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Table of Content

    30 January 2013, Volume 0 Issue 1
    Reviews and Monographs
    Advances of Salt Tolerance Mechanism in Hylophyate Plants
    Fu Chang, Sun Yugang, Fu Guirong
    2013, 0(1):  1-7. 
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    Halophytes are good material for study of plant salt tolerance mechanism and cloning salt-tolerant genes due to its reflecting ofplants’ adaptive strategies. In this review, molecular mechanism of halophytes in response to salt stress was reviewed in respects of transcriptionfactors, regulation of osmotic balance, ion homeostasis, regulation of redox reaction, regulation of photosynthesis and metabolic changes. Inaddition to this, the prospects of study of plant salt tolerance mechanism were also analyzed.
    Review on Research of Tobacco Premature Flowering Mechanism
    Yang Jing, Chen Jie, Guo Hongyan, Chen Jianjun, He Guangsheng, Deng Shiyuan, Wang Wei
    2013, 0(1):  8-15. 
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    Early flowering has deeply effect on the quality and yield of tobacco. With the developing of plant flowering research, theresearch of tobacco premature flowering was not just only on light, temperature, water, and nutrient, but also on molecular research. Causes oftobacco premature flowering are still not clear. This paper summarized and analyzed the influence of tobacco-flowering-time from many aspects,including physiological mechanism, molecular mechanism and external environment condition. Combining traditional research with the molecularresearch, we can expound all the problems comprehensive and find the way to solve them. It has great significance of the research of tobaccopremature flowering.
    Progress in the Improvement of Abiotic Stress Tolerance in Plants Using Transgenic Approaches
    Liu Chun, Cao Limin, Li Yuzhong, Peng Wanxia, Ma Hao
    2013, 0(1):  16-24. 
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    Abiotic stresses including drought are serious threats to the sustainability of crop yields. Use of modern molecular biologytools for elucidating the control mechanisms of abiotic stress tolerance, and for engineering stress tolerant crops is based on the expression ofspecific stress-related genes. Hence, genetic engineering for developing stress tolerant plants, based on the introgression of genes that are knownto be involved in stress response and putative tolerance, might prove to be a faster track towards improving crop varieties. Nevertheless, thetask of generating transgenic cultivars is not only limited to the success in the transformation process, but also proper incorporation of the stresstolerance. Evaluation of the transgenic plants under stress conditions, and understanding the physiological effect of the inserted genes at thewhole plant level remain as major challenges to overcome. This review focuses on the progress in using transgenic technology for the improvementof abiotic stress tolerance in plants. This includes discussion on the evaluation of abiotic stress response and the protocols for testing thetransgenic plants for their tolerance under close to field conditions.

    The Composition and Function of Small RNA—Research Overview of Three Types of Endogenous Small RNA
    Li Ze, Bai Helu
    2013, 0(1):  25-30. 
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    There are many small non-coding RNA in the organisms. They through complete or incomplete complementary base pairingwith target mRNA, leading to mRNA breakage or translational repression, so as to achieve the purpose of the regulation of gene expression.These small RNA composed the highly complex RNA regulatory networks in cells, and play an important role in the physiological activity ofthe biological process of growth and development. Endogenous small RNA including miRNA, siRNA, and piRNA three categories, the articleoutlines three small RNA biogenesis, mechanism, and function research progress.
    Advance on Research of Ginsenoside CK
    Yu Lei, Li Chenglong, Yu Shanshan,
    2013, 0(1):  31-35. 
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    Ginsenoside CK[20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol]is the main metabolite of the protopanaxadioltype of Panax ginseng C.A. Meyer in human intestine and it belongs to the rare ginsenoside. The unique biological activity of ginsenoside CKhas attracted more and more attention from the people. The studies on ginsenoside CK progressively increased recently. This paper reviewed theadvance on research of pharmacological activity and preparation methods of ginsenoside CK.
    Advances on the Role of C-type Natriuretic Peptide in Regulating Animal Reproduction Processes
    Jia Zhenwei, Zhang Jiaxin, An Lei, Wu Zhonghong, Tian Jianhui,
    2013, 0(1):  36-40. 
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    C-type natriuretic peptide(CNP), a member of the natriuretic peptide family of structurally related peptides, is a autocrine /paracrine growth factor with important roles in cardiovascular homeostasis, regulation of cell proliferation and skeletal development ; moreover,CNP also plays a key role in modulating animal reproductive processes. Notably, recent studies have revealed that CNP acts as a regulator toinhibit meiotic resumption in mouse oocytes in vivo ; additionally, CNP can enhances superovulation response by the stimulation of folliculardevelopment prior to gonadotropins treatment. In light of the findings above, CNP may be of special importance in the promotion of oocytedevelopment in vitro as one meiotic inhibitor, as well as the improvement of superovulation efficiency in domestic animals. Hence, this paperreviewed the structure of CNP, its role as one of functional molecules that modulate reproductive processes in animals and its potencial value.
    Insights into Toll-like Receptor from Marine Animals
    Sun Hongjuan, Zhou Zunchun, Cui Jun, Wang Xiuli
    2013, 0(1):  41-48. 
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    Toll-like receptors are a family of the primitive pattern recognition receptors in innate immunity system, which attract theinterest of immunologists all the time. Animals from the most ancient metazoan phylum porifera to bony fish are considered to possess TLR genes.In this paper, we collect the latest data about TLR and signaling pathway molecules from different marine phyla, the mechanisms of domainshuffling and polymorphism of TLR, which will be useful for exploring the origin of vertebrate TLR and prevent marine animals from diseases.
    Advances in Metabonomics and Its Applications
    Zhou QiuxiangYu Xiaobin, Tu Guoquan, Wang Qiang, Hu Wenjun, Li Hanguang1, 3
    2013, 0(1):  49-55. 
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    Metabonomics(or metabolomics)is an essential part of systems biology. Through the study of metabolic changes inorganisms, their physiological and biochemical status can be known and the internal principle can be discovered. The meaning and research taskof metabonomics were introduced in this paper. Generation, technological platform of metabonomics and its application in plants, microorganisms,disease diagnosis and toxicology were summarized. A review on the development trend and challenges were also given.
    Japanese Encephalitis and Its Vaccines
    Shen QiangShi Zixue, Wei Jianchao, Ma Zhiyong
    2013, 0(1):  56-62. 
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    Japanese encephalitis, an insect-borne zoonotic infectious disease, is caused by Japanese encephalitis virus(JEV)and themost important hosts of which in nature is birds and pigs. This disease occurs mainly in Asia and Central-West Pacific regions, and has becomeone of the most serious encephalitis diseases which threats to human health and affects the development of animal husbandry particular in pigfarming industry. Published date from WHO showed that an estimated 50 000 cases occur in this region, resulting in at least 10 000 deaths and15 000 cases of neuropsychiatric sequelae. However, there is no effective antiviral therapeutic is available to treat JE, and vaccination is the mosteffective method to prevent JEV infection. This paper introduces the epidemiological characteristics and enphalitis disease, focuses on reviewof JE vaccines in the market and the latest research process. The present paper would provide useful reference for use and development of JEvaccines in China.
    Techniques and methods
    Several DNA Molecular Marker Technologies and Its Application of Tea Germplasm Research
    Yan Changyu, Li Jiaxian, Huang Hualin, Wu Hualing, Qiao Xiaoyan, He Yumei
    2013, 0(1):  63-67. 
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    The principles and features of several DNA molecular markers in recent years are introduced, such as ACGM, SRAP, TRAP,SCAR, RSAP and SCoT. The application of several DNA molecular markers tea germplasm research were reviewed, and prospects of severalDNA molecular markers applied to tea germplasm research were analyzed.
    Research Progress on the Detection Methods of D-amino Acids
    Li Huixin, Ju Jiansong, Zhang Linlin, Xu Shujing
    2013, 0(1):  68-72. 
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    As we all know there are 20 different kinds of basic amino acids in nature. In addition to glycine, each amino acid has twoenantiomers D-and L-type. D-amino acids have long been considered to be unique to microorganisms, which utilize some D-amino acids toconstruct the peptidoglycan layer of their cell wall. However, several D-amino acids have been detected in many organisms including mammalsbecause of the advance of analytical techniques in the last two decades. It was realized that D-amino acids might play significant roles in livingthings and as the important component of medicine, pesticide and food. In order to get a better analyzing and understanding the important role ofD-amino acids, the analytical methods for D-amino acids detection are constantly developing. Herein, several methods include enzymatic method,Gas chromatography, HPLC and biosensor assay were introduced.
    Research report
    Cloning, Expression and Function Preliminary Research of RPS14 in Arabidopsis thaliana
    Fan Yanrong, Liu Ying, Zhang Feiyun
    2013, 0(1):  73-77. 
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    RPS14(Ribosomal protein S14)is a ribosomal protein of Arabidopsis thaliana. It is a structural component of 40S ribosomalsubunit and belongs to the S11P ribosomal protein family. It is responsible for RNA post-transcriptional processing. We extracted the total RNAof Arabidopsis thaliana. The RPS14 full-length gene was obtained by RT-PCR. Through inserting the gene fragment into pEASY-T3 vector byT-A cloning, we successfully constructed a recombinant vector pGEX-6P-1-RPS14. The GST-RPS14 protein was expressed in BL21(DE3)strain and molecular weight was approximately 43 kD. We constructed the recombinant vector pET-28a-AK6 in the same way and obtained theapproximately 26 kD HIS-AK6 protein. We demonstrated the interaction between RPS14 and AK6 using pull-down, SDS-PAGE and Westernblot. Therefore, we speculate that they together participate in the nucleic acid metabolism and play a role in the RNA post-transcriptionalprocessing, thereby inhibiting growth of stem cells and making plants show dwarf phenotype.
    ZjAPX Gene Improving Resistance to NaCl and Drought Stress in Arabidopsis
    Huina, Meng Yuping, Hao Ziqi, Li Qian, Cao Qiufen
    2013, 0(1):  78-82. 
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    In order to investigate the function of ZjAPX(Zizyphus jujuba ascorbate peroxidase )in response of plant to osmotic stress.In this study, ZjAPX gene was introduced into Arabidopsis. Wild type(WT)and transgenic type of Arabidopsis were used to the NaCl osmoticstress and drought stress. These results suggested that transgenic seeds germination and seedlings growth were both better than the wild-type.At 10 days to be treated with NaCl osmotic stress and drought stress, real-time quantitative PCR detection showed that the transcription level ofZjAPX in transgenic plants was significantly higher than wide type. These results suggest that high expression of ZjAPX increased plant NaClosmotic stress and drought tolerance.
    Cloning and Sequence Analysis of HbNTL1 Promoter from Hevea brasiliensis
    Kang Guijuan, Li Yu, Zeng Rizhong, Nie Zhiyi
    2013, 0(1):  83-88. 
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    Membrane-bound NAC transcription factors(NTLs)are a group of NAC transcription factors with transmembrane motif(TMs)in the C-terminal region, play important roles in plant growth and development, hormone regulations and stress responses. A 1 718 bp5' regulatory sequence of HbNTL1 was cloned from leaf genomic DNA of H. brasiliensis based on the cDNA sequences using Genome Walkingmethod. Sequence analysis showed that it consisted of a typical core promoter region of eukaryotic, and the transcription start site(TSS, A)located 206 bp upstream of the initiation codon ATG. The promoter contains the basic elements :TATA-box, CAAT-box, and some hormoneresponsive elements as well as and multiple stress-induced elements, such as, ABRE, DOFCOREZM, MYBCORE, MYCCONSENSUSATHSEand W-box. It is suggested that HbNTL1 gene may play important roles in response to various stresses in rubber tree as a stress-related NACtranscription factor.
    Analysis on Gene Expression in Rice Under Endophytes Alcohol Extraction Treatment with Gene Microarray Technique
    Xiao Jun, Yang Tao, Yang Zhen, Wang Hong, Wang Liping, Chen Xun, Zhao Ying, Wang Na, Gong Na
    2013, 0(1):  89-95. 
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    In order to explore the promoting growth and disease resistance mechanism of endophytes alcohol extraction on rice, the geneexpression in rice under the treatment of RD was analyzed by gene microarray technique. 1 171 different expressed genes were identified in thisstudy, including 671 up-regulated genes and 500 down-regulated genes. The explanation on the results of gene microarray through Go annotationsystem shponse to stimulus, transcription regulation, physiological process, cellular process and metabolic function. Significant difference wasfound among the pathway change, including 39 up-regulated pathways and 24 down-regulated pathways. It can be concluded that the geneexpression of rice under the treatment of endophytes alcohol extraction RD was ascribed to the co-effect of various aspects and multi level.

    Development of a Reference Plasmid for the Quantitative Detection of Genetically Modified Rice Huahui-1
    Li Liang, Wang Jing, Zang Chao, Sui Zhiwei, Zhao Zhengyi
    2013, 0(1):  96-101. 
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    With the development of genetically modified organism (GMO) detection techniques, the reference plasmids have been themost effective and important materials for GMO quantification as a positive control. But many of them are used in researching, not developeda certified reference material. Here, we outline a reference plasmid, developed according the Technical Norm of Primary Refernce Material(JJF 1006-1994), including preparation, analysis of purify, homogeneity, stability, commutability, characterisation and uncertainty. Resultssuggested the reference molecule is stable for 6 months and particularly suitable for the rapid identification of GMO components in unknownsamples and can reduce cross contamination.

    Study and Application of a Rapid Screening Method forGM Maize Seeds
    Wu Mingsheng, Yun Xiaomin, Song Ge, Niu Qian, Peng Ruidi
    2013, 0(1):  102-106. 
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    A rapid and high efficient screening method of GM crop seeds is the important technical support for strengthening regulationof the agricultural GMO. In this paper, a rapid screening method of GM maize seeds was established by integrating the DNA extraction method ofhot alkaline and the multiplex real-time fluorescent PCR technology. The results showed that the rapid screening method can not only ensure thedetection accuracy and sensitivity, but also greatly reducing false-positive incidence and improve the efficiency of the GMO screening, and whichis suitable for the rapid detection of large numbers of samples.
    Cultivars Identification of Chinese Cabbage Using Multiplex EST-SSR Markers
    Zhao Xin, Wang Yong, Lan Qingkuo, Chen Rui, Li Oujing, Yu Jinghui, Zhu Zhu, Guo Yongze
    2013, 0(1):  107-110. 
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    In this research, 12 Chinese cabbage cultivars identification method was studied by EST-SSR molecular markers technique.The results showed that there were 21 pairs of EST-SSR primers showing the amplification and 21 pairs of EST-SSR primers showing the highpolymorphism among 30 pairs of screened SSR primers, which were designed from expressed sequence tags(ESTs)in GenBank. In orderto identify Chinese cabbage high performance, using the identification results and the mutual position difference bands of 10 pairs of highlypolymorphic primers on 12 Chinese cabbage varieties cultivars, make up 2 sets of multiplex EST-SSR markers. The polymorphic rate were 88.9%and 97.0%, polymorphism information content was 0.910%, and results indicate that the 2 sets of multiplex EST-SSR markers could be used toindentification 12 Chinese cabbage cultivars high performance.
    Establishment of Effective Regeneration System of Brassica napus L.in vitro
    Yang Changyou, Yuan Zhonghou, Zheng Xiaomin, Zhang Tao
    2013, 0(1):  111-115. 
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    We examined the effect of the seedling age of sterile seedling, the days and concentrations of growth regulators in precultivation,the concentrations of 6-BA and NAA on rape tissue culture with HC8(Brassica napus L.). We established the technical systemof regeneration of HC8 in vitro with high frequency. The results showed that the best seedling age and the optimal day of pre-cultivation wasfive days for rape tissue culture with HC8, and the optimal concentration of 2, 4-D was 1.0 mg/L. The best medium for callus induction anddifferentiation of cotyledons were MS+2.0 mg/L 6-BA+0.05 mg/L NAA+3.5 mg/L AgNO3 or MS+3.0 mg/L 6-BA+0.1 mg/L NAA+3.5 mg/L AgNO3.With the medium, the ratios of the highest level of callus induction, regeneration and differentiation were 100%, 88% and 108.33%, respectively.The best medium for callus induction and differentiation of hypocotyls were MS+4.0 mg/L 6-BA+0.05 mg/L NAA+3.5 mg/L AgNO3. With themedium, the ratios of the highest level of callus induction, regeneration and differentiation were 95.24%, 81.82% and 104.55%, respectively.The best medium for rooting of regenerated plant was MS basal medium supplemented with 0.5 mg/L NAA with the ratio of the highest level ofrooting(90%).
    Establishment and Application of Two-dimensional Gel Electrophoresis for Proteome from the Phloem of Tetraploid Robinia pseudoacacia
    Zhang Sheng, Zhao Zhong, Liu Zhaojun, Zhang BoyongZong Jianwei, Zhang Lingling,
    2013, 0(1):  116-122. 
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    This paper used the method of TCA/acetone to extract all proteins of phloem in tetraploid Robinia pseudoacacia. A twodimensionalelectrophoresis system for proteomic analysis of tetraploid Robinia pseudoacacia was established by optimizing the parametersincluding the pH gradient of IPG strip, the gel concentration, the loading quantity of sample and isoelectric focusing conditions. The resultsshowed that :Use the TCA/acetone method to extract all phloem protein of tetraploid Robinia pseudoacacia, choose 17 cm IPG strip withpH4-7, load protein samples of 550 μg, followed staining with colloidal Coomassie Brilliant Blue and use 12% SDS-PAGE for tetraploid Robiniapseudoacacia phloem total proteins in two-dimensional electrophoresis while isoelectric focusing IEF hours increased from 60 000 Vh to 80 000Vh can get a clear background with more protein points, high resolution and good repeatability. Using the established system of two-dimensionalelectrophoresis can dug protein points from the gel directly and send them to mass spectrometry. Use this system to analysis differences ofhardwood cuttings rooting callus phase protein expression in tetraploid Robinia pseudoacacia. Filter out 83 differential protein spots, amongwhich, 15 were higher, 22 were novel, 22 were lower and 24 were lack of expression.
    Cloning and Expression of Bloom Helicase Mutant
    Zhang Jinbiao, Xu Houqiang, Luo Heng, Xu Qinghe, Chen Xiang, Li Kun
    2013, 0(1):  123-128. 
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    Bloom syndrome(BS) which is caused by mutation of BLM is an autosomal recessive disorder characterized by genomicinstability and the high rate of many types of cancer. Disease-causing missense mutations have been identified at 1 036 cysteines localized in theRecQ-Ct domain. In this work, with bioinformatics and molecular biology technology, blm gene which 985 and 1 036 amino acids were mutated was obtained by two cycles of recombinant PCR, and cloned into expression vector pET-28a, and transformed into the strain E. coli BL21(DE3).It was purified that double mutation BLM helicase is the high purity(>95%), and identified by Western blotting. They could be pivotal forunderstanding the pathogenic mechanisms of BS.

    The Production and Application of Ezrin Polyclonal Antibody
    Zhang Hongling, Wan Jun, Yan Fei, Zhang Xinsheng, Cao Wei, Tao Wei, Huang Laiqiang
    2013, 0(1):  129-133. 
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    Constructed Ezrin prokaryotic expression vector pET-28a(+)-ezrin, the recombinant plasmid was transformed into E. coliBL21(DE3), induced Ezrin protein was purified through Ni-chelating affinity chromatography and purified protein was used to immunize NewZealand rabbits and Kunming mice, antiserum was removed. The titer and specificity of anti-sera were determined by ELISA, Western blottingand immunofluorescence. The specificity of the anti-Ezrin antibody was identified by Western blot and immunofluorescence. The results showedthat anti-sera antibody reacted with antigen and detected a specific band of 82 kD, and the size was agreed with the predicted molecular mass,localization dectected by inmmunofluorescence was in good agreement with the references. Finally, the anti-surem was purified by protein G.These results suggested that the Ezrin polyclonal antibody revealed high sensitivity and specificity.
    Prokaryotic Expression and Purification of GST-Smad4 Protein
    Mei Zhu, Yang Yutao, Xu Zhiqing
    2013, 0(1):  134-138. 
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    It was to express human Smad4 gene in prokaryotic cells and purify the GST-Smad4 fusion protein. The full-length Smad4 genewas amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1-Smad4 was transformedinto E.coli BL21(DE3)and exogenous protein was induced by IPTG. After purification using MagneGST particles, the GST-Smad4 fusionprotein was further identified by Western blot. Results showed that the recombinant plasmid pGEX-4T-1-Smad4 was constructed successfully.When BL21(DE3)cells transformed with pGEX-4T-1-Smad4 were cultured at 30℃ and induced with 0.2 mmol/L IPTG, GST-Smad4 proteinwas obtained in a large quantity in supernatant. The purified GST-Smad4 was further identified specifically by Smad4 antibody. Therefore, itproved that GST-Smad4 fusion protein was successfully expressed and purified, and could be used for further study of the function of Smad4.
    Expression of Human Glucagon-like Peptide-1 in Transgenic Tobaccos
    Liang Hongwei Chen Faju Wang Yubing Li Fenglan, Lu Hai
    2013, 0(1):  139-143. 
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    Human pancreatic glucagon-like peptide-1(hGLP1)is a short peptide hormone with special attention in the treatmentof diabetes, is considered as the most promising candidate medicines. Based on design composition of hGLP1 gene and construction of plantexpression vector, hGLP1 gene was introduced into tobacco through Agrobacterium tumfaciens-mediated method. The PCR amplificationand Southern blot analysis indicated that the hGLP1 gene have been integrated into the genome of six lines of the regenerated tobaccos. GUShistochemistry staining showed that the reporter gene fusion with the target gene was expressed in transgenic tobacco plants. Western blotanalysis confirmed that hGLP1 fusion protein was detected in two lines of transgenic tobacco leaves. The fusion protein demonstrated a certainbiological activity in reducing blood sugar by elementary animal experiments.
    Expression of Zearalenone Mimicking Epitope Peptide by Phage Display System
    Xu Fuyong, Xu Ling, Liu Renrong, Qiu Xuemei, Zhu Lixin
    2013, 0(1):  144-150. 
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    The research was designed to study the expression of Zearalenone(ZEN)mimicking epitope by p Ⅷ phage display systemand verify its reactogenicity. An expression vector pC89-CZEN was constructed by linking the oligonucleotide contained ZEN mimicking epitopesequence and Enterokinase cleavage sites with the pC89S4 phagemid which had been double digested by EcoR I and BamH I. Engineered bacteriapC89-ek-zen was obtained after pC89-CZEN being transformed into competent XL1-Blue cells. The phage particles displaying Zearalenonemimicking peptide were acquired after pC89-ek-zen being super infected by KM13 phage. The optimal expression conditions were also explored.The reactogenicity was detected by ELISA. ZEN antibody binding capacity(before and after Enterokinase digestion)were also compared. Theresults show that the expression vector pC89-CZEN was constructed successfully. The optimal expression conditions were explored by ELISA.The binding effect is influenced by bacteria concentration when helper phage KM13 and IPTG added, and the final concentration of IPTG in themedium, induced expression temperature and time. The binding efficiency after enzyme digestion was higher than before.
    Study on Antimicrobial and Antitumor Activities of Marine-derived Fungus Penicillium herquei FS83
    Li Haohua, Chen Yuchan, Wang Lei, Zhang Weimin
    2013, 0(1):  151-155. 
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    The mycelial and broth extracts of a marine-derived strain of the fungus Penicillium herquei FS83 were tested for theirantibacterial activities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli by filter paper method, andinvestigated for their antifungal activities against Trichoderma viride, Aspergillus niger, Aspergillus flavus, Alternaria alternate and Colletotrichumgloeosporioides by growth rate method. Minimum inhibitory concentrations(MICs)of the culture extracts were determined by serial dilutionmethod. Furthermore, the extracts were evaluated for their cytotoxic activities against NCI-H460, MCF-7, and SF-268 tumor cell lines by theMTT method. The results showed that the mycelial extract displayed significant inhibitory effect on S. aureus and B. subtilis with inhibition zonediameters of 18.1 and 17.3 mm respectively, and the same MIC of 1.56 mg/mL, and also exhibited satisfactorg antifungal activities against allthe test fungi with inhibitory rates of over 50% and MICs in the range of 0.78-3.12 mg/mL. The broth extract showed a remarkable antibacterialeffect on B. subtilis with an inhibition zone diameter of 16.4 mm and an MIC of 1.56 mg/mL, and also displayed antifungal activities on all thetest fungi with MICs in the range of 1.56-6.25 mg/mL. The mycelial extract had a strong cytotoxic activity against the three tumor cell lines withIC50 values between 55.9-63.5 μg/mL, and the broth extract showed a strong cytotoxic activity against MCF-7 cell line with an inhibition rate of84.3% at a concentration of 200 μg/mL.
    Cloning and Sequence Analysis of Clusterin cDNA of Bufo japonicus formosus
    Yuan Jinqiang, Xu Yue, Yang Xianyu, Zhuge Hui, Zhang Shufang
    2013, 0(1):  156-161. 
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    To study the polypeptide effective components included in Venenum Bufonis, the plasmid cDNA library of adult Japanesetoad Bufo japonicus formosus skin was screened. As the result, the clusterin cDNA was obtained(GenBank accession number :JX035891).The transcript is 1 616 bp in length containing 30 bp 5', 254 bp 3' untranslated region(UTR)and an open reading frame(ORF)of 1 332bp encoding a polypeptide of 443 amino acids. There is a signal peptide consisted of 20 amino acid residues, six N-glycosylation sites, tencysteine residues for disulfide bond and two coiled-coil domains in the putative gene product. The homologous analysis indicates the similarityof B. japonicus formosus with Xenopus laevis was 65%, and 41%-48% with other species. The functional characteristics of CLU of B. japonicusformosus seem to be similar as that of human.
    Isolation and Identification of PQQ Producing Strains Using Methanol-utilizing Bacteria
    Xu Wen, Xu Ran, Zhang Liping
    2013, 0(1):  162-165. 
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    The aim was to isolate Pyrroloquinoline quinine producing strains from endophytic bacteria of Polygonum cuspidatumand strains of myxobacteria. Three kinds of media with methanol as single carbon source were used for microbial fermentation of the strains.Spectroscopy and HPLC analysis of the fermentation product were performed. Through preliminary screening and complex screening we got 134strains of methanol-utilizing and 4 strains of Pyrroloquinoline quinine producing. The Pyrroloquinoline quinine producing strain 083114 wasdetermined to be 64.34 mg/L. The 16S rRNA sequence analysis showed that the phylogenetic relationship between strain 083114 and Bacillusacidiceler is the most similar. Therefore, it proved that Pyrroloquinoline quinine producing bacteria were in endophytic bacteria of Polygonumcuspidatum and myxobacteria.
    Prokaryotic Expression of CP Gene of Sorghum mosaic virus and Preparation of Polyclonal Antibody
    Wang Hongxing, Gong Dian, Sun Yujuan, Zhang Yuliang, Wang Jianhua, Liu Zhixin
    2013, 0(1):  166-171. 
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    Sugarcane mosaic disease caused by Sorghum mosaic virus(SrMV)is now recognized as one of the most widely distributedviral diseases of Saccharum. It is an important limiting factor for sugarcane production. According to the sequence of coat protein(CP)gene ofSrMV, two primers were designed, and a DNA fragment about 987 bp was amplified by RT-PCR. The PCR product, after digested with restrictionenzymes, was inserted into prokaryotic expression vector pET32a and the recombinant plasmid pET32a-SrMVCP was transferred into E.coliRosetta(DE3). Induction with IPTG, recombinant protein was got and SDS-PAGE analysis and western blot showed that CP was expressedsteadily in soluble proteins. With addition of IPTG up to 0.1 mmol/L and culturing for 4 h more in 37℃ is thought to be the best inducingconditions to express CP. The fusion protein was then purified with Ni2+-NTA agarose affinity chromatography, and used to immune rabbits forantiserum preparation. The optimal titer of the antiserum was determined to be 1∶1 000 tested by indirect enzyme-linked immunosorbent assay(ID-ELISA). Western blotting confirmed that the antiserum reacted strongly and specifically to the CP of SrMV-HN field sugarcane sampleswere detected using the antiserum by a methed of ID-ELISA.
    Isolation,Identification and Cytotoxic Activities of Endophytic Fungifrom Taxus chinensis var. mairei
    maireiZhang, Qingbo, Chen Yuchan, Li Haohua, Pan Qingling, Wang Lei, Li Dongli, Tao Meihua, Zhang Weimin
    2013, 0(1):  172-177. 
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    Plate method was used to investigate the diversity of endophytic fungi associated with Taxus chinensis var. mairei Cheng etL.K in Guangdong province,and morphological and molecular methods were combined to identify the endophytic fungal strains. A total of 22isolated strains of endophytic fungi were identified and most of them belonged to the genera of Colletotrichum,Guignardia,Botryosphaeria,Arthrinium,Xylaria,Chaetomium,Alternaria,Phomopsis,Helminthosporium. MTT method was used to evaluate their cytotoxic activities,which showed the extracts of 6 strains had inhibition rates over 50% against at least one tumor cell line at 100 μg/mL,among which the extractsof strains A233 and A240 displayed inhibition rates over 90% against the MCF-7 cell line. Therefore,the endophytic fungi from Taxus chinensisvar. mairei were rich in species diversity. Some strains exhibiting good cytotoxic activities deserve further research on their bioactive metabolites.
    Microbial Diversity of the Crude Oil and Characteristics of a Hydrocarbon-degrading Bacterium
    Wang Zhonghua, Liang Jinger, Yang Jianqiang, Zhou Jun, Li Chenghua, Li Taiwu
    2013, 0(1):  178-185. 
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    The biodegradation bacterium system was obtained through adding nutrition, and the IR Technology, GC analysis and UVmethond were used. The analyse showed high degradation ability for crude oil. The diversity of bacterial communities in crude oil was researchedusing 16S rDNA library approach. A high efficiency bacterium was obtained from the biodegradation bacterium system, which was identified asBacillus pumilus by automated cellular fatty acid identification system and phylogenetic analyses based on the 16S rDNA sequence. GC analysisshowed that the low-carbon chain alkanes and high-carbon-chain alkanes as well as aromatic hydrocarbons can be degraded.
    Screening of the Oxalate-degrading Lactobacillus Strains Using Protolast Fusion Technology
    Zhang Yu, Mao Shuhong, Lu Fupin, Nie Shijian
    2013, 0(1):  186-190. 
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    Bifidobacterium lactis with oxalate-degrading capacity can efficiently reduce the oxalate in vivo, and can be used to prevent and treat kidney stone diseases. While Bifidobacterium lactis is poorly oxygen-tolerant, that can not be employed as microbial ecological agents. In this study, protoplast fusion was studied between Bifidobacterium lactis with oxalate-degrading capacity and oxygen-tolerant lactobacillus acidophilus. Under the optimum conditions of protoplast fusion with PEG 6000 concentration 50%, fusion time 7 min, fusion temperature 30℃ , concentration of CaCl2 0.02 mol/ L and the concentration of MgCl2 0.5 mol/ L, the fusion rate reached 7.6%, and a oxygen-tolerant fusant showing the level of oxalate degradation at 13.4% was obtained.
    Enhanced Carotenoids Production by a Rhodotorula sp. Under Stress Conditions
    Shi Xinyi, Liu Dan, Chen Lili, Chen Xin
    2013, 0(1):  191-197. 
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    Carotenoids, displaying yellow, orange, and red color, are a series of polyene compounds with important physiologicalhealthy function. They are found widely in microorganisms, including algae and plants. The effects of four factors, including low temperature,treatment time at low temperature, acid treatment and high-concentration salt treatment, on the carotenoids production by a Rhodotorula sp. wereinvestigated. Based on this, response surface analysis method was used to elucidate the interaction of the four factors. The results showed thatthe effects of low temperature, acid, high-concentration salt treatment and the interactive effects of low temperature-acid, low temperature- highconcentrationsalt, treatment time at low temperature-acid on carotenoids production are significant. The maximum carotenoids yield of 31.04 mg/Lwas obtained at the low temperature of 15 ℃ for 48 h, with pH 3.5 and 2 mol/L NaCl, which indicates that the carotenoids production can beenhanced under stress conditions.
    Comparison of Four Methods for Measuring the Biomass of the Cellulase Production Process
    Li Chen, He Ronglin, Wu Gaihong, Ma Lijuan, Lu Fuping, Chen Shulin
    2013, 0(1):  198-202. 
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    Biomass of the cellulase production process with insoluble cellulose as the substrate was measured using mycelial proteindetermination, nucleic acid determination, acid washing and drying method as well as cellulose analyzer application, respectively. Measurementresults of fermentation broth samples indicated that the precision of nucleic acid determination method was the highest with RSD value at 6.32%.This method and cellulose analyzer application method could be used to determine the biomass of the cellulase production process with insolublecellulose as the substrate more accurately. The precision of acid washing and drying method was poorer, but it could be used for routine analysisof biomass due to its simple operation. The mycelial protein determination method was proved to be poorer neither in precision nor in operationsimplicity, and it could be used as the complement of the other three methods. This study provides a basis for selecting method in measuring thebiomass of the cellulase production process with insoluble cellulose as the substrate.
    Determination Methods for Lipase Activity and Its Application in the Screening of Microbial Lipase
    Wang Huan, He Laping, Zhou Huanjing, Zhang Yiming, Li Cuiqin, Tao Han
    2013, 0(1):  203-208. 
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    Four common lipase activity determination methods including Rhodamine B plate, olive oil emulsification, ρ-NPP for lipasehydrolytic activity and ρ-NPP for lipase synthetic activity were compared. The result showed that the Rhodamine B plate method was suitable forthe qualitative and preliminary quantitative estimate of lipase activity and the other three methods were suitable for the quantitative determinationof lipase activity but only the ρ-NPP method has a good reproducibility. Moreover, there is no relationship between the hydrolytic activity andsynthetic activity of lipase. So lipase activity determination method should be chosen in screening the lipase-produced microbial. Namely,hydrolytic activity determination methods should be chosen in screening the hydrolytic lipase-produced microbial, otherwise, ester synthetaseactivity assay should be used. Based on the principle, desired lipase-produced microbial were screened.