Biotechnology Bulletin

Research Progress of Flowering Gene Regulatory Networks in Arabidopsis thaliana

Li Jing, Gu Huiying, Wang Zhimin, Tang Qinglin, Song Ming

2014, 0(12): 1-8. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.001

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Flowering is one of the most important phase changes during the vegetative to reproductive growth in the life cycle of higher plants. And in recent years, flowering regulation has become a hot research in plant molecular biology. In current study, the gene regulatory network controlling flowering in Arabidopsis thaliana has grown up to a intricate web of crosstalk, feedback, and redundancy, bound tightly with other developmental processes by “process integrators”. The paper only briefly contextualizes the genetic pathways involved in regulating flowering, and focuses on the integration of signals at the shoot apical meristem(SAM), the spatial-temporal regulation of flower development, and finally functions that floral-related genes have in processes other than flowering time or flower development in A. thaliana. Also we discuss about the prospects of future research works, according to the present research status．

Effect of Vegetable Cultivation on Microbiological Properties of Soil

1Liu Yiqian, Yi Xinxin2, Xu Quanming2, Zhong Lianquan

2014, 0(12): 9-17. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.002

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Soil microbial organic system is one of the important indicators of soil quality. Vegetable cultivation effects the number of soil microorganisms, summarized various factors of vegetable cultivation affecting the quantity of soil microbial properties, provide a reference for the sustainable development of agriculture.

Bio-detoxification of Ochratoxin A by Microorganism

Jia Xin, Xu Shihan, Liang Zhihong, Huang Kunlun

2014, 0(12): 18-23. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.003

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Ochratoxin A(OTA)is a class of polyketides, which are secondary metabolites produced by Aspergillus spp. and Penicillium spp., having a strong toxicity. In recent years, there are a large number of domestic and international researches on OTA biological detoxification, mainly on screening strains of OTA detoxification, including bacteria, molds and yeasts and so on . In addition, exploring different microbial detoxification mechanism is also a research hotspot. In this paper, these two aspects of progress were summarized and discussed.

Progress on Oocytes Extracts-mediated Derivation of Pluripotent Stem Cells from Somatic Cells

Shen Xinyi, Song Kun, Yang Lishan, Xiao Xiong, Zhang Dapeng, Yang Bo, Li Yuemin

2014, 0(12): 24-28. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.004

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Oocytes extracts-mediated derivation of pluripotent stem cells from somatic cells provides a new way for reprogramming. Morphologies, genes, and functions of somatic cells will be changed and somatic cells tend to be reprogrammed into stem cells after in-vitro co-incubation with oocytes extracts. The present situation, influence factors, existing problems, and prospects of somatic cells reprogramming using oocytes extracts were described in this review.

Advance Research on Factors of Fructose Utilization in the Wine Yeast Saccharomyces cerevisiae

Zhao Wenying, Jia Wanli

2014, 0(12): 29-32. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.005

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Yeasts with a high fructose consumption capability are very important for winemakers to solve problems associated with sluggish or stuck fermentations. Residual fructose at end of fermentation also can cause undesirable sweetness and risk of microbiological spoilage in wines. The fructose consumption capability is dependent on the yeast’s genetic background and on external conditions. It was reviewed on the level of the transporters cross the cellular membrane, hexose phosphorylation, and the effects of external factors(glucose concentration, ethanol stress, temperature and nitrogen resources availability). It is of significant importance in winemaking to find out and control the key factors that can have an effect on fructose utilization at end of fermentation.

Advance in the Research of Phospholipase C Gene Family

Wang Fawei,Wang Qi,Deng Yu,Dong Jinye,Wan Nan, Li Xiaowei, Li Haiyan

2014, 0(12): 33-39. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.006

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Phospholipase C(PLC)is a key member from phospholipase family, it cleaves phosphatidylinositol 4, 5-bisphosphate(PIP2) into diacylglycerol(DAG)and inositol 1, 4, 5-trisphosphate(IP3). In animals, PLCs are recognized as key components of signals through specific targets, such as protein kinase C or Ca²⁺-dependentsignaling networks. In plants, PLCs were characterized to regulate several abiotic and biotic stresses, but the mechanism of it are still unknown. In this review, we focused on the classification, structure, and the functions in different signal transduction.

Myxobacteria ：Natural Pharmaceutical Factories

Zhao Tingfeng, Gong Guoli

2014, 0(12): 40-46. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.007

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In recent years, drug development of myxobacteria and its resistant metabolic products has become one of the hot topics of researches study. Myxobacteria is an kind of important natural products producer. There is rich diversity in both the chemical structure and biological activity of these secondary metabolites. The diversity and broad-spectrum activity of metabolites have great potential in drug development field. This review describes the unique cell behavior in myxobacteria and its outstanding ability of producing secondary metabolites, and also expounds the progress of epothilone drug development. Finally, we discuss the potential of developing myxobacterial secondary metabolites into new drugs.

Principle of Whole Genome Amplification Technology and Its Progress

Pan Xiaoming, Liang Xingguo

2014, 0(12): 47-54. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.008

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The rapid developments of DNA array and next generation sequencing technologies had greatly promoted molecular biology techniques to be used in various fields. However, the limited quantity and quality of sample DNA may affect large scale genetic analyses. To overcome these problems, the whole genome amplification(WGA)technology was employed to amplify trace amounts of genomic DNA to sufficient quantity to meet the requirements of high throughput analyses. The highly efficient and reliable WGA methods also helped to realize large scale of genetic analyses based on even single cell. Moreover, as the single cell level research continues, more efficient WGA approaches were developed. This paper summarized the general WGA methods frequently used before and newly developed methods on basic principle level as well as the advantages and disadvantages of each method, which helped to make good use of the powerful WGA techniques.

Research Advance of RNAi in Anti-influenza Virus

Han Qinggong, Zheng Yushu

2014, 0(12): 55-60. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.009

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The RNAi technology has been widely used in drug and vaccine development of the prevention and treatment of various kinds of human diseases pathogeny,and has made great achievements.Antigenic drift and antigenic shift extremely easily occurred in influenza virus, which had put forward a bigchallenge for the development of anti-influenza virus drugs and vaccines.The appearance of RNAi technology gived good ideas for the prevention and control of influenza.This paper summarized the research status and prospects of using RNAi technology to develop anti-influenza virus drugs and new influenza vaccine at home and abroad in recent years, in order to provide references for the research for the prevention and control of influenza.

Advances of Real-time PCR Technology in the Field of Gut Microbiota

Zhao Jie, Ma Chen, Xi Xiaomin, Zhang Heping

2014, 0(12): 61-66. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.010

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Real-time PCR is one of the most effective methods, which rapidly accurate quantify nucleic acids. Compared with metagenomic, real-time quantitative PCR can quantify microorganism . Real-time PCR, a simple, fast, efficient, specific and high-through put method, is widely used in the field of gut microbiology. The technology in studies of intestinal microbes and disease has a wide range of applications. This paper reviews research progress about real-time PCR in the field of intestinal microbial recent years.

Establishment of a DNA Extraction Method Suitable for RAPD Analysis of Trace Arnounts of Camellia japonica L.

1Fan Jing,Li Zhongfang,Gao Minghui,Fu Qinchao

2014, 0(12): 67-72. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.011

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To explore a simple and high yield method for preparing DNA suitable for RAPD analysis, trace fresh and dried leaves of Camellia japonica L. were used as materials. Moreover, the improved CTAB, SDS and Triton X-100 methods without using liquid nitrogen were compared. The efficiencies of different DNA extraction methods, the effects of material preservation and material amounts on DNA qualities were compared based on the absorbance spectrum and ratio of absorbance at 230 nm, 260 nm and 280 nm by Ultraviolet Spectrophotometry. Results showed that dried materials were more suitable for DNA extraction than fresh materials. The improved CTAB protocol for isolating DNA from dried leaf tissues gave satisfied results, with the ratio of A260/A280 ranged from 1.595 to 1.736. Furthermore, the DNA yield reached 230 to 295 μg per gram of leaf. Finally, clear amplified bands were detected by RAPD-PCR. High quality and high yield genomic DNA could be prepared from 100 mg dried leaves of Camellia japonica L., an increase in materials leads to the increased in DNA amount, however, impurity was also increased.

Preliminary Application of PCR-DGGE to Analyzing Microbial Diversity of Ulva lactuca L. and Dictyota dichotoma

Feng Xuezhen, Wu Shanguang, Lu Yuan

2014, 0(12): 73-77. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.012

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To study the microbial diversity of Ulva lactuca L. and Dictyota dichotoma. As a new DNA fingerprinting technique, denaturing gradient gel electrophoresis(DGGE)can be used to analyze the microbial diversity in different environmental samples. Ulva lactuca L. and Dictyota dichotoma will be studied with respect to microbial diversity by applying cultivation-independent molecular methods DGGE. The results showed, the profile of DGGE showed that Ulva lactuca L. and Dictyota dichotoma had different bands. The structural diversity of the microbial community was examined by the Shannon index of general diversity H. Quantity one analysis showed that, V3 region of 16S rRNA gene was isolated from 25 DNA fragments of Ulva lactuca L. and 16 of Dictyota dichotoma. DGGE similarity and UPGMA analysis showed that ：the similarity of Ulva lactuca L. was higher than the Dictyota dichotoma(P<0.05).. Shannon indexes of two kinds of algae all showed Ulva lactuca L. were significantly higher than the Dictyota dichotoma(P<0.05). The algae microbial diversity of Ulva lactuca L. was richer than Dictyota dichotoma.

PCR-RFLP Analysis of mtDNA to Identify Four Kinds of Sturgeons

Dong Chuanju, Liu Yuanyuan, Liu Xiaoyong,Song Yingnan,Xu Peng,Sun Xiaowen

2014, 0(12): 78-85. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.013

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With the high efficient and accurate, molecular identification methods are widely used in some easily confused species. So we carried out the research in some miscible species of sturgeons with the application of PCR-RFLP. With the help of one pair primers, we amplified one region of mtDNA in 8 kinds of sturgeons’which contain Acipenseridae sinensis, A. schrenckii, A. baeri, A. ruthenus, Huso dauricus, H. huso, A. stellatus, A. gueldenstaeti. When digested with the restriction enzyme of Taqα I, Ava II, Eag I-HF and Nae I then test the results of PCR and enzyme digestion reactions with the density 3.0% of agarose gel, A. sinensis, A. ruthenus, H. dauricus, H. huso can be identified from other sturgeons. This method just need one pair of primers which is specific for binding to the mitochondrial DNA to amplify genomic DNA, then digested the products of PCR with different enzymes. So it is convenient to operate and can identify the species accurately in a relatively short period of time. On the basis of a little fin, we can identify common sturgeon species credibility and accuracy and also greatly improved the work efficiency.

Identification of Oyster Mushroom Spherical Virus in Pleurotus ostreatus by Deep Sequencing

Wang Shaojie,Wang Nian,Shi Yingchun,Hu Xiaoyan,Zhou Tao

2014, 0(12): 86-91. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.014

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To identify viruses in malformed Pleurotus ostreatus samples collected in Shunyi district in Beijing, deep sequencing of small RNA from total RNA was performed. Consequently, there were 3 075 small interfering RNAs(siRNAs)pairing to the complete sequence of Oyster mushroom isometric virus(OMSV)(NC_004560.1). These siRNAs were located in most regions on genome of OMSV, some of which formed hot-spots. By putting these siRNAs together, three contigs with length more than 500 nucleotides were assembled. According to the sequence of one of these contigs, seq22, a pair of primers was designed to amplify partial coat protein gene via RT-PCR. The results showed that cloned sequence was similar to the reference sequence and to the contig with identities of 91% and 99%, respectively. These results confirmed OMSV in the collected mushroom samples. Meanwhile, the related data acquired from deep sequencing was compared to the predicted siRNAs obtained by four softwares, showing that they were different from each other. The difference was discussed.

Optimization of Fermentation Conditions for Neomycin-producing Strain Streptomyces fradiae FIM-S38

Zhou Jingming, Chen Hong, Zhang Zhulan, Sun Fei, Zhang Yin, Zhou Jian, Fang Dongsheng

2014, 0(12): 92-96. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.015

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It was to optimize the fermentation conditions of neomycin producing by Streptomyces fradiae FIM-S38. The single factor experiments and orthogonal experiments were adopted to optimize the fermentation medium and conditions of FIM-S38 in shake flasks. The constituents of medium, the filling volume of shaking flask, the rotation speed and the temperature were the major factors that affected the yield of neomycin. The composition of glucose 6.0%, soluble starch 3.5%, soybean meal 2.0%, MgSO4 0.1% and CaCO3 0.3% was selected to be the medium formula, and the optimal fermentation parameters were as follows ：the filling volume 50 mL for a 500 mL shake flask, initial pH at 7.0, shaker’s rotating speed 250 r/min, temperature 35℃, fermentation time 6 d, and the titer of neomycin was 50% higher than that in control.

Optimization of Refluxing Extraction Technology of Anthocyanins from Purple Sweet Potato by Response Surface Method

Liu Qian, Qi Jiying, Han Jing, Wan Yue, Zheng Shibin

2014, 0(12): 97-104. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.016

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Response surface methodology(RSM)was used to optimize the refluxing extraction process of anthocyanins from purple sweet potatoes as a result of getting the optimum technology. The results of single-factor test showed that the best ethanol concentration was 70%, extraction temperature was 60℃, extraction time was 60 min, liquid-material ratio was 7∶1. Based on these results, the significant parameters were further optimized using Box-Behnken design, RSM and regression analysis. The optimum parameters were ：ethanol concentration 74%, extraction temperature 66℃, extraction time 56 min, liquid-material ratio 8∶1. Under such conditions, the extraction efficiency could reach 4.64 mg/g, and have a smaller relative error at 0.85% compared with the theoretical value(4.68 mg/g). The study showed that the processing parameters achieved from these experiments were accurate and can be applied to the extraction and production of anthocyanins from purple sweet potatoes.

Construction and Characterization of Yeast Two-Hybrid cDNA Library Derived from Roots of Gossypium barbadense Inoculated with Verticillium dahliae

Yang Jun,Zhang Yan,Wang Weiqiao,Rong Wei,Wang Xingfen,Ma Zhiying

2014, 0(12): 105-110. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.017

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A yeast two-hybrid cDNA library derived from roots of Gossypium barbadense cv. Pima90-53 inoculated with severe virulence Verticillium dahliae was constructed based on SMART^IM technique and homologous recombination reaction. Detection of the library indicated that its capacity and titer were 2.82×10⁶ and 5.02×10⁸ cfu/mL, respectively. The result from colony PCR showed that the length of insert cDNA fragments ranged from 500 to 1 500 bp on average and the recombination rate was 93.33%. These results demonstrate that the library meets the demands of the standard cDNA library, which will be useful for screening interaction proteins, determining signal transduction components and analyzing the structure and function of disease-resistant proteins in the future.

Bending Spore of Corn Leaf Spot Fungus REMI Albino Mutant Screening and Determination of Pathogenic Transformation

Xia Shuchun,Wang Xuewu,Zhang Ruqin,Yan Honghai

2014, 0(12): 111-116. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.018

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To investigate bending spore corn leaf spot fungus pathogenic mechanism and genetic variation characteristics, this study using restriction enzymes mediated gene integration technology(REMI)of Curvularia lunata genetic transformation, received 109 stable mutant strains. PCR detection results showed that the plasmid pUC-JS has successfully imported mutant strains, into the son has both single copy and copy, albino strain single copy insert frequency was 40%. In addition, an albino corn bending spore bacteria leaf spot REMI contrast with wild strain of the mutant strains colony color, and also significantly weaker than wild on pathogenic strains. Plasmid save, sequencing and analysis of blast speculated that inserts may result in the loss FAD3 gene function.

Insecticidal Test of the Extracts from Tomato Stems and Leaves to Lipaphis erysimi Kaltenbach

Yang Liu,Chen Yufei

2014, 0(12): 117-120. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.019

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Insecticidal Test of the extracts from tomato stems and leaves to Lipaphis erysimi Kaltenbach were explored in this paper. Through the insecticidal test of the extracts by using different solvent, the biological detection of indoor concentration gradient of ethanol extracts from tomato stems and leaves, the insecticidal effect test in field plot, the insecticidal test by using different concentration of ethyl acetate, the result showed the extraction solvent was ethanol, half lethal concentration of indoor biological detection was 10.5 mg/mL, the insecticidal effect by using high concentration of extracts in field plot was ideal, the solvent extraction was ethyl acetate. The half lethal concentration of indoor biological detection of ethyl acetate was 6.51 mg/mL.

Bioactivities of the Alcohol Extracts from Three Plants Against Dioryctria splendidella Larvaes

Zhou Jiechen Yan Ruikun Wang Shengjie He Yuanhao Tan Yiming Zhou Guoying

2014, 0(12): 121-127. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.020

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In order to study the bioactivities of ethanol extracts from Phytolacca americana, Citrus maxima peels and Macleaya cordata against the larvaes of Dioryctria splendidella preliminarily in laboratory, contact toxicity, antifeedant activities and body weight gains of D. splendidella larvaes were determined after treated with 3 ethanol extracts. The results indicated that 3 ethanol extracts with different concentration had some bioactivities against the larvae of D. splendidella, and the higher the extracted concentrations, the more obvious of the bioactivities to the larvaes were. When the concentration of Citrus maxima peels extracts was 100 mg/mL, there were the strongest bioactivities. The contact toxicity corrected mortality, antifeedant rate and growth inhibiting rate reached to 80.00%, 99.28%, and 96.32%, respectively. The control effect in the forest was 55.70%, the same as the pesticides thiacloprid. The ethanol extracts from Phytolacca americana had the lowest bioactivities, but the corrected mortality of ethanol extracts being high concentration were more than 70%, antifeedant rates and growth inhibiting rates were more than 90%.

Effects of Mutations in IQ Motif of AtIQM1 on Its Calmodulin Binding

Huang Zhangke, Zhang Yi’neng, Mo Zhongzhen, Zhou Yuping, Huang Xiaoling, Tian Chang’en

2014, 0(12): 128-132. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.021

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To confirm the effects of mutation of key amino acid residues in IQ motif of AtIQM1 on its calmodulin binding, the LQ was deleted or L was mutated to S through the mutagenesis technology in vitro. Then calmodulin binding of the mutant protein iqm1 was analyzed via yeast two-hybrid assay. The self-activation was not observed when the recombinant plasmid pGADT7-IQM1CDS, pGADT7-IQM1Del113-114, pGBKT7- CaM5 or pGADT7-IQM1L113S was transformed into the yeast AH109 strain, respectively. Blue colonies were achieved on the selective agar plates (SD/-Ade/-His/-Leu/-Trp/X-α-Gal)when pGADT7-IQM1CDS and pGBKT7-CaM5 were co-transformed into AH109 yeast strain. However, the yeasts which were co-transformed with pGADT7-IQM1Del113-114 and pGBKT7-CaM5 or pGADT7-IQM1L113S and pGBKT7-CaM5 were not able to grow on the selective medium(SD/-Ade/-His/-Leu/-Trp). The results suggest that the LQ or L in IQ motif is required for AtIQM1 to combine with CaM5.

Influence of Piriformospora indica on Host Plant Selection by Aphid Lipaphis erysimi(Kaltenbach)

Liu Jinhua, Wang Ting, Gao Qikang

2014, 0(12): 133-140. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.022

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In order to confirm the influence of Piriformospora indica on host plant selection of L. erysimi. Aphid dual-choice assay was taken under greenhouse and artificial control environment and the amounts of aphids on the P. indica-colonized B. napus was significantly less than the P. indica-free B. napus. Volatiles emitted by plants collected by dynamic headspace systems, the quality and quantitative differences of volatiles emitted by B. napus were analyzed through GC-MS. The result indicated that P. indica inoculated B. napus and P. indica-free B. napus had the same volatiles, but the quantity of total volatiles, α-pinene, terpinolene, camphor, β-thujene, cedrene and β-caryophyllene emitted by B. napus with P. indica were significantly higher than P. indica-free B. napus in unit time. Through the four-sector olfactomete method, we confirmed that the α-pinene significantly attracted the L. erysimi, while the camphor significantly repelled the L. erysimi. And the mixture of camphor and α-pinene(2∶1)was also significantly repelled the L. erysimi. Terpinolene, β-thujene, cedrene and β-caryophyllene had no effect on the L. erysimi.

Preparation of the Polyclonal Antibody Against Helicoverpa armigera FK506 Binding Protein by Immunizing with pcDNA3-FK506BP

Zhu Yan, Zhang Fuchun, Ma Ji, Huang Li’na, Liu Xiaoning

2014, 0(12): 141-146. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.023

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It was to prepare the polyclonal antibody against FK506 binding protein, which was to be to verified the specificity of the antibody by Western blot. Firstly, recombinant expression plasmid pcDNA3-FKBP12 was constructed and sequenced, and the recombinant plasmid pcDNA3-FKBP12 was then transfered into Kunming mice four times. Finally, ELISA assay, Western blot analysis and immunohistochemistry(IHC)were used to determine the titer of polyclonal antibody of FK506 binding protein and its specificity, respectively. The results showed that the titer of polyclonal antibody reached 1∶25 600, and it was able to bind specifically to both the fusion protein His- FKBP12 and FKBP12 from the different tissues of the cotton bollworm in the 3rd larvae, which laid the foundation for the subsequent experiments.

Analysis of Genomic Structure and Introns of SoNIN1 Gene from Sugarcane

Niu Junqi,Wu Chaoxing,Yang Litao,Li Yangrui

2014, 0(12): 147-152. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.024

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According to the full length cDNA and part of the promoter sequences of the SoNIN1 gene we designed primers，amplified the SoNIN1 gene form gDNA of sugarcane leaves by PCR, and analyzed the gene structure of SoNIN1 by using bioinformatics methods. The results showed that the DNA sequence of the SoNIN1 gene is 4 938 bp, containing six exons and five introns, GenBank accession number KF563902. 5’UTRs of the SoNIN1 gene contain 268 bp introns, and five introns contain low temperature, drought, and hormonal stress response related cis- acting elements, which may play a role in transcription and expression regulation of SoNIN1 gene. According to phylogenetic analysis and exonintron gene structure, the NIN proteins can be classified into 2 classes’type I and type II.

Research on Isolation of Trichoderma from Waste of Walnut Peel and Adaptability

Yang Weimin, Ran Cuixiang, Gao Xingsheng, Shi Yabo, Zhao Linfang

2014, 0(12): 153-160. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.025

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The peel derelict of hazardous substances from walnut fruit picking and processing has given a rise to the concern around the world. Walnut peel was used as the raw material and was left to mildew after its moist heat sterilization(temperature 121℃, 30 min)treatment, and then breeding strains were isolated from the walnut peel. The dominant species were to be gained through disproveal experiments, and adaptability of the species through detection changes in the main nutrient content of the peel around the mildew was researched . The results showed that the four walnut strains were isolated, which fall into Aspergillus flavus, Aspergillus niger, Penicillium and Trichoderma. Trichoderma strains is the most dominant strain among all the strains. The change of peel nutrients after mildew as follows ：soluble sugar content decreases by 6.03%, fat content decreases by2.97% ；proteins decreases by 7.68%, cellulose content decreases by 64.7%. In a conclusion, Trichoderma is adaptable to walnut peel and has decomposition effects, and can be developed as a new bio-fertilizers and biocides.

The Isolation of Beta-amylase-producing Bacteria and Cloning， Expression of Beta-amylase Gene in Escherichia coli

Li Meng, Chen Lifei, Yang Jianlou, Ma Chunling

2014, 0(12): 161-167. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.027

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A bacterium which can degrade starch was isolate from the soil near the starch factory, was identified as Bacillus cereus by means of morphology and physiological-chemical characterization as well as 16S rDNA sequencing. Beta-amylase gene was cloned from the strain by PCR, and gene sequence was analyzed, which can encode a signal peptide which contains about 30 amino acids. The beta-amylase gene was cloned and expressed in Escherichia coli BL21(DE3). The results showed that recombinant bacteria can produce beta-amylase, and enzymatic activity increased by 53.9% compared with the parent.

Identification and Molecular Characterization of a Phage from Streptomyces fradiae

Bai Yu, Xu Chunyan, Su Jianyu

2014, 0(12): 168-172. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.028

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It was to identify and explore the molecular characterization of a phage SF1, which was isolated from Streptomyces fradiae fermentation broth during the failed industrial production of tylosin. Phage SF1 was characterized by observing the shape of plaque, titering on double-layer agar culture plate. The morphology of SF1 was observed by the electron microscopic observation after negative staining. The sensitivity of the phage to chloroform and isopropyl alcohol was tested to detect its envelope. The structural proteins analysis of phage SF1 was carried out by SDS-PAGE. Restriction fragment length polymorphism(RFLP)analysis of genomic DNA was further performed to characterize the phage. Results showed that the plaque(11-15 mm in diameter)is neat and transparent. SF1 has a polyhedral head(about 24-30 nm in diameter)and a long tail(about 52-64 nm long and 3-5 nm wide). It is insensitive to chloroform and isopropyl alcohol. The genome of SF1 consists of double-stranded DNA and is approximately 35 kb. Capsid protein analysis shows a higher level of 14 major protein bands ranging from 14.8-59.5 kD, which is presumed to be the structural proteins of SF1.

Streptomycin-resistant Rational Screening for High-yield Neomycin Producing Strain

Chen Hong, Zhang Zhulan, Sun Fei, Zhang Yin, Zhou Jingming, Zheng Rong

2014, 0(12): 173-176. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.029

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NTG mutagenesis and streptomycin resistance screening was applied to screen neomycin high producing strain. A number of streptomycin-resistant(str)mutants were obtained from original strain of Streptomyces fradiae FS1109 treated with three different dosage of NTG under the selection pressure of resistance to streptomycin(the lethal concentration is 3 μg/mL). Streptomycin-resistant mutants strain Str63 was obtained with high neomycin yield by replica plating method and the neomycin productivity could be more than 50% higher and the component of C was lower than that of the original strain in the rotation-flask experiments, the rate of the positive mutation was larger than that of the negative mutation.

Cloning and Prokaryotic Expression of p38β MAPK from Lutjanus sanguineus

Xia Hongli,Cai Jia,Lu Yishan, Jian Jichang, Wu Zaohe

2014, 0(12): 177-183. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.030

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Humphead snapper(Lutjanus sanguineus)p38β MAPK(Ls-p38β)was cloned using RACE method. The GenBank accession number is KJ502277. The full-length cDNA of p38β MAPK was 1 628 bp containing an open reading frame of 1 086 bp, encoding 361 amino acids with an estimated molecular weight of 41.6 kD and a theoretical pI of 5.74. Amino acid alignment indicated that Ls-p38β possessed a Thr-Gly-Tyr(TGY)dual phosphorylation motif of p38 MAPK family and shared 97% similarity with Oreochromis niloticus p38β. The prokaryotic expression vector pET-p38β was constructed and then transformed into BL21(DE3). SDS-PAGE and Western blotting analysis indicated that the recombinant protein of Ls-p38β was successfully expressed and molecular weight of expressed fusion protein was 60 kD.

Comparison on Muscle Growth and Expression of Muscle Growth- related Genes Among Three Mandarin Fishes

Xu Miaoyang,Zhao Jinliang,Li Chuanyang,Qian Yezhou,Wu Chao,Qian De

2014, 0(12): 184-189. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.031

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Muscle samples of Siniperca chuatsi ♂×S. scherzeri ♀ F1 and their parents at five developmental stages in 90 days after hatching. The skeletal muscle growth characteristics were identified by numbers and diameters of muscle fibers through paraffin sections.Meantime were obtained and the mRNA expression of myostatin, MyoD, myogenin in muscle was detected by quantitative PCR. The results showed there were significant differences in growth among three mandarin fish. Siniperca chautsi grew the fastest, F1 secondly and S. scherzeri kept the last. At different developmental stages, numbers of muscle fibers in three mandarin fish showed no significant difference, while the average diameters of the muscle fibers showed positive correlation with growth rate. Growth differences among three mandarin were mainly caused by muscle hypertrophy. The myostatin mRNA expression peak appeared in D20, then the expression showed a downward trend. MyoD mRNA expressed highly in D10, D20 and decreased in following days. The myogenin mRNA expression was the lowest in D20, followed by an obviously increase in Siniperca chuatsi, and F1 secondly, while S. scherzeri stable. It showed correlations between muscle growth and gene expression in three mandarin at different growth stages.

Construction of Eukaryotic Expression Vector for the CD151 and CD163 Receptor Proteins of Porcine Reproductive and Respiratory Syndrome Virus

Ruan Zheng,Li Jie,Liu Kaiwu,Wang Lianfang,Hua Juan,Hu Xiaoming,Liu Jie,Huang Haijun

2014, 0(12): 190-194. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.032

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In order to study the interaction mechanism between the PRRSV and cell receptors, the CD151 and CD163 code fragments were obtained from PAM cells by PCR. These PCR products were digested with Sal I and Kpn I, then inserted into pIRES2-EGFP vectors separately to construct eukaryotic expression plasmid pIRES2 EGFP - CD151 or pIRES2 EGFP - CD163. These vectors were identified by the methods of restriction enzyme digestion, PCR and sequencing. Marc 145 cells transfected with CD151 or CD163 or CD151/CD163 expressed strong green fluorescence, which was further confirmed by western blot. The successful establishment of eukaryotic expression vectors of CD151 and CD163 provides the material foundation for the further exploring the synergistic effect of CD151 and CD163 in the process of PRRSV infection, especially to the further study of PRRS virus invasion and the host identification.

Construction of a Humanized DCF1 Gene Prokaryotic Expression Vector and Protein Purification

Wang Qian, Yang Mei, Feng Ruili, Wang Jiao, Wen Tieqiao

2014, 0(12): 195-200. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.033

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In order to further study the role of human DCF1 gene, the target fragment DCF1-TAT was amplified from plasmid pcDNA3.1- DCF1-TAT by PCR, and then cloned into a prokaryotic expression vector pET32a to construct the recombinant plasmid pET32a-DCF1-TAT,which was then transformed into Escherichia coli Rosetta(DE3)cells to express the fusion protein and induced to express by isopropyl-β-D-thiogalactoside(IPTG), and meanwhile the expression condition was optimized. The inclusion body was dissolved by urea after the optimization of dissolution condition, purified by Ni ion affinity chromatography, and renatured by dialysis. SDS-polyaerylamide gel electrophoresis(SDS- PAGE)and Western blot analysis were used to detect the fusion protein. The results indicated that the recombinant expression vector pET32a- DCF1-TAT was constructed and expressed in Rosetta successfully. The fusion protein existed in the form of inclusion body, and the target protein was obtained with high purity through the dissolution of urea and purification of affinity chromatography. SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.

Expression and Activity Assay of Human Aldehyde Dehydrogenase2 in Escherichia coli

Huang Juan, Wang Hehua, Zhang Xiaoji, Pan Boyu, Chen Xiaoping, Wu Yuanxin

2014, 0(12): 201-206. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.034

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The commercial acetaldehyde dehydrogenase is mainly extracted from animal cell and hepatocellular mitochondria. Source is limited and the cost is high. So the micobiological fermentation was used to obtain large amounts of aldh2, which had been cloned into the expression vector pET32a with 6His tag that was advantageous to purify the recombinant protein by nickel-chelate chromatography, then the recombinant was transformed into E.coli BL21(DE3). After induced by IPTG, the recombinant protein had been expressed and their molecular masses were determined to be approximately 55 kD by SDS-PAGE. Through the optimization of inducing expression conditions, the results showed that the condition(37℃, 1.0 mmol/L IPTG, induced for 6 h)is the optimal. Because all target protein were almost expressed in the form of inclusion body, the bioactive acetaldehyde dehydrogenase was obtained by means of degeneration, purification and renaturation for the inclusion body, and measured the concentration of the renaturated protein by the Bradford method that was 77 μg/mL. Experimental data showed that the final yield of enzyme was 14.49 U/L. Activity tests showed that the protein has the acetaldehyde dehydrogenase activity of about 1.449 U/mL. By constructing a recombinant vector pET32a-ALDH2 and optimizing the expression condition, aldh2 in prokaryotic expression host get a lot of expression. In addition, the activity of renaturation protein was improved by the dialysis renaturation method.

Study on Synthesis of Ethyl Butyrate by Butyric Acid Fermentation Broth Extraction Coupling with Esterification

Zhang Shexi, Chen Shaopei, Yang Jing, Wang Jufang

2014, 0(12): 207-213. doi:10.13560/j.cnki.biotech.bull.1985.2014.12.035

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Ethyl butyrate is one of the short chain ester, which is widely used in food, flavor, chemical, medicine and energy industry. In order to synthesize ethyl butyrate using butyric acid fermentation broth, butyric acid extraction from fermentation broth and estrification catalyzed by Novozym 435 in the extractive phase was studied. Tri-n-octylamine(TOA)+ cyclohexane were adopted as extractive agent and esterification reaction medium. Under the optimum conditions, the esterification reaction in the extractive phase had approached equilibrium in 5 h, the corresponding esterification yield was 81.42% and productivity was 65.96 mmol/(L?h). Good operational stability of Novozym 435 in this reaction system was observed. Ethyl butyrate yield relative to butyric acid was 0.65 mol/mol, which ethyl butyrate was synthesized by 100 mL butyric acid fermentation broth extraction-esterification coupling. These results suggest that synthesis of ethyl butyrate by butyric acid fermentation broth extraction-esterification coupling could realize the scale-up industrial production.

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