Table of Content

22 April 2013, Volume 0 Issue 4

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Review

Drought，Salt and Temperature Stress-induced Metabolic Changes in Plant

Liu Chun, Cao Limin, Zhou Dongsheng, Peng Wanxia

2013, 0(4):  1-7. 

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Plants regularly face adverse growth conditions, such as drought, salinity, chilling, freezing, and high temperatures. These stresses can delay growth and development, reduce productivity, and, in extreme cases, cause plant death. Plant stress responses are dynamic and involve complex cross-talk between different regulatory levels, including adjustment of metabolism and gene expression for physiological and morphological adaptation. In this review, information about metabolic changes in response to drought, extreme temperature, and salinity stress were summarized.

The Cis-elements of Responsing Jasmonates in Plant

Huang Shiwei, Ma Shu, Tian Yun, Lu Xiangyang

2013, 0(4):  8-13. 

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Jasmonates is a kind of basic plant hormone, widely exists in living organisms, as a signal molecule, it plays an important role in the process of the plant development, senescence and stress signal response. The promoter a DNA sequence which is located in gene 5'end ATG upstream and responsible for the regulation of gene transcription. At present, a lot of induced expression related cis-elements has been identified. This paper mainly introduces the research progress of the of plant promoter’s cis-elements which is responsing jasmonates.

Recent Advances in ANL Andenylating Enzymes

Li Lili, Lin Shuangjun

2013, 0(4):  14-20. 

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The ANL adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular non-ribosomal peptide synthetases（NRPSs）. Members of this family catalyze a two-step reaction ：the initial adenylation of a carboxylate to form an acyl-AMP intermediate, the second step commonly for formation of a thioester. With more members of the adenylateforming family of enzymes being identified, 10 conserved and important sequence motifs for catalytic activity have been revealed. The comprehensive studies of the structures and kinetics provide insight into the role of the rotation of the C-terminal sub-domain in the catalytic cycle. Such a rotation, a common strategy used by ANL adenylating enzymes, can present different conformation of the active sites to catalyze different partial reactions. In this paper，we describe the three types of ANL adenylating enzymes and summarize the research course of the ANL superfamily enzymes，conserved regions and conformational dynamics of enzymes in the process of the enzymatic reactions.

Overview of the Protection Effect of Zinc on Mammalian Cell Damage

Zheng Juanjuan, Zhang Yu, Xu Wentao, Huang Kunlun

2013, 0(4):  21-26. 

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Zinc is one of the most important nutritional trace elements of human body. It is essential to the structure and function of a large number of macromolecules and cofactor to more than 300 enzymes. We discussed the involvement of zinc in antioxidant defense, DNA integrity and apoptosis regulation, which protect cells against toxic substances, such as heavy metals, hydrogen peroxide and alcohol.

Application of Ribosomal RNA Technique in Identification to Aquatic Animal’s Bacterial Pathogeny

Li Li, Xie Xuyang, Cao Yanchao, Yan Lina

2013, 0(4):  27-32. 

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Bacterial disease is the most common disease which caused high mortality in aquaculture, people have pay more attention to the identification of pathogens. As the microbial ribosomal RNA（rRNA）database is getting more sophisticated, rRNA analysid has been widely recognized as the main basis for classification of bacteria, rRNA become a powerful tool for identification of pathogen. In this paper, we reviewed the character, research method and the application of rRNA gene in detection of aquatic anmiml’s pathogenic bacteria, and analysised the existing problems in the application.

Character and Application of Killer Yeast

Wang Xianghong, Jia Renjie, Li Jing, Chi Zhenming

2013, 0(4):  33-38. 

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The killer toxin produced by killer yeast can inhibit and kill the sensitive yeast strain, and the killer yeasts have immunity to the killer toxin produced by themself. When the killer yeasts are used in industrial production, they can clean up environment and improve the quality of productions. Some marine killer yeast may use in marine aquaculture to against the infection of the marine pathogenic yeast. In addition, the killer toxin produced by killer yeast can be a potential antifungal agent to against infection of pathogenic yeast. This composition discusses the variety, mechanism of action, applications, and research advancement of killer yeast.

Screening and Preparation of Aptamer and Its Analytical Application

Liu Tengfei, Yang Daifeng, Deng Jinhua, Dong Minghui, Deng Qingqing

2013, 0(4):  39-48. 

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Aptamer is a kind of synthetic oligonucleotides by in vitro screening of systematic evolution of ligands by exponential enrichment（SELEX）, which can bind to different targets, such as small organic molecules, proteins, drugs or antibiotics etc., with high affinity and specificity. Due to their special characteristics of a wide range of targets, good stability, simple preparation and easy modification in vitro, aptamers have been widely used in disease diagnosis, clinical therapy, bioanalysis, drug development and other fields. The SELEX method using pesticides as targets has been preliminarily applied in the determination of pesticide residues. Based on recent literatures, this paper describes the finding history and merits of aptamers, the principle as well as separation methods in aptamers sieving process by use of SELEX. Applications of aptamers in pesticides, antibiotics, metal ions, biotoxins, pathogenic microorganism and proteins were also summarized. Furthermore, the prospect of aptamers based detection techniques were proposed.

Applications of Nanobiosensor in Biomedical Field

Dun Wentao, Li Mian, Li Yan, Li Cong, Zhao Zhonglin, Yuan Chao

2013, 0(4):  49-54. 

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The development of novel nanobiosensors was accelerated by the combination of nanomaterials and biological sensing elements, which could recognize chemical or biological molecules selectively. Nanobiosensors would have significant impacts on human and it also have many advantages compare to conventional biological procedures. The developmental trends of nanobiosensor are miniaturized, reliable, and sensitive. The selective sensing instruments have focused on combining nanomaterials with biomolecules for analytes’ detection. Herein, we reviewed the various nanomaterial-based biosensors which use different biological recognition elements for biomedical applications.

Research Report

Real-time PCR Cloning and Expression of EhKCR1 from Eutrema halophilum

Xu Xiaojing, An Huiling, Wei Baiyang, Zhou Yijun, Feng Jinchao

2013, 0(4):  55-62. 

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A cDNA was cloned from Eutrema halophilum（Shangdong ecotype）using in silico cloning and RT-PCR methods. This gene was predict coding β-ketoacyl-CoA reductase, named as EhKCR1, with GenBank accession number JQ389855. The full length is 1 152 bp and contains a complete ORF with 954 bp encoding 318 amino acid. The conservative domain, hydrophobicity and second structure of EhKC1 protein were predicted by bioinformatic analysis. EhKCR1 has the putative NADH binding motif［G（X）3GXG（X）3A（X）3A（X）2G］and the essential catalytic motif（Y（X）3K）. Alignment of predicted amino acid sequence of EhKCR1 with other plants and phylogenetic analysis of various EhKCR1 homologues found that EhKCR1 was very close to that from Arabidopsis thaliana and Brassica napus, but far from to EhKCR1 of monocotyledon. The results of Real-time PCR indicated that EhKCR1 was induced by ABA and drought. Therefore EhKCR1 may be connected with the strong stress tolerence of Eutrema halophilum.

Establishment of Alfalfa Tissue Culture System and Research on Agrobacterium-mediated Transformation System in Alfalfa

Zhou Xiaofu, Lü Jie, Miao Lu, Gao Feng, Xu Hongwei

2013, 0(4):  63-68. 

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In order to establish high-efficiency alfalfa genetic transformation system，the influence factors of Agrobacterium-mediated transformation system were studied. The callus and adventitious bud of alfalfa were induced by culturing leaves as explants in 8 different kind of medium，named M1-M8, respectively. The results showed that M7 medium（MS+1 mg/L 2，4-D + 2 mg/L CH + 0.25 mg/L KT + 2 mg/L agar） was the best one which can induced the much more ammount of callus and adventitious bud of alfalfa than others. The results showed that the most suitable conditions for kanamycin was 75 mg/L; OD600 of agrobactium concerntration value for 0.45-0.6.

Research on the Regeneration System from Hypocotyls and Cotyledon of Brassica napus L.

Hao Xiaoyun, Shen Haitao, Li Hongbin

2013, 0(4):  69-74. 

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To study the changes in the hypocotyls and quizzing cotyledon differentiated and plant regeneration frequency under different concentrations of hormone combinations, hypocotyls and cotyledons with petiole of Brassica napus L.（wild-oil 19）were taken as explants. The results showed that, after pre-cultured for 4 days using 1.5 mg/L 2, 4-D and transferred to differentiated medium of hypocotyls and cotyledons with Petiole, the derived callus initiated earlier and developed faster with a higher induced frequency. Among them, the hypocotyls transferred to the MS +3 mg/L 6-BA +0.1 mg/L NAA as differentiated medium reached the highest regeneration frequency of 86.67% ；However, the regeneration frequency of cotyledon explants was much lower than hypocotyls with a final regernation frequency of 46.67% using MS with 4 mg/L 6-BA and 0.3 mg/L NAA as differentiated medium. In this study, the high frequency regeneration system of Brassica napus L.（wild-oil 19）was established.

Cloning and Analysis of the Polyneuridine Aldehyde Esterase in Rauvolfia yunnanensis

Guo Chuan, Cao Fuxiang, Liu Jike, Feng Yanzhi, Li Meng

2013, 0(4):  75-80. 

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According to some identified cDNA sequence of the polyneuridine aldehyde esterase（PNAE） gene in plant species on the NCBI，pairs of primers were designed，and the partial intermediate sequence of the PNAE cDNA was obtained using RT-PCR technique in Rauvolfia yunnanensis’ fresh leaves，and then by the technique called rapid-amplification of cDNA ends（ RACE），the sequence in both two flanks of the known sequence was acquired. The full length sequence of the cDNA of PNAE in Rauvolfia yunnanensis was 1 004 bp and that included an open reading frame（ORF）which encoded 264 amino acids. Sequence analysis showed that for PNAE gene the homologies of amino acid sequence among each species was a little low ranging from 40% to 60%，demonstrating PNAE gene wasn’t a gene that its complete sequence is relatively conservative. The further analysis of all the known PNAE in plant species found that there are two conservative domains in amino acid sequence，revealing that PNAE gene has two highly conserved domains among different plant species.

RNA Editing Restores Mutations in the Chloroplast Gene accD of Takakia lepidozioides

Wan Ping

2013, 0(4):  81-84. 

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RNA editing highly occurs in Takakia lepidozioides chloroplast. The reason underlying the phenominon remains unclear. In this study, with comparative genomic approach, we analized the sequences of the gene accD and its protein products in mosses Physcomitrella patens, Syntrichia ruralis, and Takakia lepidozioides. We find that RNA editing restores mutations in the gene accD of T. lepidozioides, T. lepidozioides aquires the same amino acid sequence as its homologs in Physcomitrella patens and Syntrichia ruralis. The high occurrence of RNA editing in T. lepidozioides chloroplast is an important stratigy to restore DNA mutations. RNA editing enhences T. lepidozioides the adaptation to survive under the high radiation enviroment.

Construction of a Genomic DNA Library of Chlamydomonas reinhardtii

Wang Chaogang, Chen Aina, Hu Zhangli, Yu Shanshan

2013, 0(4):  85-89. 

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The genomic DNA isolated from Chlamydomonas reinhardtii CC-849 was digested with BamH I and Bgl II. The 6-12 kb genomic DNA fragments were recovered from agarose gel and concentrated to 200 ng/μL. Then they were inserted into λDNA ZAP expression vector, packed by packaging extracts of λ phage and transformed into Escherichia coli XL1-Blue. Finally, the genomic DNA library was obtained with an average insert size of 9 kb. Results showed that the titer of genomic library was 2.12×10⁵ pfu/mL and contained 4.26×10⁴ clones. After amplified, the titer of genomic library was up to 9.5×10⁶ pfu/mL.

Analysis of Total RNA in CMV Infected Tobacco by Ion Pair Reverse Phase High Performance Liquid Chromatography

Qiao Wenjie, Lei Rong, Jiang Hongshan, Hu Fan, Li Zhihong, Zhu Shuifang

2013, 0(4):  90-95. 

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To study the impact of plant virus on host plant at the RNA level gene expression, in this study differential expressing of total RNA induced by cucumber mosaic virus（CMV）infection in tobacco through ion pair reversed phase liquid chromatography（RP-HPLC） was analyzed. Ion pair RP-HPLC has been applied for RNA isolation, purification and analysis in recent years, and has the advantages of direct injection, good reproducibility, and rapid analysis. Suitable ion pair reagents as the hexylamine/1, 1, 1, 3, 3, 3-Hexafluoro-2-propanol（HA/ HFIP）system was optimized to achieve a acceptable total RNA separation, the separation peak differences between healthy and diseased plant were observed, but needed further experiments to verify. Therefore, this paper provided an alternative method for study the viral effects on gene expression of host plants at the RNA level and plant virus pathogenic mechanisms.

Optimizing Method of Preparation of Protoplast in Fusedum oxysporum f. sp. capsicum

Mi Baobin, Zhang Jixiang, Yang Yuhong, Mao Zhenchuan

2013, 0(4):  96-100. 

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Several conditions in preparing the protoplast of Fusedum oxysporum f. sp. capsicum were optimized. Effects of the incubation time, enzyme system, the enzymolysis time, temperature and rotate speed were discussed. It was found that when keeping other parameters as : spore cultured for 14 hours, 15 mg/mL of Drislase, 0.7 mol/L NaCl and 140 r/min, 32℃ for 4 h, the minimum consummation of protoplasts can be obtained and the regeneration rate of the protoplast was 32.3% on regeneration medium SR.

Isolation of Strain Producing Complex Lignocellulase and the Optimization of Culture Conditions

Yu Junjie, He Ronglin, Wu Gaihong, Zhang Can, Chen Shulin, Zhang Tongcun

2013, 0(4):  101-109. 

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Based on enrichment culture, initial screening, and further screening by spotting vaccination, a group of degradation lignocelluloses strains were isolated from soil. Then strains were cultured in the flask to develop the repeated screening, and enzymes activity was tested, finally a filamentous fungus 26-6 was screened. Then colonial morphology of the strain on PDA plate was observed, spore morphology was also observed by scanning electron microscope. According to the ITS rDNA sequence analysis, the fungi was initially identified as a Penicillium oxalicum strain and named Penicillium oxalicum EH26-6. Culture optimization was then carried out in flask scale and finally the optimal culture condition was determined as corn cob for carbon source, corn steep powder for organic nitrogen source, pH5.0, temperature 30℃ , inoculation 10%. The research provided an alternative strain for the low cost production of composite cellulose enzyme.

Cloning and Characterization of L-aspartate-α-decarboxylase from Corynebacterium glutamicum

Shi Zengxiu, Cui Wenjing, Zhou Li, Zhou Zhemin

2013, 0(4):  110-115. 

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L-aspartate-α-decarboxylase gene panD from Corynebacterium glutamicum was cloned and efficiently expressed in Escherichia coli BL21（DE3）. The α and β subunits of PanD were observed by Tris-tricine SDS-PAGE, indicating that the PanD was successful processed by self cleavage. The specific activity of the recombinant enzyme was as high as 2.60 U/mg（156 mmol/g?h）. The optimum temperature was 55℃ and the optimum pH was 7.0. Under the condition of 80℃ , the half-life of PanD was approximately 40 min. Under the condition of 37℃ and pH7.5, the Km value was 4.26 mmol/L, the V_max value was 35.97 nmol/min, the k_cat value was 1.02/s and the catalytic efficiency（k_cat/K_m）was 0.24 L/mmol?s.

Molecular Cloning and Bioinformatics Analysis of Outer Membrane Protein H Gene from Vibrio alginolyticus Strain HY9901

Zhou Zejun, Pang Huanying, Ding Yu, Jian Jichang, Wu Zaohe

2013, 0(4):  116-122. 

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Primers for PCR cloning were designed according to the whole genome sequence of vibrio alginolyticus published in GenBank. The outer membrane protein H（OmpH）gene of V.alginolyticus strain HY9901 was amplified by PCR and cloned into pMD18-T vector to investigate the possibility of OmpH as a candidate antigen for vaccine production. Sequence analysis revealed that OmpH gene（GenBank Number ：JX855924）is 573 bp and encodes a putative protein of 190 amino acids. The predicted molecular weight（MW）of OmpH was 21.1 kD with an estimated pI of 9.02. Using SignalP 4.0 and TMHMM Server 2.0 software, and it was predicted that the OmpH protein did not contain a signal peptide or a transmembranous region. This protein had one N-glycosylation site, six phosphorylation sites, one endoplasmic reticulum targeting sequence and three microbodies C-terminal targeting signals predicted by SoftBerry-Psite software. To further analyze the evolutionary relationship among OmpH, a molecular phylogenetic tree was constructed using MEGA5.0 software. In this tree, the OmpH protein showed high genetic relationship with Vibrio parahaemolyticus and Vibrio harveyi. Using bioinformatices softwares and methods, the B-cell preponderant epitopes of OmpH were localized in the regions of 5-10、106-110、120-125、158-163 and 175-180. The three-dimensional structure of OmpH was determined using SWISS-MODEL work-space and it had a similar structure with Skp protein of Escherichia coli. These results can provide a basis for further studies on the immunogenecity of OmpH and vaccine preparation.

The Effect on Phosphatidylserine Biosynthesis of Gene sdaA，sdaB，pgpB in Escherichia coli

Tong Xinwei, Yang Hongzhe, Li Yu, Lu Fuping

2013, 0(4):  123-128. 

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The gene sdaA, sdaB, pgpB were deleted in Escherichia coli by the technology of Red homologous recombination system, resulting in the cut-off of part of the L-serine and PGP catabolic pathway, then the levels of phosphatidylserine（PS）could be elevated. To assess the effects of sdaA, sdaB, pgpB deletion on PS accumulation, both the recombinant and recipient strains were cultivated in LB-containing flasks. Then the samples were taken after 1 000 minutes and quantification of PS performed with HPLC. The results showed that the recombinant E. coli cells produced PS one fold more than the wild-type strain, suggesting the effectiveness of targeted alteration of the metabolic pathways of PS for improvement of PS production. Cost-effective large-scale production of PS should find important applications in various industrial fields.

Application of Pichia pastoris Original Constitutive Strong Promoter GCW14 in Candidn antarctic Lipase B Yeast Surface Display

Zhang Xuanwei, Ye Yanrui, Liu Xiaoxiao, Lu Liuliu, Lin Ying

2013, 0(4):  129-135. 

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To compare the transcription activity of original promoter P_GCW14 with other promoters, Candidn antarctic lipase B（CALB） was displayed on the cell surface of Pichia pastoris using P_GCW14, P_AOX1, P_GAP and P_TEF1 as promoter respectively. Anchor protein was a cell wall protein named G_cw14p, which was obtained originally from P. pastoris. In shake-flask cultivation, the activity of P_GCW14 was equal to that of _PAOX1.Under control of P_GCW14 and P_AOX1, the highest lipase hydrolytic activities of recombinant yeasts reached 694.8 U/g（Dry cell weight）and 684.3 U/g（Dry cell weight）after cultivation for 48 h and 120 h, respectively. The fermentation period of recombinant yeast using P_GCW14 as promoter was shortened significantly. While the highest lipase hydrolytic activities of recombinant yeasts controlled by constitutive promoter P_GAP and P_TEF1 reached 266.7 U/g（Dry cell weight）and 449.2 U/g（Dry cell weight）respectively after cultivation for 48 h. This means that the activity of P_GCW14 was higher than that of P_GAP and PTEF1. Furthermore, replacing the α-factor with signal peptide from protein G_cw14p, the lipase hydrolytic activity increased about 4%.

ELISA Expression and Identification of Recombinant A Domain of Adhesin Fnbp A from Staphylococcus aureus in Bovine Milk

Su Yan, Wang Shimin, Li Ying, Wei Haina, Shao Jungao, Zhang Baojiang, Yin Tao

2013, 0(4):  136-139. 

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Fibronectin binding protein A plays an important role in Staphylococcus aureus early infection, and is also a potential target of immunization. We subcloned the A domain of FnbpA into the prokaryotic expression vector pGEX and transformed it to the BL21 host cell. After removing the GST tag the purified purpose protein was analyzed by SDS-PAGE and approximately 63 kD exogenous protein was observed by SDS-PAGE. The purified protein without GST tag was emulsified by Freund’s adjuvant and injected rabbits subcutaneously to produce hyperimmune serum. The antibody titer and the binding capacity between the S. aureus and rabbit serum were tested by ELISA. The result indicated the antibody titer could reach 6.7×10⁶ and the antibody could recognize the antigen of S. aureus in vitro.

Optimization and Construction of the Intracellular and Extracellular Proteomic Map of Lysobacter yanansis sp. nov.

Wang Feifei, Wu Kunyi, Guo Ling, Cui Langjun, Ren Jing

2013, 0(4):  140-146. 

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In this study, we separated the extracellular protein excreted by Lysobacter yanansis sp. nov. through the two-dimensional （2-D）electrophoresis, and analyzed the effect of isoelectric focusing programs and loading volumes on 2-D proteomic maps. The results showed that a clearly 2-DE map was obtained when the 150 μg loading volumes and isoelectric focusing programs Ⅱ were used. Furthermore, 2-D proteomic maps of extract intracellular and extracellular protein from Lysobacter yanansis sp. nov. were constructed. The separated protein spots in gels were analyzed by PDQuest. 100 spots of extracellular proteins and about 204 spots of intracellular proteins had been separated by 2-DE.

Screening of Prolific Micromonospora carbonacea in Antibiotics Production by Heat Mutagenesis

Li Jin, Huang Yunhong, Li Luming, Peng Weimeng, Long Zhonger

2013, 0(4):  147-151. 

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Heating mutagenesis effect, combined with gentamicin resistance screening, was used in breeding of prolific Micromonospora carbonacea in antibiotics production from the strain of Micromonospora carbonacea JXNU-1. The overproducing strain JXNU-1-16-R4 was screened with the production of antibiotics is 98.53% more than that of the original strain.

Effects of New Oxygen Fertilizer on Soil Bacterial Diversity Under Waterlogging by PCR-DGGE Method

Wang Jing, He Gang, Wang Lei, Du Linqian

2013, 0(4):  152-157. 

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We used Macropanax rosthornii soil as research subject, and water as the stress factor. The experiment was tested 36 days under the following three different conditions：A just was waterlogged；B was waterlogged with new oxygen fertilizer；C was normally watered without new oxygen fertilizer. Amplifying 16S rDNA genes of bacteria by PCR, subsequently the products of PCR were analyzed by DGGE on the sixth, twelfth, eighteenth, twenty-fourth, thirtieth and thirty-sixth day. The results indicate that at the same stage, with the prolongation of processing time, condition A and condition B’s DGGE bands similarity are reduced ；on the twelfth, eighteenth and twenty fourth day, the DGGE bands of condition B are more and brighter than condition A. The Shannon index of B is higher than A, and the difference presents the trend of “low-highlow”. The DGGE bands of B and C owns lower similarity during the same period. The diversity of different conditions presents differently during the detection time. Under the condition A, the similarity of soil bacteria diversity is generally lower at each detection time. Under the condition B, the similarity of soil bacteria diversity is higher and stable at each detection time. Under the condition C, the similarity of soil bacteria diversity is lower at each detection time. The research reveals the new oxygen fertilizer has a significant effect on waterlogged soil bacterial diversity.

The Determination of the Taxonomic Status of Two Soil Rhizobia from Southern Tibet

Shan Huihui, Li Zheng, Han Suzhen

2013, 0(4):  158-166. 

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Polyphasic taxonomy was adopted to determine the taxonomic status of two soil rhizobia from southern Tibet. Two Gramnegative, aerobic, rod-shaped bacteria, designated strains CNU8561007 and CNU85000012. The morphological studies demonstrated that the two test strains had similar features. The slight features differences between CNU8561007 and CNU85000012 might be related to the fact that they were isolated from different geographic regions. According to comparison of 16S rRNA gene sequences, strains CNU8561007 and CNU85000012 were identical and their closest phylogenetic relatives were Rhizobum yanglingense, R.loessense, R.mongolense and R.gallicum. Phylogenetic analysis based on four housekeeping genes（recA, atpD, glnII, and danK）which were in broad agreement with 16S rRNA gene sequence similarities indicated that the two isolates were closely related to Rhizobum yanglingense. The DNA G + C mol% and DNA -DNA hybridization provided supporting evidence that the two test strains belongs to the Rhizobum yanglingense and the BOX-PCR fingerprint profiles demonstrating that the two isolates were not clones of one strain.

Preliminary Research for Inhibitory Effect of Essential Oil from Cuminum cyminum Against Pathogenic Microorganisms on Fruit and Vegetable Storage

Nie Ying, Li Shuying, Qi Xiaoyu, Yang Fan, Mu Taihua, Tang Xuanming

2013, 0(4):  167-171. 

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Yellow essential oil of Cuminum cyminum was obtained by steam distillation estraction method with the yeild of 2.7%. 13 kinds of components was identified by GC-MS.Cuminylalcohol and an unknow compound are the first main component（42.68%）, and cuminal is followed by the content of 27.66%. Moreover, the antifungial activity of Phytophthora infestans, Penicillium expansum, Rhizopus stolonifer, Fusarium solani was tested, and the minium inhibition concentration of essential oil to these 4 kinds of fungis was 0.4‰ , 0.6‰ , 0.3‰ , 0.3‰ . The inhibitory concentration of Cuminum cyminum extract to the spore of Penicillium expansum and Rhizopus stolonifer is 0.16 ‰ , 0.8 ‰ respectively. Also, essential oil extracted from Cuminum cyminum also showed stronger antibacterial activity of Bacillus subtilis than the of Escherichia. All of above these provide that Cuminum cyminum essential oil with high antimicrobial activity could be developed as a new kind of essential oil germicide on fruit and vegetable preservation.

The Quantitative PCR Technology for Three Oil and Gas Indicating Bacteria and Its Preliminary Application in Oil and Gas Field Soils

Shao Mingrui, Xu Kewei, Tang Yuping, Zhao Kebin, Fu Bo, Liu He

2013, 0(4):  172-178. 

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In this paper, the methane-, butane- and alkane-oxidizing bacteria contents in oil and gas field soil was determined by the established real-time quantitative PCR（qPCR）technologies targeting the pmoA, bmoX and alkB gene respectively, and the background field soils were compared as the control. The pmoA and alkB gene copies decreased by 1-2 orders of magnitude with the sampling depth increased. The gene quantification results of DNA samples extracted by manual method indicated the pmoA and alkB gene copies in oil and gas field soils were slightly higher than in background field soils. The bmoX gene copies in gas field soils was 2.75×10⁵copies/g, which was 0.5-1 order of magnitude higher than that in background and oil field soils. The results of DNA samples extracted by kit method showed the pmoA and alkB gene copies in oil field soils were 3.09×10⁴ and 2.56×10⁶ copies/g, respectively, which were higher than that of gas and background field. The bmoX gene copies in gas field soils is 1 order of magnitude higher than that of background field soils, and 0.5 order of magnitude higher than that of oil field soils. It is concluded that the established qPCR method of the three oil and gas indicating bacteria provided new detection means for microbial prospecting of oil and gas.

Cloning and Bioinformatics Analysis of MCL-1 cDNA of Bufo japonicus formosus

Zhang Shufang, Yuan Jinqiang, Huang Hui, Zhuge Hui, Xu Yue, Yang Xianyu

2013, 0(4):  179-184. 

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It was to clone mcl-1（myeloid cell leukemia-1）gene from Bufo japonicus formosus and analyze the sequence of the gene. The plasmid cDNA library of adult Japanese toad Bufo japonicus formosus skin was screened by bacterial colony PCR. The sequence was analyzed by bioinformatics method, and the structure and function of MCL-1 were predicted. Result showed that the full length cDNA of mcl-1（GenBank accession number ：JX219482）was 1 090 bp. The 5'- and 3'-untranslated regions are 19 bp and 432 bp, respectively, while the ORF is 639 bp encoding 212 amino acid residues. Homologous analysis indicated the similarity of Bufo japonicus formosus with Xenopus laevis was 60%, and 38%-55% with other species. The phylogenetic tree of MCL-1 amino acid sequences was consistent with morphological taxonomic characters. The analysis indicates that the structural characteristics of MCL-1 of B. japonicus formosus seem to be similar with those of human. This report would become the basis for the further studies on MCL-1.

Ammonia Eexcretion Related Genes Expression of Common Carp Under the Stress of Carbonate Alkalinity

Zhao Lan, Xu Peng, Sun Xiaowen

2013, 0(4):  185-193. 

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Common carp（Cyprinus carpio）is one of the most important aquaculture species in China and with certain alkalisaline tolerance. In order to understand the common carp genes expression in different alkali-saline water, we studied the candidate ammonia excretion related genes ：Rh（Rhesus blood group-associated glycoprotein）, AQP（Aquaporin）, PRL（Prolactin）, CA（Carbonic Anhydrase）, NKA（Na⁺/K⁺ transporting ATPase）, NHE（Sodium Hydrogen Exchanger）expression at a rising carbonate alkalinity gradient in a certain pH value by quantitative PCR method. Results showed that with the rise of carbonate alkalinity value, the overall expression of these genes in gill, kidney and intestine are up-regulated compared with freshwater controls. And AQP3A, PRLRA, NHE7 are mainly expressed in gill ；PRL are mainly expressed in kidney ；RHAG, RHBG2, RHCG1 are mainly expressed in intestine under the stress of carbonate alkalinity. The changes of these genes expression in different level of carbonate alkalinity can provide insights into the mechanism of osmotic regulation for fish.

Purification，Tag and Method for Detection of Grass Carp Serum IgM

Tian Yuanyuan, Ye Xing, Zhang Lili

2013, 0(4):  194-200. 

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Protein A affinity chromatography was used to purify immunoglobulin IgM from grass carp serum, and the purity and molecular weight of the product was analyzed by SDS-PAGE. Rabbit polyclonal antibodies of the purified grass carp IgM were prepared and detected by ELISA. The results showed that molecular weights of the light chain and heavy chain of the purified grass carp IgM were about 25 kD and 75 kD, respectively, and high titers of rabbit anti-grass carp IgM antiserum was obtained. Horseradish peroxidase was used to label the rabbit anti-grass carp IgM antibody. The labeled antibody was used to detect the antibody levels in serum of grass carp after immunizing by capsid protein VP4 and VP5 of grass carp hemorrhage virus isolated from Guangdong province（GCRV-GD108）. The results revealed that the labeled anti-grass carp IgM antibody could be used for the rapid detection of the IgM levels in grass carp serum. In this study, method to detect IgM levels in grass carp serum was established, which would provide a convenient method of serological testing for further study of pathogen invasion mechanism, grass carp immune defense mechanism and evaluation of grass carp vaccine effectiveness.

Screening and Identifying of GPS2 ：a Novel Interactive Protein of LOH12CR1

Wang Yongjun, Wang Yingwei, Shi Xiaoliu, Lin Ruoheng, Zhang Wenting, Shen Hongwei,

2013, 0(4):  201-205. 

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Loss of heterozygosity of the short arm of chromosome 12 is a frequently observed in a wide variety of haematological malignancies and solid tumours, suggesting the presence of tumour suppressor genes in this region. LOH12CR1 is one of the genes identified in 12p12. To explore the normal function of LOH1CR12, yeast two-hybrid system was used to screen proteins interacting with LOH1CR12, and the transcriptional regulator GPS2 was identified as a LOH1CR12- interacting protein. Furthermore, GPS2 was confirmed to interact with LOH1CR12 in vivo in HEK-293 cell by co-immunoprecipitation, and the co-localization of GPS2 and LOH1CR12 in nucleus was observed by immunofluorescent co-localization.

Preparation and Analysis on Biological Characteristics of Monoclonal Antibodies Against CP4-EPSPS for Sandwich ELISA

Li Zhongpeng, Yu Hansong, Hu Yaohui, Shi Shengfeng, Liu Yunhui, Duan Yongjie, Li Xiaoyu, Wang Yongzhi, Li Qiyun

2013, 0(4):  206-209. 

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In order to obtain specific monoclonal antibody against CP4-EPSPS protein for sandwich ELISA, the CP4-EPSPS gene was cloned into a bacterial expression vector and transformed into E.coli strain Rossetta. The target protein was expressed efficiently. After purification, the target protein was obtained. Purified CP4-EPSPS was used to immunize BALB/c mice and two monoclonal antibodies（1A5 and 8A3）were generated. These antibodies can recognize nature and denatured CP4-EPSPS. Overlay ELISA showed that they recognize different epitopes on CP4-EPSPS, so they can be used in sandwich ELISA to detect CP4-EPSPS.

Research Report

Study of the Renaturation and Purification of LL-37-haFGF Fusion Protein

Shen Juan, Jin Xiaobao, Ding Jing, Zhu Jiayong

2013, 0(4):  210-214. 

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A new multifunctional healing peptide was designed and developed for the first time, human antimicrobial peptide LL-37 modified haFGF fusion proteins, which can promote healing and fight infection. This paper discussed renaturation conditions and purification conditions of the new fusion protein named recombinant LL-37-haFGF was expressed by Escherichia coli and identified of effects on the refolding of eight kinds of small molecules added to inclusion bodies were preliminary investigated. The result showed supplementation of 0.3 mol/L arginine can greatly improve renaturation efficiency of LL-37-haFGF. The pure product of was obtained with nickel affinity chromatography and CM cation chromatography. Through this study, we obtained 5.9 mg target protein of high purity 95.43%, laying the function for its functional study, and also providing the reference for the preparation of similar multifunctional polypeptide.

Comparative Study Based on the LAPS System of Protein Chip Surface Modification Technology

Zhou Shuang, Jia Yunfang, Sun Xiaoxuan, Xing Keli

2013, 0(4):  215-220. 

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The surface of chips were modified by 3 - aminopropyl - trimethoxysilane（（3-aminopropy1）trimethyoxysilane APTES）and glutaraldehyde（Glutaraldehyde, GA）, levodopa（L-DOPA）modification of the chip carrier surface. The two kinds of different types of modification of the protein chips prepared a comparative study. XPS detect surface modification effect of the activator. AFP antibodies were fixed on the modified film base, then adding AFP solution, and photocurrent detected by LPAS system, selecting reactive protein probes as monitoring indicators. The results showed the silicon protein modified by levodopa fixing preferably, having a higher reaction activity and a large detection limit, though the amplitudes were small.

Highly Sensitive and Selective Detection of Hg2+ in Aqueous Solution with Mercury-specific DNA and SYBR GREEN I

Wu Jikui, Wei Biwen, Lin Li, Mao Fang, Zhou Dongxiang, Xiong Zhenhai

2013, 0(4):  221-224. 

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A novel “signal-on” assay for sensitive and selective detection of Hg2+ in aqueous solution based on mercury-specific DNA and a molecular light switch complex, SYBR GREEN I, was reported in the present work. This Hg²⁺ specific sensor system composed of two labelfree DNA probes with five T-T mis-matches, which could form stable DNA duplexes by thymine-Hg²⁺-thymine（T-Hg²⁺-T）in the presence of Hg²⁺. The fluorescence of SYBR GREEN I is weak in aqueous solution, and significant fluorescence can be observed when intercalating into DNA duplexes. By monitoring Hg²⁺-dependent fluorescence intensity enhancement, highly sensitive and selective determination of Hg²⁺ has been achieved. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of Hg²⁺ in the range of 5-100 nmol/L. A detection limit of 2 nmol/L Hg²⁺ was achieved. The presence of other metal ions did not interfere with the detection of Hg²⁺. This approach is simple and rapid, which can be used to monitor the Hg²⁺ concentration in drinking water.

Information

The Exploration of Bio-technology Development Trend from the Bio-industry Development During the First Ten Years

Liu Xiansheng, Guo Jiwei, Luo Xu

2013, 0(4):  225-228. 

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The bio-industry’s development is important in the first decade of the 21st century. It can lay a good beginning and foundation for the boom of the bio-technology development. We analyze the bio-technology development from the bio-industry’s R&D input and economic output in the first ten years of the 21st century of the competition, economy, bio-safety and the command of bio-technology.