Table of Content

11 August 2013, Volume 0 Issue 8

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Review

Research Advances on the Mechanism of Glyceraldehydes-3-phosphate Dehydrogenase in Plant

Lu Qian, Mi Xiaoju, Cui Jizhe

2013, 0(8):  1-6. 

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Glycolysis and the Calvin cycle are critical metabolic pathways of energy supply and carbon fixation in plants. Glyceraldehyde-3-phosphate dehydrogenase plays a central role in glycolysis and the Calvin cycle. Recent research results disclosed that the function and regulation of GAPDH turned out to be quite diverse and complex. This article provides an overview of the latest progress on the GAPDH type and function, the specificity interaction of GAPDH and NAD（P）, PRK/GAPDH/CP12 complex and regulation, and the mechanism of GAPDH response to stress in plants.

Progresses of SNPs Studies in Aquaculture Animals

Zhang Xiaomeng, Ma Pu, Wang Hongdi, Wang Xiuli

2013, 0(8):  7-11. 

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Single nucleotide polymorphism（SNP）is the most widely distributed molecular marker in genome and has become the popular issue in molecular biology. In this paper, some progresses of SNPs in aquaculture animals including disease, growth trait and reproductive performance were reviewed. We hope to provide the reference on breeding and molecular marker-assisted selection in aquaculture animals.

Research on Gene Promoter of Fish

Du Xiaoxi, Gao Xianggang, Chen Panhai, Wang Yang, He Chongbo

2013, 0(8):  12-16. 

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Promoter is an important component of gene and controls the transcription and expression of gene. It is widely recognized that the investigation of promoter could be used in changing and improving the characteristics and performances of organisms. In recent years, the research in fish promoter was extensive and made us a better understanding about promoter. In this paper, we summarized the characteristics of fish promoter and also the application in metabolism, growth development, immune of fish and the application of dual promoter in transgenic fish.

Advances in Cellular and Molecular Basis of Hematopoietic Stem Cell Niche

Liu Jiajia, Zhang Yiting, Peng Hang, Liu Pengxia

2013, 0(8):  17-22. 

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Hematopoietic stem cells（HSCs）are thought to reside in specific three-dimensional spatial structure, know as the hematopoietic microenviroment or niche. Studies on stem cells have shown that stem cell function is controlled by intrinsic genetic programs within the stem cell and by extracellular cues from the niche. The niche provides the extrinsic molecular mechanisms that control stem cell maintenance, self-renewal and differentiation. Nevertheless, the structure as well as cellular and molecular basis for niche activity, have long remained a ‘black box’. Elucidating the mechanisms holds many promises for future clinical applications and regenerative medicine. We reviewed the remarkable progress recently made in characterizing the composition of the niche and the molecular crosstalk between HSCs and the cellular constituents of the niche.

Processing in Whole Tumor Cells Antigens for Cancer Immunotherapy

Fang Li, Hu Wei

2013, 0(8):  23-27. 

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Whole tumor cell（WTC）as tumor antigens is a very promising alternative in tumor immune protection and immunotherapy, which can theoretically eliminate some important limitations in vaccine development. Whole tumor cells express a whole array of tumor associated antigens（TAAs）, and simultaneously can induce CTLs and CD4⁺ T helper cell activation. In this paper, some recent proceedings and current preparing methods of whole tumor cell antigens were reviewed including some types of WTC antigens, DC cells loaded by WTC antigens, and modification and immune enhancement of WTC antigens.

Research Progress of Magnetosome Formation Genes and Proteins

Liu Xinxing, Yun Hui, Xie Jianping, Huo Zhuanzhuan, Wu Haiyan, Yang Yingjie

2013, 0(8):  28-35. 

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Magnetotactic bacteria are able to synthesize nano-scale crystals called magnetosomes which contain a lipid bilayer membrane. Based on their bio-compatibility and well-dispersibility, these crystals are potentially to be widely applied in molecular biology, immunology, medicine, information storage, and the removal of heavy metals in polluted waters, so do the distinct theoretical significance in geology. Due to the unknown information for magnetosome formation and chain assembly, of which the main researches are focusing on Magnetospirullum, the majority of magnetotactic bacteria in the environment and their magnetotactic particles are difficult to study. Metagenomics focuses on the environmental samples to exploit the microbial sequences and functions with the pure culture waived, which can be applied in the research of environmental magnetotactic bacteria. The essay will briefly introduce the recent researches on genes and proteins referring to the magnetosome synthesis and chains assembly. Meanwhile, some researches on magnetotactic bacteria and magnetosomes using metagenomics technology were exhibited, and some viewpoints and prospects were put forward.

Bioremediation of Cyanide Contamination and Its Applications

Fang Shumei, Liang Xilong, Li Chunyan

2013, 0(8):  36-42. 

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The cyanide contamination of soils and water sources by anthropogenic activities caused severe threaten to lives of human, animals, and plants. So, the treatment of contaminants and remedy of environment are significantly important to environmental protection. Biological degradation may be a potentially efficient, cost-effective, environmentally friendly alternative to the previous conventional processes. Many microbial organisms can use cyanide as carbon and nitrogen source converting it into ammonia and carbonate which are less or no toxic products. This paper reviewed the research progress on the microbial remediation of cyanide contamination in terms of the major source of cyanide in soil and water, recent advances, affecting factors, the degradation mechanisms and the current applications in the microbial remediation of cyanide contamination.

Research Report

Comparison on Stress-resistent Phenotypes Between Arabidopsis pumila and Arabidopsis thaliana and Expression Analysis

Zhang Tingting, Zhou Yaping, Zeng Youling

2013, 0(8):  43-50. 

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In this study, the data of stress-resistent phenotypes were compared between Arabidopsis pumila and Arabidopsis thaliana and the expression of some stress-responsed genes was examined by Real-time PCR in Arabidopsis pumila subjected to the salt, mimic drought, ABA and cold stress. Some results were as followed：（1）The water-retention capacity of Arabidopsis pumila was stronger than Arabidopsis thaliana under the same condition with the determination of relative water content between two species.（2）Arabidopsis pumila showed more stressed resistence than Arabidopsis thaliana as model plant under salt, mimic drought, heat and ABA stress by the observation on the growth phenotypes and the statistical analysis of relative root length between two species. Arabidopsis pumila didn't present a better growth phenotype than Arabidopsis thaliana after a cold stress treatment, then a normal growth condition.（3）The expressions of some stress-responsed genes in Arabidopsis pumila, such as RD22, RD29A and ADH, had significant increases in most case under the treatments of salt, mimic drought, cold and ABA.

Cloning，Functional Sequence Analysis and Prokaryotic Expression of Cotton GhAO3 cDNA

Ran Maofei, He Yi, Qian Wenjie, Wang Fei, Li Hongbin

2013, 0(8):  51-56. 

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A cotton ascorbate oxidase(GhAO3)full-length open reading frame（ORF）cDNA was cloned from elongating fiber tissue（15 DPA）by RT-PCR method, GhAO3 ORF contains 1 758 bp nucleotides and codes a protein of 585 amino acids. Through sequence function analysis and homology sequence alignment, GhAO3 protein includes three multi-copper oxidase domain and transmembrane signal sequence with a high conservation. Phylogenetic tree analysis showed that GhAO3 has a similar relationship with GmAO protein. Prokaryotic expression vector pET32a-GhAO3 was constructed and transformated into E. coli BL21（DE3）. Recombinant GhAO3 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD, and had a higher enzymatic activity.

Cloning and Sequence Analysis of CBF1 Gene in Vitis amurensis

Liu Yang, Sun Jiadi

2013, 0(8):  57-62. 

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Bioinformatic method was used for the comparative analysis of CBF1 gene sequences in cold acclimation, conservative domains among the sequences were determined for degenerate primer designing, the defined PCR conditions and Reverse PCR clone were adopted to clone the full length CBF1 genes from genes of Vitis amurensis which is in cold acclimation. Sequence analysis showed that the ORF lengths of CBF1 gene is 753 bp and has no intron in coding domain, and encoded proteins of 251 residues. Furthermore, their molecular weights and isoelectric points of 27.503 kD and 3.11 were predicted from the calculated pI values. Similarity of amino acids to many plant which is Hordeum vulquare and Oryza sativa is 80.7%, it have a highly conserved sequence in AP2/EREBP, this demonstrate we clone transcription factor CBF1 genes of Vitis amurensis . It is named ViCBF1 which is registered in GenBank, number is DQ517296.

Research Report

Transcriptome Library Construction and Sequencing from Chemically Induced Aquilaria sinensis

Wu Hongqing, Wang Lei, Tao Meihua, Gao Xiaoxia, Bai Ling, Zhang Weimin

2013, 0(8):  63-67. 

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Modified guanidinium isothiocyanate-CTAB method was used to isolate the total RNA from five-year-old Aquilaria sinensis treated by chemical induction one year ago. The mRNA of A. sinensis was enriched from the total RNA and broken into short fragments, and then the cDNA library was established for RNA-Seq. As a result, 54 685 634 clean reads was obtained after sequencing with a total length of 4 921 707 060 nt. The value of Q20 was up to 97.45%, exhibiting good sequencing quality. After the initial assembly of sequence data, these clean reads were assembled to 190 109 contigs, which were then assembled to 83 467 unigenes with a total length of 58 569 625 nt and an average length of 702 nt after the further assembly. The value of N50 was up to 1 120. There were 1 691 unigenes longer than 3 000 nt, accounted for 2.03% of all unigenes. The good quality of assembly showed the information on the transcriptome of A. sinensis was well preserved, which laid the foundation for digital gene expression analysis associated with agarwood formation of A. sinensis.

Study on Phylogenetic Relationships of Ten Urticaceae Species Based on nrDNA ITS Sequences

Hou Xindong Yin Shuai Sheng Guilian Cheng Dandan Lai Xulong

2013, 0(8):  68-73. 

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In this study, we collected 12 Urticaceae plants from Lushan mountain area to carry out the study of internal transcribed spacer（ITS）sequences, and thus further explore the feasibility as a study of the Urticaceae Phylogeny using ITS sequences, as well as the similarities and differences of traditional plant taxonomy and molecular systematics. We obtained the sequences of nrDNA ITS from 10 species of Urticaceae by PCR amplification and clone sequencing. For phylogenetic analyses using MEGA 4.0 software, both NJ and MP trees of Urticaceae were constructed with U.marirei as outgroup. The two methods produced trees with absolutely congruent topology. These phylogenetic trees showed three separate lineages：（1）Elatostemeae, （2）Boehmerieae, （3）Urticeae. The results suggested that it is feasible to research the phylogenetic of Urticaceae plants using ITS sequences. The results were basically consistent about the phylogenetic using molecular techniques and traditional morphology classification.

Studies on the Genetic Diversity and Relationship of Manglietia hainanensis Dandy by ISSR

Wei Xiaoling, Cao Fuxiang, Chen Jian

2013, 0(8):  74-77. 

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Inter-simple sequence repeat（ISSR）technique was applied to detect 4 populations and 36 samples of Manglietia hainanensis Dandy，and 106 bands were amplied by 10 primers，including 87 polymrphic bands. The polymorphic percentage is 82.08%. The variation range of alleles per locus(Ne)was 1.416 6-1.625 7；variation range of Nei's gene diversity(H)was 0.222 8-0.339 2；variation range of shannon's information index(I)was 0.315 1-0.491 0；coefficient of gene differentiation(Gst)of the 4 populations was 0.197 1. Based on the genetic identity，dendrogram of 4 populations was constructed by UPGMA.

Optimization for ISSR Reaction System of Erythropsis kwangsiensis by Orthogonal Design

Dai Wenjuan, Luo Wenhua, Ma Husheng, Fu Zhihong, Tang Wenxiu, Zhao Bo, Pan Bo, Huang Shixun

2013, 0(8):  78-82. 

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Abstract: To optimize the inter simple sequence repeat（ISSR）reaction condition for Erythropsis kwangsiensis genomic DNA, the concentrations of MgCl₂, dNTPs, Taq polymerase and template DNA were studied with an orthogonal experimental design, and the optimal anneal temperature of primer and cycles were determined through gradient PCR. The optimal PCR system for ISSR analysis was 1×PCR buffer, 2.5 mmol/L MgCl₂, 0.15 mmol/L dNTPs, 0.06 U/μL Taq polymerase, 3 ng/μL template DNA, 0.2 μmol/L primer in 25 μL reaction solution. The augmentation procedure was pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 45 s, annealing at 52℃ for 45 s, extension at 72℃ for 1.5 min, reaction with 30 cycles, and extension at 72℃ for 7 min and holding the samples at 4 ℃. The system was applied in the amplification of 10 varieties of Erythropsis kwangsiensis, and indicated the suitability and stability of the system.

The Technique for Agrobacterium-mediated Transformation via Germinating Seeds of Soybean

Liu Zhimin, Xiong Hewen, Xie Hao, Qin Yutao, Zhao Yanran, Guo Bei

2013, 0(8):  83-87. 

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Germination seeds were used to research the genetic transformation technology of Agrobacterium-mediated in soybean. The growing point, cotyledon node and hypocotyl of germination seed were used as explants, and the plasmid that carried with the resistance to the herbicide glyphosate gene EPSPS and glufosinate resistance screening gene Bar was transferred to soybean by Agrobacterium-mediated. Transformants were obtained by co-culture and potted plant growing. After the double PCR detection as well as the two herbicide resistance identification of transgenic T₀ and T₁ plants, the positive strains were acquired. The experiment results showed that two genes resistance to herbicides have been successfully transferred to soybean via germinated seeds by Agrobacterium-mediated transformation method, with the transformation rate of approximately 0.37%.

The Influence of Exogenous Ca²⁺ on the Physiology of Oryza sativa L. Under PEG6000 Simulated Drought

Sui Yazhen, Liu Yang, Xu Mengmeng, Liu Sixue, Wei Rongrong, Zhao Xin

2013, 0(8):  88-93. 

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In this study, the wild type of Oryza.sativa L. was used as the experimental subject. Using water culture method, under PEG6000 simulating drought condition, the physiology indexes of rice were measured, to understand the effect of Ca²⁺ on drought. The experiment result showed that, under the osmotic stress, a series of changes happened against the stress, including MDA, SOD, POD, proline, chlorophyll, the contents of which were increased. Under mild desiccation（PEG=10%）, when the concentration of Ca²⁺ was 1 mmol/L, the increase of SOD, POD and chlorophyll were most visible, meanwhile the contents of proline and MDA increased with the concentration of Ca²⁺. Under severe desiccation（PEG=20%）, the contents of proline and chlorophyll were remarkably raised, with the increased of concentration of Ca²⁺, and reached the maximum when [Ca²⁺]equals to 5 mmol/L. On the other hand, the activities of SOD and POD reached the maximum at [Ca²⁺]equals to 1 mmol/L..

Identification of Rodents by DNA Barcoding in Inner Mongolia Area

Chang Zili, Liu Fang, Wang Jianjun, Li Jianyun, Hu Yanhong, Wu Zhenghua, Wang Haohui, Li Ke, Yang Zhixian, Ma Zhidong, Fan Mengguang, Zhang Zhongbing

2013, 0(8):  94-98. 

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To study the feasibility of DNA barcoding in the identification of rodents. 59 specimens of 7 species were sequenced（barcoded）for a 657 bp region of the mitochondrial cytochrome oxidase subunit I gene（COI）in Inner Mongolia Area, China. The GC content and genetic distance was calculated using MEGA5.0, and the phylogenetic trees were constructed with Neighbor-Joining（NJ）method. The results showed that AT contents was larger in all sequences, mostly species had specified amino acid component. The average intraspecific genetic distance（K2P）was 0.6% and the average interspecific genetic distance was 23.1%. A Neighbor-Joining tree showed 7 major clusters apparent. We conclude that COI sequencing of DNA barcoding can be used to identify rodents species in Inner Mongolia.

Identification of Crocodile Production Through DNA Barcode

Guo Fengjuan, Yang Jianchun, Hu Shijia, Li Yongshi

2013, 0(8):  99-104. 

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DNA barcode has been extensively used in species identification and taxonomical research. In this paper, a batch of smuggled crocodile products was identified using DNA barcode. Three types of the crocodile products（dried crocodile meat, crocodile penis and dried crocodile skin）were selected as the samples for species identification. Mitochondrial DNA was extracted and cytochrome oxidase subunit I gene fragments of each sample was amplified using the universal primers. Alignment analysis with the data of eighteen homologous sequences of five different crocodile species in GenBank revealed that the three unknown samples had the highest sequences similarity, which was 99.6%－100%, with the sequences of Crocodylus siamensis. The genetic distance that was computed by MEGA4.0 between thoses samples and Crocodylus siamensis was 0.000-0.003, which was the minimum. The phylogenetic tree were constructed by P-distance model and Neighbor-joining method, and Varanus bengalensis was set as the outgroup. The result indicated that all of the three samples come from Crocodylus siamensis. Consequently, it convincingly demonstrated that the samples were Crocodylus siamensis products, which provides a powerful evidence for the execution of the customs.

Cloning and Tissue Expression of β-actin in the Mud Crab（Scylla paramamosain） and Its Utility as an Endogenous Control

Xu Zhen, Ma Hongyu, Ma Chunyan, Feng Nana, Li Xincang, Li Shujuan, Jiang Wei, Ma Lingbo

2013, 0(8):  105-112. 

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β-actin gene is a member of the actin family，mainly involved in maintaining cell structure，movement and physical activity in cell division, and plays an important role in quantitative genetic experiments. In this study, RT-PCR and RACE techniques were used to amplify the cDNA full-length of β-actin in the mud crab(Scylla paramamosain). Sequence analysis showed that the full cDNA of β-actin gene was 1 434 bp long, including a 1 131 bp open reading frame（ORF）, a 83 bp 5' untranslated region（5' UTR）and a 243 bp 3' untranslated region（3' UTR）. The ORF encoded 376 amino acids residues with a predicted molecular mass and PI of approximately 41.79 kD and 5.205, respectively. Multiple alignment with homologous amino acid residues was performed and it indicated a high similarity among the 18 species. Scylla paramamosain was more closely related to Gecarcinus lateralis than any other species according to the neighbor joining（NJ）phylogenetic tree. Real-time PCR results indicated that β-actin gene expressed in all eight different tissues including blood, heart, liver, stomach, gill, muscle, testis and connective tissue, with the highest expression in muscle and lowest expression in connective tissue. Owing to the significant differences of RNA expression among these tissues, this gene is not suitable used as an endogenous control.

Prokaryotic Expression of Dendrorhynchus zhejiangensis Actin Gene and Self-assembly of Recombinant Protein in vitro

Li Ye, Chen Lei, Zhou Jun, Li Chenghua, Su Xiurong, Li Taiwu

2013, 0(8):  113-118. 

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Actin is a major component of cytoskeleton. In cells, actin associates with actin binding proteins（ABPs）to form highly ordered polymerization structure on the basis of F-actin, which plays a variety of important physiological functions. According to the full length cDNA of Dendrorhynchus zhejiangensis actin, the prokaryotic expression plasmid for actin（pET-28a-A）was constructed and transformed into E.coli BL21(DE3). After IPTG induction, the recombinant proteins were highly expressed and contained in inclusion bodies. The recombinant proteins from inclusion bodies were purified by Nickel nitrilotriacetic（Ni-NTA）agarose affinity chromatography, and further dialyzed into a refolding buffer. The results of sodium dodecyl sulfate polycrylamide gel（SDS-PAGE）showed that the relative molecular weight of r-actin was 43 kD. In in vitro polymerization condition, the atomic force microscope（AFM）was used to observe and analyze the large-scale aggregate structure of r-actin during self-assembly. Our results showed that in vitro assembly of r-actin polymerized into complicated discrete structures, like filopodia-like fiber bundles, random coiled actin clusters, in addition to form disorganized protein aggregates. These data implicate that in vitro assembly of r-actin reflects its inherent thermodynamic properties of polymerization；further study would help to explore the regulatory function of actin binding proteins（ABPs）in the assembly of actin supramolecular aggregate structures.

The Construction of Rhizobia Engineering Strain of Introduction a Tandem Gene nodD-lba into Sinorhizobium fredii

Li Shanshan, Li Haiying, Yu Bing

2013, 0(8):  119-123. 

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Abstract: In this study, homology-based cloning was used to clone hemoglobin gene of lba and nodulation gene of nodD. The two genes were ordered joined into downstream of lac promoter and connected by the DNA recombinant technique to constructed expression vector pTR-P_lac-nodD-lba marked by luxAB gene. The vector was transformed into Sinorhizobium fredii with the method of triparental hybridization to construct genetic engineering strain. Western blot indicated the genes we used can express normally. Strains were inoculated soybean seedlings in order to get nitrogenase activity. The result showed engineering strains can improve the nitrogenase activity and the engineering strain with tandem genes can enhance nitrogenase activity 4% and 15.1%, respectively, by contrast with engineering strain with lba or nodD gene.

Screening and Identification of Antagonistic Actinomyces 1-g-59 Against Fusarium oxysporium f. sp. cubense

Liu Xiaoyu, Zhou Dengbo, Tan Xin, Gao Zhufen, Chen Bo, Huang Xiao, Zhang Xiyan

2013, 0(8):  124-129. 

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Five antagonistic strains against Fusarium oxysporum f. sp. cubense were isolated and screened from the rhizosphere soil of the banana garden in Meitai and Nanbao, strain 1-g-59 possessed the most prominent bioactivity. By studying the morphology, cultural, physiological and biochemical properties, cell wall components analysis, 16S rDNA and phylogenetic tree, the results showed that strain 1-g-59 was identified as Streptomyces lunalinharesii. Pot experiment showed that control effects of the fermentation filtrate of strain 1-g-59 on banana fusarium wilt disease was 64.9%.

Research Report

Construction of Recombinant Adenovirus Vector of Co-expressing MRP and EF Gene from Streptococcus suis Type 2

Wang Tao, Yu Hui, Mei Jun, Li Xingnuan, Zhou Xiaoou, Zhao Zhijun

2013, 0(8):  130-134. 

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The purpose of this study was to construct recombinant adenovirus vector of co-expression MRP and EF genes from Streptococcus suis type 2（SS2）. The specific primers were designed by the sequence of MRP and EF genes. The MRP（1 801-2 513）and EF（1 783-2 563）gene fragment were amplified from genomic DNA of SS2 by PCR technique. PCR products were cloned in pMD18-T vector. Then MRP gene, IRES element and EF gene linked into the adenovirus shuttle plasmid（pShuttle-CMV）to construct recombinant shuttle plasmid（pShuttle-CMV-MRP-IRES-EF）. The recombinant shuttle plasmid was lineared with PmeⅠand transformed into BJ5183-AD-1 competent cells to construct homogeneous recombinant adenovirus plasmid（rAdeno-CMV-MRP-IRES-EF）. Then the recombinant adenovirus plasmid was linearized with PacⅠand transfected into AD-293 cells. Finally, virus liquids of cell pack were identified by PCR. The results showed that MRP and EF genes were verified by gene sequencing. Both the recombinant shuttle plasmid and recombinant adenovirus vector plasmid were verified by enzyme digestion. Cytopathic effect（CPE）was observed after transfection linear rAdeno-CMV-MRP-EF 6 days. MRP and EF gene were also detected in the virus liquid by PCR.

Cloning and Expression of Helicobacter pylori Urease Subunit A and Its Immune Cross Protection Between Animal Antiserum and Patient Serum

Liu Wen, Tang Yanchao, Huang Rui, Cui Haiyan, Yuan Jinshan, Hu Wei

2013, 0(8):  135-138. 

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In this paper, immune cross protection from ureA antigen between rabbit antiserum and patient serum, infected by H. pylori, was tested. The ureA gene from H. pylori 26695 strain was amplified with PCR technology and cloned into expressive vector pBVIL1 and the recombinant pBVIL1-ureA was transformed into DH5α strain. The ureA antigen purified was used to immune rabbit three times and antiserum was acquired. The antiserum titre and immune cross protection were tested with ELISA. The results showed that the antigen expressed by prokaryotic system could specifically react on both patient serum and rabbit antiserum and possess a competitive immune cross reaction. As a result, it was concluded that animal antibodies against H. pylori may be applied in immune precaution and therapy of infections diseases from H. pylori.

Gene Cloning，Expression and Characterization of an HAP Phytase from Pseudomonas fluorescens 206

Li Yanan, Huang Huoqing, Yao Bin, Zhao Qing, Liu Kun, Ma Wenkang

2013, 0(8):  139-144. 

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A Pseudomonas fluorescens 206 strain producing HAP phytase was isolated from the bottom mud of artificial fish ponds in Jiaxin city. A novel HAP phytase encoding gene phyP was cloned by degenerate PCR and TAIL-PCR. The full length gene consist 1 284 bp and encodes 427 amino acids, including a putative signal peptide of 24 residues. The gene phyP was expressed in E.coli. The recombinant enzyme PhyP was purified to homogeneity by ammonium sulfalte precipitation and anion exchange chromatography. The optimal pH of recombinant PhyP was pH5.5, and the enzyme showed high stability at pH3.5-7.5. The optimal temperature of PhyP was found to be 45 ℃, and the enzyme had a higher activity at 25 ℃. These superior properties of PhyP make it advantageous for applying in feed industries, especially fisher industry.

Establishment of a Novel Prokaryotic Inducible Expression System

Liu Zheng, Gao Shuangcheng, Yang Shangjun, Sun Ning, Du Rui, Zhao Yanhong

2013, 0(8):  145-149. 

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Quorum sensing is a term to describe bacterial communications, coordination of population behaviors and regulation of gene expressions depending on population density. Marine luminous bacterium Vibrio fischeri utilizes this mechanism to synthesize signal molecules N-Acyl homoserine lactones（AHLs）which serve as co-inducers with a transcriptional activator protein（LuxR）to regulate the expression of a set of target genes in an extremely high level. Based on the hypothesis that chloroplasts of higher plants were derived from cyanobacteria in ancient times and that the transcriptional and translational mechanisms of chloroplasts are highly similar to that of prokaryotes, we constructed that chloroplast transformation vector of the reporter gene GFP under the control of LuxR-dependent activation of the lux operon. The construct was then transformed into Escherichia coli. When no signal molecule of AHLs was added to the colonies, no GFP expression was detected. However, when AHLs were added, GFP expression was strong enough to be detected by naked eyes under ultraviolet lamp, demonstrating that the combination of the GFP gene and the lux operon is an ideal chemical inducible gene expression system which can then be used for studies of gene function in Escherichia coli.

Screening of a Chitosanase-producing Strain Cellulophaga sp. and Optimization of the Fermentation Conditions

Sun Yuying, Zhang Jiquan, Wu Shiang

2013, 0(8):  150-154. 

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A chitosanase-producing bacterium M5 was isolated from Lianyungang sea area. It was identified as Cellulophaga sp. based on its morphological, physiological and biochemical properties and 16S rDNA sequence analysis and named Cellulophaga sp. M5. By single factors combination with orthogonal experimental, the optimum medium composition for producing chitosanase by Cellulophaga sp. M5 was as follows（g/L）：chitosan powder 10, yeast extract 3, ammonium sulfate 5, glucose 1.0, NaCl 5, MgSO₄ ?7H₂O 1.4, K₂HPO₄ ?3H₂O 1.4, the initial pH6.5. The optimum cultivation condition was to incubate in 250 mL flask installed with 70 mL medium of 1% inoculum size at 30℃, 180 r/min for 84 h. The maximal chitosanase activity could reach 6.67 U/mL when Cellulophaga sp. M5 was incubated in flask under the optimum cultural conditions. The activity increased by 1.5 times in comparision with initial activity of 2.67 U/mL.

Tolerance of Three Potentially Piglets Feed Probiotic and Antagonistic Pathogens Research

Sun Hongmei, Wang Tengfei, Li Piwu, Tang Weihua, Qu Lina, Wang Ruiming

2013, 0(8):  155-159. 

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It was to investigate the viability of Saccharomyces boulardii, Bacillus licheniformis and Lactobacillus plantarum in the acidic and bile salts conditions, three potentially probiotic role with the two pathogens E. coli and Staphylococcus aureus verify three probiotic antagonistic pathogens ability. The acidic and Bile salts conditions tolerance of the three potentially probiotic were determined by exposing washed cell suspensions to acidic conditions（pH1.0, 2.0, 3.0, 4.0 and 5.0）and different bile salts concentration[0%, 0.05%, 0.1%, 0.2%, 0.3%（W/V）]of physiological saline for 1, 2, 3 and 4 hours, reseparately, then three potentially probiotic separately with two pathogens 37℃ static co-culture 4 h, plate colony counting method for the determination of the probiotic viable cells. Result showed that Lactobacillus plantarum acid resistance is the most obvious, the Saccharomyces boulardii and Bacillus licheniformis also has good acid resistance. Bacillus licheniformis bile salts tolerance is the most obvious, the Lactobacillus plantarum and Saccharomyces boulardii also has good resistance to bile salts. These studies suggested that the three potentially feed probiotic have certain acid and bile tolerance capacity, possibly have a prebiotic effect in piglets role by antagonizing pathogens.

Glutathione Biosynthesis by Engineered Saccharomyces cerevisiae

Chen Jiali, Wu Liang, Wang Juan, Duan Xuehui

2013, 0(8):  160-165. 

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The GSH1 and GSH2 genes were amplified from the extracted total DNA of Saccharomyces cerevisiae S288c by PCR and expressed under the control of alcohol dehydrogenase(ADH1)promoter. With Kan^r or Hyg^r as the selective markers, two expression vectors YEplace181AK-GSH1 and YEplace181AH-GSH2 were constructed. The recombinant plasmids were transformed into an industry strain Saccharomyces cerevisiae TS013 respectively and simultaneously by electroporation, and three recombinant strains S.TS013/GSH1, S.TS013/GSH2 and S.TS013/GSH1+GSH2 were generated by screening on YPD plates supplemented with G418 or（and）Hygromycin. The transformants were cultured in fermentation media, afterwards intracellular GSH content was detected by the method of ALLOXAN. Compared with the content of control strain, the glutathione content of recombinant strains increased by 44.11%, 29.79% and 56.47%, respectively.

The Improved Intrasplenic Immunization and Semi-solid Medium to Prepare Monoclonal Antibody

Ren Ruimin ^{Wang Yunlong, Zhang Yiqing, Li Hengsi, Wang Guoqiang, Li Yulin, Wang Jichuang}

2013, 0(8):  166-169. 

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It was to utilize the improved intrasplenic immunization and semi-solid culture medium to reduce the immune dosage, clone the hybridoma simply, and shorten the period of cycle. The skin areas of mice spleen was anesthetized, then the spleen was injected with trace antigen, which could be immuned in a short period of time, cell fusion was prepared after intravenous injection. The three different methods（limited dilution, methylcellulose, soft agar）were used to get hybridomas which can secret monoclonal antibody. Results showed that compared with the traditional method of preparation of monoclonal antibody, the improved intrasplenic immunization was simpler operation, without the addition of adjuvant, and the high titer of antibody was getted in a short time. Positive rate was the highest when methylcellulose concentration in the 1.1% condition. It was suggested that these methods could be suitable for large scale preparation of monoclonal antibody.

Cloning and Construction of Expression Vector of Human Lysophosphatidic Acid Receptor LPAR1 and Its Transient Transfection into 293T Cell

Li Tiewei, Zhao Pengfei, Ma Jie, Alatan Gaole

2013, 0(8):  170-175. 

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To detect the over-expression of LPAR1 and the accumulation of cAMP in transfected 293T cells, human LPAR1 gene was cloned and the LPAR1 expressing vector was constructed. LPAR1 CDS sequence was cloned from human gastric cancer cells and sub-cloned into the pCR2.1 vector. By sequencing, digestion and ligation, LPAR1 was directionally cloned into eukaryotic expression plasmid pIRES2-EGFP. The reconstructed plasmid was identified with enzyme digestion and sequencing；the plasmid was transfected into 293T cells with lipofectamine2000. Real-time PCR was performed to assess exogenous LPAR1 expression in 293T cells. ELISA was used to detect the activity of LPAR1. These results indicate that the expression vector containing human LPAR1 gene has been established and LPAR1 gene has also been over-expressed successfully in 293T cells. Through the signaling pathways downstream of LPAR1 mediated under the stimulation of LPA demonstrates the activity of the cloned gene expression of LPAR1 protein. The establishment of LPAR1-overexpressing cell model, not only provides the foundation to research the function of LPAR1 in the next step, but also make it possibly for quantitative the concentration of LPA through the linear relationship between LPA and cAMP.

Separation of Plasma Membrane from Nasopharyngeal Carcinoma Cell Line 6-10B and Preliminary Analysis of Its Protein Profile

Zhang Pengfei, Zeng Guqing^，, Tang Can’e, Yi Hong, Liang Songping, Chen Zhuchu, Xiao Zhiqiang

2013, 0(8):  176-183. 

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To establish plasma membrane（PM）protein profile of 6-10B nasopharyngeal carcinoma（NPC）cell, and accumulate valuable data for understanding the carcinogenesis mechanism of NPC. First of all, two-phase partition in combination with differential velocity centrifugation was used to purify PM of 6-10B cell. Then, purified PM were detected the purity by Western blot and electron microscopy. Next, SDS-PAGE was used to isolate PM proteins. After that, PM proteins were identified using mass spectrometry and bioinformatics. Finally, identified PM proteins were determined relevance with other tumors in human cancers database（dbDEPD）searching. A total of 406 proteins of 6-10B cell were identified, and 255 proteins were PM and membrane-associated proteins（accounting for 62.8%）. 46 kinds of proteins in 145 identified PM proteins were tumor-related proteins, of which 18 proteins associated with tumor metastasis. Further analysis found that CD225, CD104 and p63 were oral cancer（belong to head and neck cancer）metastasis related proteins, suggesting that they may be potential metastasis associated proteins in NPC. A PM protein profile of NPC 6-10B cell has been established, which provides useful information to investigate carcinogenesis mechanism of NPC.