Table of Content

15 July 2014, Volume 30 Issue 7

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Review and editorial

Abiotic Stress-related RING Finger Proteins in Plant

Liu Lijuan, Liu Ailing, Chen Xinbo

2014, 30(7):  1-7. 

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RING finger proteins belong to a large family of the zinc finger proteins. They become the third-largest protein family in Arabidopsis. A total of 488 RING finger protein genes were retrieved in rice genome. The most typical structural characteristic is the RING finger domain they contained. RING finger proteins participate physiological and biochemical processes in plant cells mostly through ubiquitin pathway. This paper introduced the structural characteristics and classification of plant RING finger proteins, summarized the subcellular localization, and reviewed their regulation roles in response to plant abiotic stress such as drought stress, temperature stress, and salt stress.

Research Progress of MIKC-type MADS-box Protein Regulation on Flowering

Zhao Xiayun, Xian Dengyu, Song Ming, Tang Qinglin

2014, 30(7):  8-15. 

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MIKC-type proteins represent a class of MADS-domain transcription factors in plant and are defined by a unique domain structure. In addition to the highly conserved MADS-domain, they have three other domains（I, K and C）. The number and functional diversity of MIKC-type proteins increased considerably during plant evolution, culminating in higher flowering plants. They are known to play key regulatory roles in different stages of flower development. This paper summarized the plant MIKC-type MADS protein classification and structure, interactions with DNA, interactions between proteins and molecular mechanism of the interactions. It also discusses about the prospects of future research works, according to the present research status on MIKC-type proteins.

Studies of Immune Duck Plague Virus Progress

Zhao Jun, Yang Xiaopu, Song Xiaoyu

2014, 30(7):  16-19. 

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Duck plague is caused by a virus of the duck, it’s an acute and septic infectious. This disease has spread rapidly, high morbidity and mortality, it has a serious harm to the waterfowl breeding industry. The duck plague's produce and hazard was reported in the domestic and overseas .In the paper from the main antigenic protein of duck plague virus persistent infection, mucosal immunity, cellular immunity, humoral immunity and immune suppression aspects was discussed between DPV complex interactions with the host immune function, designed to duck plague vaccine design and the development of new ideas and methods.

Role of MicroRNAs in Genes Regulatory Apoptosis

Qi Renli, Huang Jinxiu, Yang Feiyun, Liu Zuohua

2014, 30(7):  20-25. 

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MicroRNA（miRNA）, a class of non-coding small RNA molecules, widely exists in the most of the animals and plants. Major function of MicroRNA is expression regulation of target gene. This regulation is primarily carried out by repression of translation in mammals. Apoptosis is programmed cell death regulated by various genes and the role of miRNAs in apoptosis is not fully understood, however, evidence is mounting that miRNAs are important in this process. In this paper, the relationship between MicroRNA and cell apoptosis, and concludes the possible regulation mechanism were reviewed.

Research Progress of Controlling Conifer Root and Butt Rots by Phlebiopsis gigantea

Li Xingchun, He Shuanghui

2014, 30(7):  26-32. 

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Conifer root and butt rots is an common forest disease in the north temperate zone, and the pathogen is Heterobasidion spp. But with the development of molecular biology, transcriptomics and genomics, we will change our mind to the genes related to controlling efficacy of Phlebiopsis gigantea. Phlebiopsis gigantea is the best antagonistic fungus to prevent the disease. This paper began with controlling Heterobasidion spp. and expounded research progress of controlling conifer root and butt rots by Phlebiopsis gigantea, which included the history of controlling disease, products based on Phlebiopsis gigantea, the mechanisms, ecological influence and process of screening.

Bacterail Pathogens Resistance to Phages During the Phage Therapy

Cai Liuti, Chen Xingjiang, Liu Yanxia, Shi Junxiong

2014, 30(7):  33-36. 

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Bacterial pathogens have been treated effectively with antibiotics for several decades, but with more and more resistant bacteria appeared, the resistance to antibiotics has become a difficulty issue in antibiotics therapies. Phages therapy, the application of the phages that infect bacterial pathogens as antimicrobials, has been expected and advocated as a promising alternative to conventional antibiotics. However, under the selection of the co-evolution between phages and bacteria, bacterial pathogens can became resistance to the phages by several mechanisms. So, at the same expecting and advocating, should we worry about the problem that whether the phage-treated pathogens will develop to resistance to the phages caused to treat difficulty at last? Here, the arguments about the bacterial pathogens resistance to the phages under the phage-bacterium co-evolution and its effect on the phages therapy were summarized and reviewed, and the phage therapy for control the bacterial pathogen was prospected.

Technique

Gene Editing Tools Mediated by CRISPR-Cas

Zuo Qisheng, Li Dong, Zhang Ya’ni, Li Bichun

2014, 30(7):  37-43. 

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Recently, with the deepening of the research and transformation of CRISPR-Cas system, it has been applied as a new gene targeting modification technology, which can be directed to silence target genes. At present, this technology has been successfully used in HEK293, mouse and zebrafish cells and generated stable gene knockout cell lines, meanwhile gene knockout models has been constructed on the models animals such as mice and fruit flies. With the advantages of simple design, short time-consuming and higher efficiency, CRISPR-Cas has become a gene targeting knockout technology with broad application prospect. In this paper, structure and function mechanism of the CRISPR-Cas, also the current applications of Cas9/gRNA were reviewed.

Advance on Virus-free Plant Tissue Culture and its Application on Crocus sativus L.

Li Jun, Gao Guangchun, Li Bai, Zhu Zhiming

2014, 30(7):  44-48. 

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The technique of virus elimination in plant tissue culture can eliminate the virus, rejuvenate, and improve yield and quality of plants. This paper summarized the virus-free methods and its application in the recent years, discussed the virus-free methods used in medicinal plant Crocus sativus L..

Study on Extracting of Extracellular Proteins in Valsa mali

Wang Haiying, He Yuanyuan , Huang Lili, Han Qingmei

2014, 30(7):  49-53. 

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An optimal protein extraction method, as much as possible to obtain high-quality extracellular proteins of Valsa mali, is helpful to thoroughly analyze the proteomics and pathogenesis of the pathogen. In this study, the three methods freeze drying system/dialysis, DOC-10%TCA precipitation and ammonium sulfate precipitation were used to extract the extracellular proteins of Valsa mali, respectively. Then the optimal concentration of ammonium sulfate needed to be screened. The proteins extracted by the three methods were quantified and separated separately through Bradford and SDS-PAGE, and then extracellular proteins extracted from pathogenicity of different isolates were also compared with SDS-PAGE. The results showed that the content and distinguishability of protein sample extracted by 70% ammonium sulfate was optimal, and four protein lanes are significantly different in the SDS-PAGE electrophoresis patterns of different isolates. So this method is the most suitable for extracting extracellular protein sample to analyse the difference of Valsa mali protein.

Development of an Internal Amplification Control in the PCR Detection for Salmonella

Yang Jin, Zeng Qingmei, Zhang Di, Liu Kun, Wang Lin, Zhang Yajun

2014, 30(7):  54-59. 

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By constructing an internal amplification control（IAC）, this study developed a PCR system for the detection of Salmonella, which could effectively indicate false-negative results. The specific primers were designed according to the conserved gene invA in Salmonella spp.. An IAC was constructed by the compound primer technology, and finally the PCR detection system was developed. The experiment indicated that the specific 385 bp DNA fragment was amplified against 33 reference strains of Salmonella spp., while 6 strains of non-Salmonella only showed the 484 bp amplified band of IAC. The detection limit of this PCR system forpurified genomic DNA was 6.35 fg/μL. The artificial contamination assays showed that Salmonella could be detected after eight hours enrichment when the original bacterial concentration was 3.2 CFU/25 mL. A large number of food samples were also tested, and the results demonstrated that the detection system could effectively avoid the false-negatives and improve the detection accuracy.

Establishment and Application of a Specific PCR Assay for Rapid Identifying Thermoactinomyces intermedius

Zhang Mingjuan, Yao Su, Li Hui, Liu Yang, Xin Chunhui, Xu Ling, Cheng Chi

2014, 30(7):  60-63. 

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This study establishs a rapid specific PCR assay to screen Thermoactinomyces intermedius strains and screens Thermoactino-myces intermedius strains from high-temperature daqu of sesame flavor liquor by the specific PCR assay. Experimental results show that the designed specific primers are specific for Thermoactinomyces intermedius, it is suitable for screening rapidly Thermoactinomyces intermedius strains. The specific PCR can save a lot of time and costs compared with the traditional identifying methods.

Detection and Application of Three Food-borne Bacterial Pathogens by Multiplex PCR

Yu Qian, Huang Mengna

2014, 30(7):  64-68. 

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Food poisoning incidents have occurred by the pollution of food-borne pathogenic bacteria frequently. PCR meet the requirements of the rapid detection of pathogenic bacteria. The three primers was designed by primer 5.0 software of Enterohemorrhagic Escherichia coli O157:H7 rfbE gene, Staphyloccocus aureus nuc gene, Salmonella hilA gene. Three amplified segments of rfbE gene, nuc gene, hilA gene were 287 bp, 354 bp, 468 bp. The specificity and sensitivity of primers were verified by single, duplex and triplex-PCR reaction, and by detecting the foods artificially infected with the three pathogens. The results showed that three food-borne bacterial pathogens were amplified purpose fragments, and no non-specific fragments were amplified. It was obvious that there were the special fragments of the artificial food infected with three pathogens by PCR test. The three primers could be used for PCR detection of target genes about three bacteria.

Research Report

Separation and Purification of Hydroxylamine Oxidase from Agrobacterium tumefaciens LAD9

Chen Qian, Ma Tao, Wang Ting

2014, 30(7):  69-73. 

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The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase（HAO）. Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe2+.

Cloning, Expressing and Functional Analysis of Na⁺/H⁺ Antiporter Gene OpNHX1 from Olimarabidopsis pumila in Xinjiang

Zhao Yunxia, Guo Danli, Wei Yanling, Huang Xianzhong

2014, 30(7):  74-80. 

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A NHX1 gene, designated as OpNHX1（GenBank Accession No. KC200248）was isolated from Olimarabidopsis pumila, a plant for stress tolerance research. The cDNA of OpNHX1 was 2 153 bp in full length, contained an open reading frame（ORF）of 1 605 bp encoding a peptide of 534 amino acids residues. Phylogenetic analysis revealed that OpNHX1 had high sequence similarities with AtNHX1 and EhNHX1 and they belong to the same clade. The expression of OpNHX1 is induced by salt, PEG, cold, and abscisic acid. OpNHX1 is detected in roots, stems, leaves, flowers and siliques of Olimarabidopsis pumila, with the highest expression in stems. Importantly, overexpression of OpNHX1 in Arabidopsis thaliana improved the survival rate of transgenic plants under salt stress. This study provides valuable information to understand more about the molecular mechanism of salt tolerance in plant. Heterologous expression of OpNHX1 in W303-1B Δnhx1 yeast Na⁺/H⁺ antiporter mutants showed functional complementation.

Effects of Aluminum Stress on Photosynthesis and Active Oxygen Metabolism in C4 Ppc Transgenic Rice

Zhang Yunhua, Meng Qingling, Ji Chenglin, Chen Lijuan, Wu Gang, Liu Xueshi, Zhang Shuxiang

2014, 30(7):  81-85. 

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The effects of aluminum stress on photosynthesis and active oxygen metabolism were investigated by chlorophyll fluorescence and enzymatic technology in C4 Ppc transgenic rice. The results showed that under Al stress, chlorophyll content, photosynthetic rate and chlorophyll fluorescence parameters Fv/Fm, φPS Ⅱ, qP and qN in C4 Ppc transgenic rice declined significantly less than those in the wild type. The results indicated that C4 Ppc transgenic rice still showed higher photosynthetic efficiency under Al stress compared to the wild type. Al treatment led to an increase in active oxygen content in rice. However, the active oxygen scavenging enzymes such as SOD, POD and CAT in C4 Ppc transgenic rice were significantly higher than those in the wild type, revealing the lipid peroxidation was lower in C4 Ppc transgenic rice. The results indicated that the introduction of C4 Ppc into rice significantly enhanced aluminum tolerance.

Recombinase-mediated Marker Gene Excision in Transient Gene Expression System of Mesophyll Protoplasts

Chi Junjie, Dai Shaojun, Wei Jianhua, Wang Hongzhi

2014, 30(7):  86-92. 

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In the present study, we used transient gene expression system of Arabidopsis mesophyll protoplasts to test and compare site-specific recombination constructs, which the expression of recombinase was induced by the promoter for heat-shock proteins Hsp18.2, developed a simple and convenient system for rapid assessment of site-specific recombination constructs.

Cloning and Analysis of Cytosolic Glutamine Synthetase cDNA from Eichharnia crassipes

Jiang Lihua, Fu Minghui, Li Yuanmei, Yan Guohua, Zheng Lijun, Cheng Xiaoli, Peng Jinping

2014, 30(7):  93-99. 

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In this study, the template cDNA which was reversely transcribed from the total RNA of Eichharnia crassipes, was subjected to polymerase chain reaction（PCR）with the degenerate primers which was designed according to sequences of the Cytosolic Glutamine Synthetase（GS1）from other plants. The full-length cDNA sequence was achieved with Rapid Amplification of cDNA End method（RACE）from the amplification product. It includes 1434 bp with the open reading frame of 1071 bp which encoded 356 coding amino acids with a predicted size of 39.3 kD and a calculated pI of 5.52. The result of sequence homology analysis showed that the deduced protein had relatively high amino acid identity with GS1s from other plants. The prediction of sub-cellular localization showed that EcGS1 is a cytosolic glutamine synthetase.

Prokaryotic Expressing and Functional Analysis of C3HC4-type Zinc Finger Protein in Nicotiana benthamiana

Wu Wenxian, Yang Xiufen, Liu Yong, Qiu Dewen

2014, 30(7):  100-105. 

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C3HC4-type RING finger genes comprise a large family in the plant kingdom and play important roles in various physiologic processes of plant life. In this study, a zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24 kD protein with 203 amino acids and contains one typical C3HC4 zinc finger domain and named NbZFP1. The entire coding region of NbZFP1 gene was then cloned into the bacterial expression vectors pGEX-6P-2 and pMAL-C2X. After IPTG induction, the recombinant protein was analyzed by SDS-PAGE, which showed that the expected protein was expressed at a high level in bacterial cells. NbZFP1 gene was cloned into the over-expression vector pBI121, and then transfered into Nicotiana benthamiana through Agrobacterium tumefacien-mediated transformation. According to PCR and Southern blot detection, NbZFP1 gene was successfully integrated into Nicotiana benthamiana genome. Regeneration plants showed type compactness, internode shorten and stem sturdy, also enhanced disease resistance against tobacco mosaic virus（TMV）.

Overexpression of OsPT8 Gene to Enhance the Pi Deficiency Tolerance of Transgenic Tobacco

Jia Hongfang, Zhang Hongying, Yin Guining, Huang Huagang, Xu Guohua, Cui Hong

2014, 30(7):  106-111. 

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Plant phosphate transporters（PTs）are active in the uptake of inorganic phosphate（Pi）from the soil and it's translocation within the plant. In this work, the key phosphate transporter gene OsPT8 in rice was transformed into tobacco（Yunyan 87）by Agrobacterium-mediated method. We set two treatments-normal P（1 mmol/L Pi）and low P（0.1 mmol/L Pi）by transgenic and wild-type tobacco as materials, detecting biomass and contents of P in shoots and roots, and analyzing the expression of phosphate transporter family gene（NtPT1 and NtPT2）. The results show that the biomass, the contents of total P and Pi of shoots and roots in transgenic line increased significantly compared with wild type under low Pi treatment, this data suggest that overexpression of OsPT8 can enhance the Pi deficiency tolerance of transgenic tobacco；the data of gene expression of NtPT1 and NtPT2 suggest that overexpression of OsPT8 enhance the Pi deficiency tolerance of transgenic tobacco caused by OsPT8 and other phosphate transporters.

The Possible Role of Alternative Oxidase in Jatropha curcas L. Responses to Salt Stress

Yu Lulu, Wu Yangchen, Wang Jingjing, Wei Qian, Xu Fei

2014, 30(7):  112-118. 

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Jatropha curcas L. is a stress-resistant oil species and can growth well in lands with low-rainfall harsh climate conditions. However, the mechanism of Jatropha curcas L. tolerance to adverse environment conditions is still unclear. In this study, the role of alternative oxidase（AOX）in Jatropha curcas L. response to salt stress was investigated. The results showed that Jatropha curcas L. was good tolerance to 200 mmol/L and 400 mmol/L NaCl stresses, and no significant differences in chlorophyll contents and water contents between treated and untreated control plants. 1 mol/L NaCl treatment obviously suppressed plant growth and leaf development, reduced leaf stomatal conductance, but no serious oxidative damages were shown. Moreover, it should be noted that AOX pathway respiration and AOX gene transcription was induced markedly under salt stresses. Compared with control plants, AOX transcription increased two-fold in response to 200 mmol/L and 400 mmol/L salt treatments, and increased three-fold under 1 mol/L NaCl treatment. Therefore, it hypothesis that AOX plays an important role in Jatropha curcas L. salt stress tolerance.

Transcriptome Analysis for Quercus liaotungensis Koidz. Based on High-throughput Sequencing Technology

Liu Yulin, Li Wei, Zhang Zhixiang

2014, 30(7):  119-124. 

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In this study, Illumina Solexa Hiseq 2000 high-throughput sequencing technology was used to get the comprehensive transcriptome from mixed samples of buds, flowers, leaves and fruits of Quercus liaotungensis. As a result, 3.8 Gb effective data was obtained. After de novo assembly by the software of Trinity, a total of 95 800 unigenes were generated, corresponding to a total of 73.57 Mb with a maximum length, average length and N50 of 11 284 bp, 768 bp and 1 373 bp respectively. Using Blastx against the public databases of Nr and Swiss-Prot with an E-value cut-off of 10^-5, 38 163 unigene were not found in any databases with a high homology. According to the KEGG pathway assignment, 67 unigene encoding nine key enzymes which may involve in starch synthesis were identified. In addition, 15 901 potential SSR loci were detected from 13 380 unigene. Of them, dinucleotide repeat and trinucleotide repeat accounted for 98.16% of all.

Cloning and Tissue-specific Expression of TCTP Gene in Inner Mongolia Cashmere Goat (Capra hircus)

Yang Limin, Yao Ruiyuan, Guo Zhixin, Bao Chaogetu, He Qiburi, Zheng Xu, Bao Wenlei, Wang Zhigang

2014, 30(7):  125-130. 

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The present study aims at cloning the CDS fragment of Translationally controlled tumor protein（TCTP）gene cDNA in Inner Mongolia Cashmere Goat and analyzing its expression pattern. TCTP gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by BLAST, while amino acid sequence was analyzed by online softwares. The tissue-specific expression pattern of TCTP was analyzed by quantitative RT-PCR. The cloned TCTP gene cDNA was 519 bp in length, including a complete ORF encoding 172 amino acids. The full cDNA nucleotide sequence has an identity from 99% to 95% with Ovis aries, Bos taurus, Sus scrofa, Homo sapiens, Macaca mulatta and Rattus norvegicus. Bioinformatics analysis showed that theoretical molecular mass of encoded protein was 19.6 kD, isoelectric point（pI）was 4.673. It contained a N-glycosylation sites, a protein kinase C phosphorylation sites and three casein kinase Ⅱ phosphorylation sites and most probably is localized in cytoplasm. The results of quantitative RT-PCR showed that TCTP gene is expressed in kidney, muscle, pancreas, liver, testis and brain. The expression was higher in liver, whereas lower in brain.

Cloning and Sequence Analysis of TpRS1 Gene from Cysticercus pisiformis

Han Lele, Li Zuolei, Gou Huitian, Sun Xiaolin

2014, 30(7):  131-136. 

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To study the function of TpRS1 gene from Cysticercus pisiformis, the open reading frame（ORF）cDNA sequence of TpRS1 gene was cloned by RT-PCR from C. pisiformis total RNA with primers derived from Taenia solium RS1 gene sequence in the GenBank database. Biochemical properties, secondary structure, signal, hydrophobicity and antigenicity in TpRS1 protein were predicted by bioinformatics tools. The results indicated that TpRS1 cDNA sequence contained an ORF of 258 nucleotides and the deduced protein consisted of 85 amino acids with the theoretical molecular weight of 9.6 kD and isoelectric point of 9.07. Analysis of secondary structure revealed 70.59% and 5.88% of α-helix and β-strands, respectively, and others were loop. The signal peptide sequence of TpRS1 were not identified. TpRS1 protein sequence showed more than 72% identity with other Taenia spp.. Phylogenetic analysis indicated that TpRS1 located in the same clade with Taenia hydatigena.

Construction and Prokaryotic Expression Sip-GAPDH Fusion Gene of Streptococcus agalactiae from GIFT Strain of Nile Tilapia（Oreochromis niloticus）

Wang Bei , Li Guihuan, Lu Yishan , Tang Jufen , Huang Yucong , Wu Zaohe , Jian Jichang

2014, 30(7):  137-142. 

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The DNA fragment encoding the Sip and GAPDH gene of Streptococcus agalactiae strain ZQ0910 isolated from GIFT Strain of Nile Tilapia（Oreochromis niloticus）were obtained by PCR. The Sip-GAPDH gene was constructed in a Sip-linker-GAPDH format with the standard 15-amino acid linker（Gly4Ser）3 by SOEing PCR technique, and the final full length product was cloned into the pET-32a（+）vector for protein expression in Escherichia coli strain BL21（DE3）. The result showed that the expression fusion protein Sip-GAPHD were about 102.01 kD and Western blotting analysis confirmed that the 102.01 kD protein was the fusion protein, because it was specifically recognized by mouse anti-His monoclonal antibody. Inducing the cells at 37℃ in 0.1 mmol/L of IPTG for 5 hours was the optimal conditions for expression of the recombinant Sip-GAPDH fusion protein.

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Sweetfish Macrophage Inflammatory Protein-2 Gene

Wang Shihui, Shi Yuhong, Chen Jiong

2014, 30(7):  143-149. 

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Macrophage inflammatory protein-2（MIP-2）is an important chemokine, which promotes neutrophils to be the sites of inflammation and eliminates the inflammatory response. In this study, the sequence of MIP-2 gene from de-novo transcriptome sequencing of sweetfish macrophages was obtained. An open reading frame was composed of 318 nucleotides, which encoded a protein of 105 amino acids with a molecular weight of 11.6 kD. Its N-terminal 19 residues were the signal peptides. Sequence alignment showed that sweetfish MIP-2 shared the highest homology with the MIP-2 from Esox lucius with 54% protein sequence identity. In healthy sweetfish, MIP-2 mRNA was mainly expressed in the spleen, kidney, brain and gills, weakly expressed in the liver, heart, muscle and intestine. Quantitative RT-PCR analysis showed that MIP-2 transcripts were significantly up-regulated in the tested tissues, especially the kidney and muscle. The results suggested that MIP-2 might play an important role in sweetfish antibacterial immunity.

Isolation, Identification and Quorum Sensing of a Serratia spp.

Zhang Caili, Zhu Suqin, Wang Ying, Sun Xiujiao, Zeng Mingyong

2014, 30(7):  150-155. 

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One quorum sensing strain was isolated from spoilage Litopenaeus vannamei. Specie was determined by 16S rRNA gene analysis and classical tests, it can not produce red pigment on medium, named it Serratia marcescens AK1.Quorum sensing was detected by reporter strains and signal molecules types were identified by thin layer chromatography. Quantitative analysis of the signaling molecule secreted by AK1 during different growth stages. The result indicates strain AK1 can induce Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136 produce purple and blue color respectively. Thin layer chromatography showed that AK1 can produce C6-HSL and 3-oxo-C6-HSL types of signaling molecules with density-dependent. They reached the maximum in the late logarithmic phase.

Isolation of a Biosurfactant Producing Strain and Property Study

Wang Rui, Wu Juan

2014, 30(7):  156-161. 

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A biosurfactant-producing strain H1 was screened with blue agar plat from polluted soil of an oil field in China. The physiological and biochemical tests and phylogenetic analysis of 16S rDNA sequence showed the similarity of 99% between strain H1 and Klebsiella pneumoniae. The experiments showed that strain H1 could grow well and produce more biosurfactant under the following conditions：sucrose as carbon source, ammonium nitrate as nitrogen source, initial pH7.0 and 30℃.

Isolation, Identification and Enzyme Properties of a Strain Producing Feruloyl Esterases

Hu Hongyu, Zhou Yanyan, Chen Jing, Hou Yunhua

2014, 30(7):  162-167. 

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Using the method of transparent zone, 37 filamentous fungi producing feruloyl esterase（FAE）were selected from rot soil samples containing wood fibers. The strain HA4087 was isolated by its high FAE activities. It was identified as Alternaria alternata by conidia morphology, 18S rDNA sequence and ITS sequence analysis. The characteristics of crude feruloyl esterase from A. alternate HA4087 were preliminarily studied. FAE activity of the crude enzyme was 86 mU/mL. The optimal temperature and pH for crude FAE was 55℃ and pH5.5, respectively. Crude FAE from A. alternate HA4087 remained nearly 90% of its maximal activity at 70℃ and was active in the pH range of 3.0-7.0. The strain producing thermostable and high active FAE provided a prerequisite for the industrialization of feruloyl esterase.

Cloning and Sequence Analysis of Transcriptional Activator Protein Gene from Aeromonas hydrophila

Li Xue, Hu Xiucai, Lan Yun, Shen Xiaojing, Lü Aijun, Zhu Aihua

2014, 30(7):  168-172. 

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Transcriptional activator protein（AhyR）gene of Aeromonas hydrophila strain Zf1 was amplified by PCR using the designed primers, resulting in a 1 063 bp DNA sequence. The sequencing analyses revealed AhyR gene encoding a protein of 260 amino acids which has autoinducer binding domain（18-168 aa）and LuxR-type helix-turn-helix（HTH）domain for DNA binding（176-241 aa）. Moreover, AhyR protein was predicted containing 13 phosphorylation sites（ie., 15S, 30T, 62S, 144S, 145S, 156S, 161Y, 173S, 184T, 188T, 200S, 227S and 231Y）. The homology modeling of 3D structure of AhyR protein was carried out based on the template retrieved from chain A of quorum sensing control repressor（PDB：3SZT_A）. AhyR protein showed the homology of 28.40% with the standard model, and the physiological activity subunit LuxR-type HTH domain profile was located in the outer surface of the C-terminal. The quality of the resulting protein structure was further checked by the Ramachandran plot.

Expression, Purification and Identification of Zta-P54 Fusion Protein in Escherichia coli of Epstein-Barr Virus

Wang Yunlong, Zhang Chunyan, Li Yulin, Cheng Lei, Wang Jichuang, Deng Lili, Mi Hai, Bai Xiaojing

2014, 30(7):  173-178. 

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It was to obtain high purity and active epstein-barr virus fusion protein and use for detection kit. The fragments of BZLF1-BMRF1 amplified by PCR from pGEX5T-BZLF1-BMRF1 was inserted into expression vector pET32a, and the recombinant plasmid pET32a-BZLF1-BMRF1 was transformed into E. coli, which was induced to express by IPTG. The expression protein Zta-P54 was purificated by DEAE Sepharose CL-6 B and Ni -NTA affinity chromatography then analysed by SDS-PAGE and immunoreactivity was proved by western blotting.Double endonuclease digestion and DNA sequencing results showed that sequencing results constructed successfully. SDS-PAGE showed that the protein was soluble expressing. The expression product Zta-P54 with the moleculor weight 60 kD was located in the cytoplasm and soluble.Expression Protein, s purity was 96.5%. Western blotting showed that frusion protein Zta-P54 possessed a well bioactivity and specificity.Therefore, It proved that the pET32a/BZLF1-BMRF1 plasmid can effectively express in E. coli.

The Construction of the EGFP Enterovirus 71

Zhu Shouhai Wang Lulu

2014, 0(7):  179-183. 

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Human Enterovirus 71（EV71）is one of the major agents causing hand, foot and mouth disease（HFMD）. An infectious full-length cDNA clone of EV71 as well as the recuse virus were obtained using the reverse genetic techniques. Then, the EGFP gene was inserted into the EV71 genome to construct the reporter virus. The results show that the EGFP-EV71 virus not only are replication-competent and infectious, but the EGFP gene are stable during passaging, implicating the recombinant virus could be used as a reporter virus for high throughput EV71 antiviral drugs screening.

Effect of Co-expressing Protein HAC1 on Expression of α-galactosidase in Pichia pastoris

Zhong Kaixin, Chen Lizhi, Li Yangyuan

2014, 0(7):  184-189. 

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To improve the production of a recombinant α-galactosidase in Pichia pastoris, the hac1 gene from P. pastoris GS115 constructed in pPic9K vector was coexpressed with α-galactosidase gene in P. pastoris X33. Results showed that the HAC1 protein under control of AOX1 promoter increased the expression level of α-galactosidase protein in P. pastoris. The α-galactosidase activity superexpressed with the HAC1 protein was up to 2.2 times than that of constitutive expression of HAC1. hac1 gene integrated into the genome of the P.pastoris was proved by PCR. The galactosidase activity of Gal-HAC1-4#, after 168 h methanol induction in 50 L high-density fermentation, reached the highest value of 6 560 U/mL, and increased by 27% compared with Gal-21#. The crude protein concentration of Gal-HAC1-4# was higher than the strain Gal-21# after methanol induced. These results showed that the galactosidase expression ability of Pichia pastoris was inproved by co-expressing protein HAC1.

Mutation Breeding of Yarrowia lipolytica and Optimization of α-Ketoglutaric Acid Fermentation Conditions

Gong Jian

2014, 0(7):  190-195. 

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A strain of Yarrowia lipolytica（ZY-4）which producing α-ketoglutaric acid（KGA）was treated for mutation by UV and NTG. Mutation strains which could accumulate much more KGA were obtained. Culture medium for mutation strain on KGA fermentation was also studied. The concentration of KGA accumulated by UV mutation strain and NTG mutation strain increased 67.8% and 110% respectively compared with original strain. The optimal fermentation medium contains glycerol 8%, NH₄Cl 5.0 g/L, thiamine 1.0 μg/L, KH₂P0₄ 1.0 g/L and MgSO₄·7H 2O 0.5 g/L. The production of KGA increased 232.4% after optimization when compared to the original strain.

Isolation and Identification of Accumulation of Quorum Signal Molecules Strains from the Trichoplusia ni Midgut

Huang Yuanyuan, Ma Hong, Jia Zhenhua, Huang Yali , Song Shuishan, Zhang Xia

2014, 0(7):  196-200. 

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Two strains named Tni-9 and Se-9 were isolated from Trichoplusia ni midgut by CV026 report system. The 16S rDNA sequences of the isolates were studied, blast analysis and the phylogenetic tree showed their 16S rDNA were highly homologous to that of Enterococcus mundtii ATCC 43186. By the physiological and biochemical characteristics, the strain Se-9 was further confirmed belong to Enterococcus mundtii .TLC analysis present that Se-9 could produce C₆-HSL and 3OC₆-HSL quorum sensing signals respecctively.

Fe₃O₄ Magnetic Dextran Nanoparticles Modified with SPA Ligand for IgG Purification

Ren Jinggang, Cheng Chao, Zhong Conghao, Zhang li, Li Rongxiu

2014, 0(7):  201-208. 

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The objective of present research is to prepare magnetic dextran nanoparticles conjugated with Staphylococcal Protein A（SPA-MDPs）for the purification of IgG. The magnetic nanoparticles were prepared by following a high-temperature solution-phase procedure, and the average particle size can be tuned from 30 to 200 nm by simply varying the concentration of NaOH during the reaction. The size of SPA-MDPs nanoparticles has a great influence on their adsorption capacity for IgG. A maximum adsorption of human IgG was calculated to be 84.85 mg/mL with a binding affinity constant of 3.48×10⁶ M^-1 when the average size of SPA-MDPs is about 101.5 nm. The experiment results also demonstrated that the SPA-MDPs nanoparticles can be recycled for 5 times with minor loss of adsorption capacity. Moreover, IgG was purified from human plasma with a purity of 93.5% and recovery of 88.7%. Thus, SPA-MDPs show promising potentials for isolation and purification of IgG.