, such as the establishment of effective insulin gene transfer system, selection of target cells resembling physiological characteristics of β cells and preventing them from the attack of own immune system, sustainability of gene expression and identification of D. mellitus susceptible genes.

Research Progress on the Prevention and Control of Agricultural Non-point Water Pollution by Microbial Technology

Li Bai, Li Jun

2015, 31(10):  99-104.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.017

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The agricultural non-point source pollution is the largest one of total non-point source pollution in China, while water pollution is the most important part. “Prevention” is better than “treatment” for preventing and controlling the agricultural water pollution due to its complexity. With the rapid development of science and technology in recent years, microbial technologies play a more and more important role in the prevention and control measures for the water pollution in agriculture. Applying microbial technologies could reduce the pollutants discharge into the receiving water by reducing the agricultural source pollution and intercepting the agricultural process pollution, therefore it is beneficial to the sustainable development of agricultural ecological environment. In this paper we review and prospect the applications of microbial technologies in the prevention and control of agricultural non-point source pollution, including “source reduction” and “process interception”, which is expected to provide the valuable reference for the control of agricultural non-point source pollution.

Virus-induced Gene Silencing and Its Application in Plant Research

Sun Wei, Xu Yi, Xu Guiying, Sun Peiguang, Song Shun, Chang Shenghe

2015, 31(10):  105-110.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.018

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Virus-induced gene silencing（VIGS）is a useful tool for the identification of gene function in plants, therefore widely utilized in plants. Compared to traditional gene disruption approaches, VIGS is simple, rapid, short-cycle, transformation-free and capable of silencing multiple gene family members. Moreover, under the certain conditions, VIGS can even be transmitted to progeny plants. Here, the principle and operation of VIGS are introduced, and its recent applications and research advances are reviewed, which conducively promotes its further application and development in plants, especially for crop breeding.

Research report

Comparison of Metabolic Differences of Trehalose in Nicotiana tabacum Seedlings Under Drought and Chilling Stress

Zhang Jianbo, Wang Shasha, Hao Dahai, Yang Huiqin, Ma Wenguang, Gao Xue, Cui Mingkun, Gong Ming

2015, 31(10):  111-118.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.019

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In order to investigate differential response of trehalose metabolism to drought and low temperature stress, and roles of trehalose in the forming of drought and chilling resistance, tobacco seedlings（cv. Yunyan 203）were treated under drought and low temperature stresses. Then the changes of trehalose content, their activities of enzymes related to metabolism（TPS, TPP and THase）and gene expression were detected in the tobacco leaves during the drought and chilling stress at 4℃. The results indicated that trehalose content increased firstly, achieved the maximum level in 2 d, and the trehalose content in the leaves under drought stress always maintained higher level than that under the chilling stress. Similar to the change pattern of trehalose content, TPS, TPP activities were increased firstly and then decreased, and showed higher under the drought stress. On the other hand, THase activity demonstrated persistent increase under the drought and chilling stress, and was higher under chilling stress than that under drought stress. The expression level of TPS, TPP, and THase genes was higher in the drought stress than the control at 0 d, but the expression level under chilling stress was lower than the control. Above results showed that drought and chilling stress induced the accumulation of trehalose, the drought stress led to relatively higher level of trehalose content, related enzyme activities and gene expression, implying that response of trehalose to drought stress was more sensitive than that to chilling stress.

A Study on OpZFP Increasing Salt Tolerance of Nicotiana tabacum

Wang Qiqi, Guo Danli, Wu Xiaoqing, Fei Chunyan, Huang Xianzhong

2015, 31(10):  119-124.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.020

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To research the role of C2H2 zinc finger protein in signal transduction under abiotic stresses and the developmental processes of plant, an OpZFP was cloned from Olimarabidopsis pumila in Xinjiang. An OpZFP was the single C2H2 gene cloned from Olimarabidopsis pumila in Xinjiang. Analysis of semi-quantitative RT-PCR indicated that OpZFP was successfully expressed in the transgenic tobacco. Interestingly, the overexpressed transgenic plants showed more tolerance to salt stress compared with the wild plants. Under high salt stress, the root lengths of transgenic plants were longer, and they contained lower MDA contents than the wild type. Further experiments revealed that the transgenic plants had a lower degree of bleaching than the wide type ones. These results indicated that the OpZFP-overexpressing transgenic plants could enhance plant resistance to salt stress.

Clone and Expression of MeASR Gene in Cassava

Hu Wei, Yan Yan, Wei Yunxie, Wang Wenquan, Xia Zhiqiang, Lu Cheng, Hou Xiaowan, Peng Ming

2015, 31(10):  125-130.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.021

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Abscisic acid-stress-ripening induced protein（ASR）plays an important role in response to abiotic stresses. In the present study we isolated a first ASR gene designated MeASR from cassava. Sequence analysis showed that the open reading frame of MeASR gene contained 330 bp, encoding 109 amino acids. Multiple sequence alignment and phylogenetic analysis indicated that MeASR protein contained the conserved domains as ASR family had, and had a close genetic relationship with SlASR4 of tomato. Subcellular location assay showed that MeASR protein was localized in nucleus. Real-time quantitative PCR assay revealed that expression of MeASR was induced by osmotic stress and ABA treatments. These results suggested that MeASR might function as a transcription factor to involve in response to drought stress and ABA-signal regulation in cassava.

Cloning and Prokaryotic Expression of Dd-Gpx from Ditylenchus destructor

Liu Yang, He Xufeng, Liu Jianxi, Ding Zhong

2015, 31(10):  131-139.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.022

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To further understand the structure and function of glutathione peroxidase, a glutathione peroxidase gene was cloned from plant-parasitic nematode（Ditylenchus destructor）using RACE method, and the gene（DdGpx）was transferred into vector of pET-41b to have the fusion expression. The full-length cDNA of DdGpx was 950 bp containing an open reading frame of 681 bp and encoding 226 amino acids. Bioinformatics analysis indicated that the N terminal of the protein had obvious signal peptide, belonging to the secretory protein. The prokaryotic expression vector pET-41b-DdGpx was constructed and then transformed into BL21（DE3）, there was a apparent band in 60 kD. Mass spectrum analysis indicated that there were 7 peptides matched with D. destructor GPX protein, implying that the recombinant protein of Dd-GPX was successfully expressed

Gene Cloning and Cold-induced Expression Analysis of the Gene Encoding Gibberellin 20 Oxidase from Jatropha curcas

Wang Haibo, Zou Zhurong, Gong Ming

2015, 31(10):  140-148.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.023

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Gibberellin（GA）20 oxidase, a rate-limiting enzyme in GA biosynthesis, determines the amount of active GA forms, GA₁ and GA₄. Based on our previous transcriptome data of chilling-hardened Jatropha curcas, a new gene encoding gibberellin 20 oxidase（named JcGA20ox）was cloned by RT-PCR from the root of J. curcas seedlings（GenBank accession number：KJ670150.1）. The full-length cDNA of JcGA20ox was 1 307 bp, containing an entire open reading frame（ORF）of 1 131 bp. This ORF encoded a polypeptide of 376 amino acids with the molecular weight of 43 kD and the pI value of 6.7. Bioinformatics analysis showed that the JcGA20ox protein contained a Fe²⁺ and 2-oxoglutarate dioxygenase domain（Fe2OG_OXY）, belonging to the 2-ODD family. Semi-quantitative RT-PCR analysis revealed that JcGA20ox expressed differently in different tissues, with abundant and remarkably cold-induced expression in stem, but scarcely in leaves.

Gene Cloning, Sequence Analysis and Expression Studies of Vitellogenin Gene in Geocoris pallidipennis

Liang Huifang, Zeng Fanrong, Mao Jianjun

2015, 31(10):  149-156.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.024

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It was to study Vitellogenin（Vg）and the function of Vg gene in G. pallidipennis. In this study, the gene cloning, sequence analysis and expression studies of Vg gene of G. pallidipennis were carried out by RT-PCR, RACE and ELISA. The full-length cDNA of the Vg gene was 5 667 bp（GenBank accession number：KP688587）, encoding 1848 amino acids residues, and there was a signal peptide of 19 amino acids in N-terminal. There were 2 conserved polyserine regions and 1 RXXR cleavage site in amino acid sequences. Close to the C-terminus there was a GLAG motif followed by 5 conserved cysteine residues, and a DGYR motif was located in the 18th residue of the GLAG upstream. The deduced amino acid sequence of the Vg gene showed a high similarity with the Vg sequences from other hemipterainsects. Typical features of Vg were found through sequence analysis. The results indicated that the cDNA obtained was Vg gene from G. pallidipennis. The Vg detected by ELISA showed it increased gradually and reached the peak on the 22th day after adult emergence, then decreased. The results also indicated that the egg production was closely correlated to Vg expression.

Gene Cloning and Expression Characterization of Gene for a Heat Shock Protein 60 from Japanese Scallop Mizuhopecten yessoensis

Kong Fandong, Shi Jingyun, Bao Xiangbo, Gao Xianggang, He Chongbo, Liu Weidong

2015, 31(10):  157-164.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.025

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In order to investigate the molecular mechanism of high temperature stress in Mizuhopecten yessoensis, as well to find effective ways to solve the problem of summer mortality. The HSP60（heat shock protein 60）as a member of the highly conserved heat shock protein family, not only can help other protein fold correctly and restore the natural conformation in the condition of stress, but also can be used as a danger signal to the natural immune system. Specific primer was designed based on transcriptome sequence of HSP60, and the full sequence of cDNA of heat shock protein 60（MyHSP60）from Japanese scallop Mizuhopecten yessoensis was cloned by RACE. The cDNA sequence was 3 368 bp in length, including 68 bp 5' UTR, 1 569 bp 3' UTR and 1 731 bp open reading frame encoding 576 amino acids. Using gene walking the promoter region of MyHSP60 gene sequences was amplified and sequenced, the sequences of promoter region of MyHSP60 gene was 1 236 bp, which contained 2 TATA boxes, 4 CCAAT boxes, and 3 heat shock elements. The phylogenetic tree based on amino acid sequence matched with the traditional species trees based on basic anastomosis. The Real-time PCR result showed that HSP60 in all tissues of M. yessoensis was expressed, the expression in the tissues after heating increased extremely significantly in kidney, and significantly in hepatopancreas, mantle, hemolymph and muscle（P<0.05）, indicating that the gene plays a certain role in the process of heat stress.

Identification of Endophytic Fungi in Bupleurum and Screening of Antimicrobial Activities

Li Lingling, Luo Hechun, Zhang Xianshu

2015, 31(10):  165-170.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.026

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In order to determine the main distribution of endophytes and their broad-spectrum antimicrobial activity, 27 strains of endophytic fungi were isolated from the stems, roots and leaves of wild bupleurum at the Ganzi Tibetan Autonomous Prefecture of Sichuan Province. Identification of these strains was determined by their morphological features. They were classified as 9 fungal genera in which Aspergillus and Penicillium were the dominant. The antimicrobial activities from endophytic fungi were investigated by using the agar diffusion method and the cylinder plate method, and Bacillus subtilis, Escherichia coli and Staphylococcus aureus as indicator microorganisms. The results showed that 26 strains, accounting for 96.30% of all the isolates, had antimicrobial activities to 2 indicators or 3. Endophytic fungi of CHG-01, CHG-04, CHJ-10, CHY-01, and CHY-03 had remarkable antimicrobial activities to 3 indicators respectively. There is natural antimicrobial substance in the secondary metabolites of endophytic fungi from the wild bupleurum, which provides a new source for searching novel antimicrobial products.

Screening and Identification of Eosinophilic Fungal Strain Producing Lipase and Optimization of Its Lipase-producing Condition

Zhao Jun, Zhao Shuqin, Yang Xiaopu, Liu Xiaoli

2015, 31(10):  171-176.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.027

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This experiment was devoted to screen the strains of high-yield lipase, identify the genera, and define the optimal fermentation conditions. The strains were preliminary screened from greasy soil nearby Lanzhou urban area through Victoria blue and rhodamine B plates. The strains were re-screened through a copper soap spectrophotography measuring lipase activity while using emulsified olive oil as the substrate, finally a fungal strain LZ-6 with high-yield lipase was obtained. It was identified as Actinomucor elegans through morphological observation and molecular identification. The fermentation conditions for lipase production optimized by the single factor experiments and the orthogonal experiments with strain LZ-6 were：yeast powder 1%, olive oil 2%, 0.05% MgSO₄·7H₂O, 0.1%（NH₄）₂SO₄, 0.2% K₂HPO₄, fermentation temperature at 30℃, pH5.0, 2% inoculums volume, and fermentation time of 5 days; under the optimal condition, the lipase activity of strain LZ-6 reached 13.35 U/mL, it increased 1.78 times as before optimization.

Screening of Pseudomonas aeruginosa Strains Highly Producing Rhamnolipid

Zhang Cuikun, Chang Dongmei, Yang Hongjiang

2015, 31(10):  177-183.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.028

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This work aims to screen strains highly producing rhamnolipid from multiple sources, analyze the characterization of fermentation and physicochemical characteristics of rhamnolipid. CTAB（cetyltrimethyl ammonium bromide）methylene blue plate was used for primary screening the strains synthesizing the rhamnolipid. Then the strains were identified by analyzing 16S rRNA sequences, and the property of rhamnolipid was analyzed by TLC（thin layer chromatography）and FTIR（Fourier transform infrared spectroscopy）. Total 163 strains with a dark blue halo around the colony were selected for further analysis of producing rhamnolipid. Among them, 10 strains producing 12.2-17.7 g/L rhamnolipid were identified as Pseudomonas aeruginosa. Moreover, strain B12 yielding highest rhamnolipid was selected and used for the optimization of carbon resource, including glycerol, rapeseed oil, peanut cake or sunflower seed cake, and rapeseed oil was recognized as the optimal carbon source for the synthesis of rhamnolipid. Fermentation temperature was also evaluated at 35℃, 37℃ and 40℃, so the production of rhamnolipid was the highest of 26.8 g/L at 37℃. In addition, rhamnolipid produced by strain B12 was purified and analyzed by TLC and FTIR. In conclusion, strain B12 could synthesize a relatively high level of rhamnolipid and could be a candidate strain for industrial production.

Screening, Identification and Characterization of Strains Coupling Nitrogen Removal with Sulfide Removal

Huang Jun, Zhang Shiying, Wang Fanfan, Li Yuehan, Wu Peng, Song Yinling

2015, 31(10):  184-190.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.029

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In order to promote the engineering application of denitrification and desulfurization process, 2 highly-active autotrophic denitrifying bacterial strains H3 and H7 were screened from stable UASB with denitrification and desulfurization, and the strains were identified and characterized. Two strains were both gram negative bacteria, the 16S rDNA sequence analysis revealed that strain H3 and H7 had a similarity of 99.6%, 98.9% with Pseudomonas stutzeri CONC12 and P. stutzeri 19smn4 respectively. Combing the morphologic, physiobiochemical characteristics and the analysis of its 16S rDNA, 2 strains were identified as P. stutzeri. The results of growth and denitrification of 2 strains demonstrated that the optimal initial pH for H3 and H7 growth were 6.94 and 6.88 respectively, for denitrification were 6.77 and 6.56 respectively. The optimal temperatures for growth were 30.4℃ and 30.6℃, for denitrification were 31.4℃ and 31.2℃ respectively, both of H3 and H7 were non-resistant strains to salt.

Identification and Denitrification Characterization of a Psychrotrophic and Aerobic Nitrite-bacterium

He Tengxia, Li Zhenlun

2015, 31(10):  191-198.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.002

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The phylogeny and denitrifying characteristics of a newly isolated strain Y-9 were clarified. The psychrotrophic and aerobic nitrite-bacterium, strain Y-9, was successfully identified based on the analysis of its morphology, phospholipid fatty acid and 16SrRNA gene sequence analysis. Additionally, factors such as temperature, shaking speed, pH, incubation quantity, carbon source and nitrite content affecting the aerobic denitrification ability of Y-9 strain were investigated. Results showed that the Y-9 was identified as Pseudomonas putida which combined with three methods to identify the newly isolated strain could offer taxonomic assignment with higher confidence. A psychrotrophic and aerobic nitrite-denitrifying strain Pseudomonas putida Y-9 exhibited high removal efficiency of nitrite and total nitrogen. The nitrite and total nitrogen removal efficiency was up to 100% and 77.13% with 100 r/min shaking speed at 15℃. The optimal conditions of culture were determined, including temperature 15℃, shaking speed 100 r/min, pH 7, inoculated quantity 1 mL（OD₆₀₀, 0.5）bacterial suspension within 100 mL denitrification broth medium and carbon source sodium citrate. Low concentration of nitrite nitrogen could be beneficial for the strain Y-9 to perform denitrification at 15℃. Strain Y-9 was a psychrotrophic nitrite-denitrifying bacterium with optimum temperature of 15℃ and strain Y-9 could tolerate high concentration of nitrite nitrogen, which has a great potential for removing nitrogen from aquaculture water environment, especially in the cold circumstance.

Rapid Mutation Breeding Schizochytrium Strains Producing High-yield Docosahexenoic Acid by Atmospheric and Room Temperature Plasmas（ARTP)

Yuan Jun, Zhao Ben, Sun Mengyu, Wang Wu, Yang Hailin

2015, 31(10):  199-204.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.030

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This work aims to establish an expeditious method of mutation breeding strain producing high-yield docosahexaenoic acid（DHA）. The original strain Schizochytrium sp. ATCC 20888 was treated by Atmospheric and Room Temperature Plasmas（ARTP）, and screened with 100% lethal concentration of 2, 2&-Dipyridyl. The mutants were cultured by rotation-flask fermentation, and the strains with high-yield DHA from mutants were screened and selected by phosphoric acid-vanillin reaction and gas chromatography. The results showed that the conditions of mutation breeding Schizochytrium were ARTP for 15 s at the gas flow rate of 10.0 L/min and radio-frequency output of 100 W, and the concentration of 2, 2&-Dipyridyl was 100 μmol/L, so the strain with high-yield was obtained. The DHA-yield of mutant D32 increased significantly up to 7.31g/L, 29.8% higher than those by the original strain. Compared with the original strain, the main saturated fatty acid（C14:0 and C16:0）by D32 decreased significantly（P < 0.005）, while the unsaturated fatty acid content increased significantly（P < 0.005）. The results of subculture test showed that the mutant strain had stable hereditary character after 5 generations. Therefore, this method is not only prompt and efficient, but also provides a reference for the mutation breeding strains of producing other polyunsaturated acids.

Antifungal Activity of Litsea cubeba Oil Against Candida albicans

Liang Qing, Li Wenru, Shi Qingshan, Zhang Weimin

2015, 31(10):  205-210.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.031

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The work aims to investigate the antifungal activity of Litsea cubeba oil against Candida albicans, and illustrate its antifungal mechanism. L. cubeba oil was extracted by distillation and the compounds were identified using gas chromatography/mass spectrometry（GC/MS）. The minimum inhibitory concentration（MIC）and minimum fungal concentration（MFC）were determined by agar dilution method, and the antifungal kinetics of L. cubeba oil was studied. Finally, scanning and transmission electron microscopy（SEM and TEM）were used to observe the cell ultrastructure alteration after treated with L. cubeba oil. The main compounds of L. cubeba oil were D-Limonene（26.51%）, citral（11.94%）and verbenol（11.84%）. The MIC and MFC were both 1.25 μL/mL. Concerning antifungal kinetics, the oil only prolonged the lag phase of C. albicans, but not completely killed the cells while the concentration of oil below the MIC. Moreover, results of SEM indicated that C. albicans was destroyed easily at the neck between the mother cell and daughter bud after treated by L. cubeba oil. The results of TEM showed that the essential oil damaged the cell wall or the cell membrane, leading to the leakage of macromolecules and lysis. Conclusively, L. cubeba oil had an excellent antifungal activity against C. albicans, the dividing cells showed much more sensitive to L. cubeba oil, and the high concentration of the oil（5.0 μL/mL）led to irreversible damages of the cells. The targets of L. cubeba oil to fungi were the cell’s wall and outer membrane, which caused the large molecules leakage and organelles deformation, and finally led the cells to death.

Effects of PFOS on the Activities of Antioxidant Enzyme in Regenerated Dugesia japonica

Miao Zili, Yuan Zuoqing

2015, 31(10):  211-215.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.032

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Perfluorooctane sulfonate（PFOS）, a persistent organic pollutant, has been a typical predominant perfluorinated chemicals（PFCs）in the environment. In this study, we used regenerated planarians（Dugesia japonica）to evaluate the toxicological effects of PFOS on the activities of antioxidant enzyme. The results indicated that PFOS stimulated planarians to generate the stress responses. In the early stage of regeneration, PFOS stress caused oxidative damage and lipid peroxide of planarians, with the concentration of PFOS increasing, oxidative damage degree of planarian intensified; at the 5th day of regeneration, the body produced oxidation reaction to eliminate oxygen free radicals; in the late stage of regeneration, the oxidation reaction of planarian was no longer significant because of drug tolerance.

Preparation of Recombinant Human Plasminogen Activator in Rabbit Mammary Gland and Detection of Expressed Products

Lu Rui, Song Shaozheng, Qi Zhengqiang, Ge Xin, Shao Bin, Cheng Yong

2015, 31(10):  216-221.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.033

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Human tissue-type plasminogen activator（ht-PA）is a major biomedicine used in the clinical treatment of thrombotic diseases. There have been some reports on transgenic animal mammary gland as bioreactor producing ht-PA, however, there has been no report about recombinant human-tissue plasminogen activator（rhPA）expressed in transgenic animals. Here we report the preparation of transgenic rabbits by microinjection of fertilized eggs with the constructed mammary gland-specifically expressed vector of PCL25/rhPA while using F, E, and K₁ loci deletants of human t-PA as coding sequence driven by the goat beta-casein/CMV chimeric promoter. PCR and sequencing PCR products were used to identify the transgenic rabbits from candidates, and ELISA and fibrin agarose plate assay（FAPA）to analyze the expressed products of the transgenic rabbits. Results showed that 5 rabbits（2♂, 3♀）were transgenic rabbits and rhPA were expressed in their milk of 2 rabbits with maximum content of 400 μg/mL, and its biological activity was 150-200 times of alteplase. This study implies that rhPA can be expressed specifically in the mammary glands under the regulation of goat beta-casein/CMV chimeric promoter, and the expressed recombinant protein possesses the high activity of thrombolysis.

Efficient Expression and Biological Activity Detection of Fusion Protein of Onconase with Human Serum Albumin in Pichia pastoris

Yang Ganggang, Ma Chengkai, Shi Shihui, Zhang Quanyi , Wang Ze, Lü Zhongyuan, Wang Xuyang, Xu Xiaoya, Cui Qingqing, Zhang Jihong, Ding Yi, Xu Cunshuan

2015, 31(10):  222-229.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.034

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Onconase（ONC, also known as ranpirnase or P-30 protein）has weak RNase activity and significant toxic effect on various tumor cells and solid tumors. In order to obtain ONC owing highly efficient expression, we utilized the advantage of human serum albumin（HSA）expression in Pichia pastoris, had a fusion of HSA and ONC which was expressed in P. pastoris, then separated and purified the expressed protein. We designed the fusion gene according to the amino acid sequence of HSA-ONC and synthesized it based on the codons of P. pastoris, HSA-ONC gene was inserted into vector of pPIC9, pPIC9K and pPICZα-A to form the recombinant plasmid pPIC9/HSA-ONC, pPIC9K/HSA-ONC and pPICZα-A/HSA-ONC, then the linearized recombinant plasmids were transformed into P. pastoris X-33, GSS115 and SMD1168 respectively. The expression conditions were optimized in the shake flask and 10-L bioreactor. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, and the biological activity was detected by SRB assay. The highest rHSA-ONC production reached 235 mg/L in pPICZα-A/X-33/HSA-ONC combination under the condition of pH7 and 23℃in 1-L flask after induced 10 d, better than other combinations. In the 10-L bioreactor, when the induction was performed in the low basic salt medium of pH 7, and induced 10 d under 0.25% ethanol, the highest rHSA-ONC production could reach 2.02 g/L. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, the purity was ≥ 95% and the yield was > 70%. The results of detecting biological activity showed that the rHSA-ONC inhibited the proliferation of various tumor cells in vitro. Conclusively, the expression level of rHSA-ONC was twice as that of rONC at the same molar dose, and rHSA-ONC inhibited the proliferation of various tumor cells in vitro.

The Expression and Immune Response Characteristics of TLR2 at the Early Development Stage of Rana chensinensis

Wang Ze, He Tianyi, Lu Yuyan

2015, 31(10):  230-235.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.001

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It was to study the expression and immune response characteristics of TLR2 at the early development stage of Rana chensinensis. Immunohistochemistry and RT-PCR were used to locate and quantify the TLR2 mRNA from stage 11 to stage 42 of Rana chensinensis. Tadpoles at stage 29 were injected with antigen LTA, LPS, ZymoA and Poly I:C intraperitoneally. The expression changes of TLR2 mRNA were detected by RT-PCR within 24 h. TLR2 is widely distributed at the early development stage of Rana chensinensis. The relative expression of TLR2 mRNA increased form 0.187±0.021 to 0.373±0.031 at stage 25, and then returned to 0.170±0.020 progressively. The expression of TLR2 mRNA had no significant change in the LPS group and the Zymo A group（P>0.05）. TLR2 mRNA of the LTE group peaked twice at 4 h与12 h after antigen injection（P<0.01）; and the PolyI：C group peaked at 8 h（P<0.01）. The expression character of TLR2 matches with its living environment at the early development stage of Rana chensinensis.TLR2 did not responses to the challenge of LPS and ZymoA timely at the stage 29, on the other hand, TLR2 recognized and responsed to LTA and PolyI:C group effectively. TLR2 of Rana chensinensis possesses the recognition and response characteristics to PAMPs both of fish and mammals.

Effect of pH Heterogeneity in Large-scale Bioreactor on Fed-batch Culture Process of CHO cells

Liu Jintao, Wang Xingyi, Fan Li, Deng Xiancun, Liu Xuping, Tan Wensong

2015, 31(10):  236-241.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.003

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In order to study the effect of pH heterogeneity in large-scale bioreactor on cell culture process of CHO cells, we established a scale down model consistent of stirred tank reactor and plug flow reactor to simulate the pH heterogeneity of large-scale bioreactor based on the mixing characteristic. The results showed that the scale down process with 30 s residence time has no statically difference with the control process. However, significant effect on cell growth, cell metabolism and protein production were found when increased the residence time of PFR. Cell growth rate decreased accompanied by tremendously increase of ammonia and lactate when increased the pH heterogeneity. In addition, the titer, sialic acid content and bioactivity of antibody fusion protein were also decreased when increased the pH heterogeneity.

A Study on the Regulation Mechanism of ERK1/2 Promoting Proliferation and Inhibiting Apoptosis in Esophageal Squamous Carcinoma

Liu Fengxia, Liu Wenjuan, Li Jianyong, Chen Shenguo

2015, 31(10):  242-248.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.035

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This study aims to explore the role of ERK1/2 MAPK in the regulation mechanism of cell proliferation and apoptosis concerning of esophageal squamous carcinoma（ESCC）. The results of plate colony, cell apoptosis and cycle revealed that the inhibition of ERK1/2 MAPK pathway eliminated the clone formation and proliferation of Eca109 cells, promoted cell apoptosis, and slowed down cell cycle; in addition, the inhibition of ERK1/2 MAPK pathway reversed the changes of proliferation, apoptosis and cycle induced by miR-21 overexpression. The results of qRT-PCR and Western blot showed that the inhibition of ERK1/2 MAPK pathway downregulated endogenous miR-21 expression, and also reversed the activation of ERK1/2 MAPK signal pathway induced by exogenous miR-21. The findings demonstrated that the inhibition of ERK1/2 MAPK pathway hindered Eca109 cell proliferation, promoted cell apoptosis, and retarded cell cycle via downregulation of miR-21 expression in Eca109 cell.

Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening

Yuan Meng, He Zhixu, Shu Liping, Yuan Jiakan, Liu Feng, Li Yan

2015, 31(10):  249-254.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.004

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It was to construct the knocking out zebrafish hoxb4 gene model by the efficient gene editing system CRISPR/Cas9 system. Three 20 bp sgRNAs which were designed to insert into linearized plasmid pT7-gRNA, such as：192#（sense strand ）, target 244#（antisense strand）, target 313#（antisense strand）.Then gRNAs were transcribed in vitro. The Cas9 mRNA was transcribed using Cas9 expression vector as templates. Following completion of transcription, the poly（A）tailing reaction and DNase I treatments were performed. Both the gRNA and the Cas9-encoding mRNA were then purified and microinjected into the one cell stage of zebrafish embyos. Targeted genomic loci were amplified from genomic zebrafish DNA using primers then were tested by T7 Endonuclease I assays. Finally, the PCR products were constructed into pMD19-T simple vector, positive clones by colony PCR and Sanger sequencing to detect mutations. Results showed that SgRNA oligonucleotide duplexes successfully connected to the target site of plasmid pT7-gRNA and the sequence is correct, which targeted Exon 313 ^# site can successfully edit hoxb4 genes in zebrafish. T7EⅠ detect the knockout efficiency up to 26.5%. And four kinds of positive mutants were obtained by sequencing. The CRISPR/Cas9 system of knocking out the hoxb4 gene is successfully constructed and its mutations were identified and sequenced. The experiments provide a reliable knockout method for the study of the HOXb4 gene function.

An Integrated Gene Regulatory Element for Tumor-specific Targeting Therapy

Zhou Peijie, Gao Weiqiang, Fang Yuxiang

2015, 31(10):  255-262.  doi:10.13560/j.cnki.biotech.bull.1985.2015.10.036

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The key for a successful tumor-specific gene therapy is to regulate the therapeutic gene specifically and efficiently expressing in tumor cells. By the construction of an embedded expression regulatory element, a high efficiency as well as tumor-specific expression of target genes in tumor cells was achieved by integrated regulation at transcriptional, post-transcriptional and translational levels simultaneously. It was shown that the integrated gene regulatory element improved expressional specificities in prostate cancer cell line LNCaP up to 420% and 480% using enhanced green fluorescent protein（EGFP）and luciferase as reporter gene respectively in vitro. By survival assay in vitro, LNCaP cell survival was specifically suppressed by HSV-1 TK expression under the control of this regulator. These results confirmed that this element could be applied in tumor-specific targeting therapy.