Table of Content

14 November 2013, Volume 0 Issue 11

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Review

Progresses on the Studying of Rice Leaf Albino

Zhang Hongzheng, Cheng Zhijun, Wan Jianmin

2013, 0(11):  1-7. 

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Rice leaf albino is a kind of common mutation, caused by chlorophyll deficit or impaired chloroplast development. Non-lethal Rice leaf albino mutant provide important materials on revealing photosynthetic mechanism, and available germplasm to genetic breeding. With the continuous development of molecular biology, more and more rice genes are being cloned, and the mechanism of pigment metabolism is gradually clear. This review briefly summarized current progresses in rice leaf albino study, involving in their origination, cytological characteristics, molecular mechanism and potential values in breeding utilization.

Research Progress in Plant Rboh Genes Function and Activity Regulation Mechanism

Liu Qiuyuan, He Haohua, Hu Lifang

2013, 0(11):  8-13. 

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Plant respiratory burst oxidase homologue is also named NADPH oxidase，which is the homologue of mammalian phagocyte gp91^phox -the main catalyze subunit of NADPH oxidase complex. Plant Rboh genes play a significant role in plant development and stress response，and its activity is regulated by Ca²⁺，protein phosphorylation，Rac protein and ABA. This article provides an overview of the plant ^phox genes function and its activity regulation mechanism based on the current researches.

Research on nrDNA ITS Pseudogenes in Plants

You Huan, Zhou Atao, Yue Liangliang, Cun Dongyi, Li Min, Ding Yuanming

2013, 0(11):  14-18. 

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The internal transcribed spacer(ITS)of nuclear ribosomal DNA has been widely used for re-constructing phylogenies. In recent years, the occurrence of ITS putative pseudogenes is well documented for many groups of plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part. In this review, we briefly summarize the formation mechanism of pseudogenes, characteristics of ITS pseudogenes and the applications of the ITS pseudogenes on the phylogenetic study. In addition, the future prospect of scientific researches on ITS pseudogenes was viewed.

Progress in Research of Dehydrins Response to Abiotic and Biotic Stresses

Dai Jinran, Ye Hui, Chen Suiyun

2013, 0(11):  19-25. 

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Plant dehydrins, or group 2 late embryogenesis abundant proteins, are highly hydrophilic disordered protein. They play a fundamental role in plant response and adaptation to water stress. Dehydrins typically accumulate in maturing seeds or vegetative tissues under salinity, dehydration and cold stresses. Based on the conserved motifs, dehydrins are classified into five sub-classes. The K segment, which represents a highly conserved 15 amino acid motif, can be found in all dehydrins. Since the first dehydrins were discovered in 1980s, the important roles of dehydrins during cellular dehydration causing by abiotic stresses were widely discussed, but their precise function remains unclear. Recently, it has been reported that in addition to the contribution in water stress, dehydrins have a pleiotropic effect on biotic stress response. They may interfere with JA signal pathway, therefore dehydrins may relate to defense response. This review outline the current structural, physico-chemical and functional characterization of plant dehydrins.

Research Progress of Rhizopus in Fermentation Industry and Environmental Science

Liu Jinmei, Zhang Fengying

2013, 0(11):  26-33. 

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Rhizopus is a genus of common fungi and widely exists in nature which is the domain strains in traditional food fermentation since thousands of years. In recent years, people found that they also can be used to produce many metabolic products used for consumers demand under different material and special conditions, such as L-lactic acid, L-malic acid, fumaric acid, lipase and other metabolic product. Its metabolites were widely used for food, medicine, environmental protection and other industries. As the special metabolites were found continuously, the related products research and development is becoming more and more concerned. This paper mainly introduced the research progress of Rhizopus in fermentation industry and environmental science.

Advance in Breeding of High Tylosin Producing Strain

Huang Wei, Xu Chunyan, Niu Chun, Zhang Ping, Su Jianyu

2013, 0(11):  34-39. 

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Tylosin is a kind of 16-numbered macrolide antibiotic which is exclusively applied in animals. As its and it could，Tylosin is widely used in livestock breeding because of its property of broad spectrum antibiotic and function of promoting animal growth. Breeding of high-yield strain is the emphasis of tylosin fermentation research. Traditional breeding methods such as physical and chemical mutagenesis are simple and mature. However，the efficiency of mutagenesis is very low，and the subsequent screening process is tedious. With the development of genetics and molecular biology，it becomes an efficiency method by using directed evolution of molecular breeding technology to improve the production capacity of tylosin. In this paper，we focused on the molecular breeding progress and combined different breeding methods to review the progress in breeding of high tylosin producing strain in recent years.

Terminase and Its Function in Packaging Process of Bacteriophage

Song Shaoyun, Jia Yanhua, Zhu Xinzhou, Pang Yi

2013, 0(11):  40-45. 

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Terminase of bacteriophage which contains small subunit and large subunit is a family of multifunctional oligomeric protein. It’s important for the packaging. Large subunit is a multifunctional enzyme and plays a major role in the packaging. Small subunit specific binds the genomic DNA of bateriophage and guides the process of packaging. This paper reviews the position of the terminase, structure, function and the role in packaging process.

Technique and Method

Application of Proteomics Technology in Study of Cell Signaling Transduction

Wang Weipeng, Miao Fangfang, Wu Dandan, Yang Jun, Wang Zhigang,

2013, 0(11):  46-50. 

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Proteomics allows for the exploration of the proteome by looking at the intracellular protein interaction and its own unique activity pattern. This opens the door to studying the cellular signal transduction with new tools and techniques. The application of these new methods, such as two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry, as used in proteome quantification and identification, have promoted the study of the cellular signal transduction effectively. This is seen more so in the research of protein interaction, molecular chaperone functioning, present passway crosstalk, and the dynamic activities of cellular signal transduction. This review presents proteomic applications and a number of the outstanding achievements accomplished in cellular signal transduction research as well as a prediction of the prospective application of these findings in the future.

Research Report

Cloning and Bioimformation Analysis of a Serine / Arginine-Rich Protein Gene SCL25 in Saccharum spp.

Liu Juan, Wu Jiayun, Liu Shaomou, Deng Zuhu, Liao Zhenyang, Fu Cheng, Li Hu, Huang Zhongxing, Lin Yanquan, Chen Rukai

2013, 0(11):  51-57. 

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A novel gene of Serine/Arginine-rich proteins, expressed in the inflorescence of Saccharum spp., was cloned. The gene with full-length of 960 bp, encoding 209 aa, might belong to the SCL subfamily of SR protein, inferred by alignment of SR protein members, named ScSCL25(gb：JQ031566). The gDNA sequence is 2 966 bp and contains five introns. Bioinformatics analysis shows that ScSCL25 protein is an unstable hydrophilic protein. There were 33 serine and tyrosine kinase phosphorylation sites. Neither signal peptides nor transmembrane domains been found in this protein sequence, which lead to prediction that it is localized in the cell nucleus. In the space structure, the ScSCL25 contained the typical RRM and RS domain of SR protein, showed high homology to the SR proteins reported in Zea mays.

Cloning and Sequence Analysis of a Chewing Cane Sucrose Synthase Gene(SoSuS1) and Investigation of Expression Profiling

Li Heping, Li Haiming, Pan Shiming

2013, 0(11):  58-62. 

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In plants, sucrose synthase is one of key enzymes for urging the sucrose into various metabolic pathways. Two EST sequences which were highly similar to sucrose synthetase genes were screened from GenBank, and the corresponding cDNA was cloned by the technologies of RACE. The cloned sucrose synthase cDNA, named SoSuS1 was 2 970 bp long(GenBank accession No. JX416283), containing the complete coding sequence(2 448 bp). The result indicated that SoSuS1 protein is a typical soluble synthetase in the cytoplasm by homoge-neous comparing of its amino acids sequences. Gene expression analysis showed that SoSuS1 was expression in stem, leaf sheath and leaf, indicating the gene plays a role in multiple tissues. The expression of SoSuS1 gene was highest in stem, and the stem was possible the main action site.

Overexpression of cyFBPase Gene Can Enhance the Drought Tolerance of Transgenic Tobacco

Guo Lina, Zhang Rui, Sun Guoqing, Meng Zhigang, Zhou Tao, Guo Sandui

2013, 0(11):  63-68. 

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Osmotic adjustment is mainly functioned under drought stress in plant. Sucrose as one kind of important soluble sugar in plant cells, is a primary osmotic regulator in plants, and its synthesis is limited by some key enzymes, such as cyFBPase. In this work, the Brassica napus cyFBPase gene was transformed into tobacco, and the gene expression level and enzyme activity were detected. The cyFBPase gene expression and enzyme activity were increased significantly in transgenic plants, with 1.88 times and 1.55 times of the wild type controls respectively, and at the same time, the sucrose content in transgenic plants leaves was also significantly increased to 1.59 times than that of the wild type. Under the condition of drought, the transgenic tobacco plants showed high drought resistance, and the sucrose content and the distribution of sucrose in root and leaf organs changed significantly. In transgenic plants, the sucrose content in the root and leaves increased by 2.49 times and 9.41 times of the plants which were treated one day, and increased by 1.78 times and 2.71 times of wild type controls under the same drought treatment. In conclusion, overexpression of cyFBPase gene can improve the ability of sucrose biosynthesis of plants, promote the sucrose transportation from leaves to roots under drought stress, and enhance the tolerance of transgenic plants to drought.

Isolation and Characterization of PtoMYB148 Transcription Factor Expressing Preferentially in Developing Xylem in Populus tomentosa

Cheng Zhanchao, Chen Yajuan, Zhang Jiewei, Wang Hongzhi, Wei Jianhua

2013, 0(11):  69-74. 

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As one of the plant’s largest transcription factor families, MYB transcription factors play important roles in transcriptionally regulating the synthesis of secondary cell wall, which is composed of cellulose, hemicellulose, and lignin. By uncovering transcription factors regulating secondary cell wall biosynthesis, it may be possible to develop novel strategies for genetic improvement of wood. In this study, the full-length PtoMYB148 cDNA was cloned in Populus tomentosa. PtoMYB148 exhibited a high level of expression in the developing and mature xylem. Phylogenetic analysis showed that PtoMYB148 was closely related to AtMYB103, AtMYB050 and AtMYB086 from Arabidopsis, among which AtMYB103 was shown to regulate secondary cell wall synthesis. Yellow fluorescent protein(YFP)-tagged fusions of PtoMYB148 was found to be targeted to the nucleus when expressed in Arabidopsis leaf protoplasts. Transcriptional activation analysis demonstrated that PtoMYB148 was able to activate the expression of the His3 and β-Gal reporter genes in yeast. Taken together, these findings showed that PtoMYB148 functions as a transcription factor, and that it may play roles during wood formation in Populus.

Isolation and Purification of Protoplast Cell from the Leaves of Torreya nucifera

Zhang Yunxuan, Miao Jiguang, Xie Mingchun, Tian Song, Dong Mingzhe, Jiang Guoyong

2013, 0(11):  75-78. 

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Isolation and purification of protoplast cell from the leaves of T.nucifera may exert great economic value for further suspension construction of somatic asexual cell and obtain secondary metabolites-kayaflavone. Enzyme methods was used to isolate the cell of T.nucifera leaf as materials successfully obtained protoplasts. The results showed that the suitable concentration for protoplast cells were：CPW+Cellulase(R-10)3.0%-6.0% and Macerozyme(R-10)5.0%-6.0%. The concentration 30%-44% sucrose obtained was employed for cell centrifugation in protoplast purification. The purified protoplast could keep on well-growth status for sustainable culture in the medium with 0.7 mol/L mannitol+50 mmol/L MES+0.5% PVP.

Polymorphism and Bioinformatics Analysis of CDS in GDF-10 Gene in Tianzhu White Yak

Li Tianke, Liang Chunnian, Lang Xia, Pei Jie, Wu Xiaoyun, Liu Jian, Qin Wen, Yan Ping

2013, 0(11):  79-85. 

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Mutation site in coding domain sequence of GDF-10(Growth differentiation factor -10)gene in Tianzhu white yak was checked by DNA pooling method, and the polymorphism and the characters of amino acid sequence were analyzed by bioinformatics. The results showed there were eight mutation(90C→G, 124C→G, 132T→C, 822A→G, 873G→T, 888T→C, 1005G→A, 1232G→C)at sequence of CDS, four synonymous mutation(90C→G, 124C→G, 1005G→A, 1232G→C)and four missense mutation(132T→C, 822A→G, 873G→T, 888T→C). NO obvious hydrophobic and transmembrane helical region, signal peptide was found in deduced amino acid sequence. GDF-10 proteins would play biological function mainly in the cytoplasm. The putative secondary structure was mixed type primarily composed of α-helix and β-extended. The deduce amino acid sequence of GDF-10 between yak and bovine, mouse and rat were high homology, which was same as their genetic relationship.

Cloning and Bioinformatics Analysis of A-FABP Gene in Yak

Qin Wen, Yan Ping, Pei Jie, Wu Xiaoyun, Li Tianke

2013, 0(11):  86-92. 

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A-FABP(Adipocyte Fat Acid Binding Protein)gene of yak was used to design a pair of specific primer based on the A-FABP mRNA sequences included in NCBI GeneBank. The CDS sequences of yak A-FABP gene was cloned by reverse transcription polymerase chain reaction(RT-PCR)from yak longissimus dorsi muscle, and were analyzed. The results showed that, the length of CDS in yak A-FABP mRNA was 399 bp and encoding 132 amino acid, which encoded a hydrophilic protein. The secondary structures were mainly composed of the helix, β turn, extended strand and random coil, containing 11 phosphorylation sites. The A-FABP and realated proteins of Bos grunnien, Bos taurus and Bubalus bubalis shared the highest homology of 97%. A-FABP amino acid sequences are preferably conserved on the evolutionary relationships, and also showed high homology of with other species. The A-FABP features cytosolic fatty-acid binding family protein functional domains, no signal peptide.

Separation and Culture of Beijing Duck Epidermal Stem Cells

Lü Chunrong, Xiong Hui, Gong Xuelian, Guan Weijun, Ma Yuehui

2013, 0(11):  93-97. 

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This experiment was conducted to study the separation methods and the biological characteristics of epidermal stem cells(ESCs). Beijing Duck epidermal stem cellswere respectively obtained by enzymic digestion and tissue piece culture, then the cultured cells were identified by immunohistochemistry. We detected the apoptosis of ESCs and studied the transfection efficiency of exogenous genes including pEGFP-N3、pEYFP-N1 and pDsRed-N1. Results showed that the ESCs obtained from the two methods both exhibited adherent growth type, small volume, high nucleus-cytoplasm ratio, high refractivity and transparent cell appearance. Immunofluorescence assay indicated that Beijing Duck ESCs expressed β1 integrin and K19, not expressed K10. The results of apoptosis showed that the apoptosis rate was only 6.5%. The study of transfection experiment indicated that the amount of positive cells and its intensity increased after 48-96 h, among which, 48 h group had the most positive cells with the transfection efficiency up to 56%. The fluorescent protein gene(pEGFP-N3, pDsRed-N1 and pEYFP-N1)expression had no obvious effect on the growth and proliferation of the ESCs. The transfection efficiency was different in the different fluorescent genes. These results indicated that the cultured cells were indeed ESCs. The cells cultured in vitro owned low apoptosis ratio, and can be used in the research of exogenous genes transfection.

Bioinformatic and Functional Analysis of Ectodysplasin-A from Common carp(Cyprinus carpio，Cyprinidae)

Wang Yangyang, Jiang Li, Cheng Anda, Zhang Baoyong, Ma Long Li Hengde, Sun Xiaowen

2013, 0(11):  98-104. 

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The Ectodysplasin-A(Eda)plays vital roles in developmental events of the skin appendages. The screening of transcripts of skin from common carp and the Germany mirror carp by Genefishing differential display, found Eda is one of the targeted different expressed in both materials, and the partial fragment of Eda gene was recovered from differential expressed products by sequencing. The specific primers contained the full length of Eda were designed with the reference sequence of Eda EST in our Carp database. The mirror carp Eda CDS consisted of 1 062 base pair nucleotides which encodes a 354 amino acid protein, has a receptor-binding region. The alignment result showed that the carp shareed the highest similarity(92.76%)with the zebrafish. However, it had the relative lower similarity with chicken(46.61%), human(50.51%), mouse(50.76%)due to evolutionarily distant relationships. The 5'-UTR regulation region of Eda shared the high similarity with the zebrafish(92.98%), but varies significantly with human(only 23.36%), mouse(31.07%). The in situ results showed that the Eda unique expression signal was detected in skin matrix which the scale formed, however weak or no expression signal was detected in other positions on skin, later disappeared in full developed scale body. All of these results showed that this gene may involve the initiation of the scales not the maintainence of the scale pattern formation.

cDNA Cloning and Prokaryotic Expression of NCCRP-1 from Grass Carp Ctenopharyngodon idellus)

Cai Jia, Dai Liping, Wang Bei, Tang Jufen, Huang Yucong, Lu Yishan, Wu Zaohe, Jian Jichang

2013, 0(11):  105-111. 

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In the present study, grass carp(Ctenopharyngodon idellus)NCCRP-1 gene was cloned using RACE method. The full length cDNA of NCCRP-1 was 900 bp containing a 714 bp open reading frame(ORF), which encoded 237 amino acids with predicted molecular mass of 27.3 kD and a theoretical pI of 5.63. Amino acid alignment indicated that grass carp NCCRP-1 possessed consensus functional domians of NCCRP-1 family and shared 88% similarity with common carp NCCRP-1. The prokaryotic expression vector pET-NCCRP was constructed and then transformed into BL21(DE3). SDS-PAGE and Western blotting analysis indicated that the recombinant protein of grass carp NCCRP-1 was successfully expressed and molecular weight of expressed fusion protein was 47.8 kD.

Fluorescence-activated Cell Sorting(FACS)of Fluorescently Tagged Cells from Transgenic Zebrafish Larvae for Gene Expression Analysis

Chen Sijie, Zhang Hefei, Zhang Cuizhen, Peng Gang

2013, 0(11):  112-116. 

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It was to optimize commonly used FACS protocols to apply to fluorescence-labeled neurons of zebrafish. lhx5:Kaede transgene line as an example was used to describe a successful protocol for isolating and expression-profiling live fluorescent-protein-labeled neurons from transgenic zebrafish brains. Numerous information of lhx5 labeling cells can be obtained by RNA profiling and would contribute to further studies. Results showed that on average, 6% of the single cell suspensions was keade-positive. The RNA extracted from selected cells was sufficient quality and quantity for microarray analysis. Microarray data showed the enrichment of both lhx5 and other molecular makers. It proved that this protocol for dissociating and sorting fluorescent-labeled neurons from central nervous system of zebrafish larve, is applicable for gene expression profiling of the target cell groups.

The Effects of Exogenous Thyroid Hormone on Gene Expression of IGF-I and IGF-IRs in Paralichthys olivaceus

Zhang Junling, Shi Zhiyi, Cheng Qi, Fu Yuanshuai

2013, 0(11):  117-123. 

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In Paralichthys olivaceus, the gene expressions of insulin-like growth factor I(IGF-I)and its receptors after thyroid hormone(TH)treatment were analyzed using real-time quantitative PCR, by administration of TH to premetamorphic larvae and cultured embryonic cells. The results in vivo and in vitro showed that the level of IGF-I mRNA dropped significantly after TH treatment, and it was up-regulated in thiourea(TU)treated premetamorphic larvae. The two IGF-IR mRNAs exhibited different expression patterns after TH treatment. The exogenous TH stimulated the expression of IGF-IR-1 mRNA, but obviously decreased the levels of IGF-IR-2 mRNA. These results reveal that TH and IGF systems are highly interrelated and provide the basic information to clarify the hormone regulation mechanisms of metamorphosis in Paralichthys olivaceus.

Segregating Pattern of AFLP Marker in G₁ Family of the Mud Crab(Scylla paramamosain)

Li Shujuan, Ma Hongyu, Ma Chunyan, Jiang Wei, Feng Nana, Xu Zhen, Liu Yuexing, Qiao Zhenguo, Ma Lingbo ¹

2013, 0(11):  123-129. 

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In this study, the segregating pattern of AFLP(amplified fragment length polymorphism)and the genetic diversity of G₁ family of the mud crab(Scylla paramamosain)was investigated using 32 selective primer combinations. A total of 1 128 loci were produced, of which 376 loci were polymorphic(11.8%), and 752 loci were monomorphic. Among 376 segregating loci, 229 loci(60.9%)segregated in Mendelian ratio while 147 loci(39.1%)segregated in distortion estimated by Chi-square test(P=0.05)．There were 259(68.9%)and 117(31.1%)loci segregating in the ratios of 1:1 and 3:1, respectively. The number of loci segregated in Mendelian ratios were 155(59.8%)for 1:1 and 74(63.3%)for 3:1, respectively. Effective number of alleles, Shannon genetic diversity index and Nei’s gene diversity index were between 1.13 to 1.73, between 0.10 to 0.56, and between 0.07 to 0.40, respectively.

Research Report

Prokaryotic Expression and Application of Recombinant Human Serum Albumin

Li Xiangrong, Feng Ruofei, Xie Jingying, Tian Wei, Yang Yanmei, Wang Dan, Ma Zhongren

2013, 0(11):  130-135. 

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This study was aimed to express and purify the protein of pGEX-4T-1-rHSA in prokaryocytes and prepare anti-HSA polyclonal antibody. The HSA gene was amplified from human embryo lung diploid total RNA by RT-PCR, with primers based on the published HSA(NM_00047.5)sequence in GenBank, then it was cloned into prokaryotic expression vector pGEX-4T-1 to construct a recombinant expression vector pGEX-4T-1-rHSA. The pGEX-4T-1-rHSA was transformed into E. coli BL21(DE3). The purified expressed protein was injected into a rabbit and the polyclonal antibody was then prepared, which were identified by the indirect ELISA and Western-blot analysis. Results showed that the recombinant protein mainly exists in the inclusion body, about 92 kD and it has good reactogenicity and immunogenicity. The ELISA titer of the antiserum was approximately 1:100 000. Western-blot analysis revealed that the purified polyclonal antibody against pGEX-4T-1-rHSA had a specific affinity for natural human serum albumin and the expressed protein.

A Study of Ig Promoter Effectively Triggering the Expression of Human Hyperplasia Suppressor Gene

He Zhiqiao, Wu Lina, Zhu Xiaohui, Ma Teng, Qiu Xiaoyan

2013, 0(11):  136-141. 

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Immunoglobulin(Ig)promoters have high promoter activity in a variety of epithelial tumor cells. In this study, PCRs were used to obtain the Ig heavy chain VH6-1 promoter. By replacing the leukemia virus promoter of cytomegalovirus(CMV)sequences of the eukaryotic expression vector(pDC316)with VH6-1 promoter, we constructed the vector named Ig promoter/human hyperplasia suppressor gene(hHSG)/PDC316. Compared with the CMV promoter, Ig promoter triggered hHSG expression more effectively, and expression of hHSG inhibited tumor cell growth and induced apoptosis. These results indicated that the Ig promoter could be used as a tumor targeted therapy vector promoter, which will have a good prospect of application in cancer gene therapy research.

Protective Effects on Dermal Fibroblasts Under the UVA Oxidative Stress by Lycopene

Yang Na, Li Ming, Hu Jiadong, Jiang Tao, Gong Sijia, Zhang Jian

2013, 0(11):  142-147. 

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The aim of this study is to test the protective effects of lycopene on the dermal fibroblasts under UNV oxidative stress. The model used in this experiment was established by exposing the mouse dermal fibroblasts to UVA(5 J/cm²)in X min and the cell proliferative activity was detected by MTT method. The activities of superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GSH-PX)and malondialdehyde(MDA)were also tested to illustrate the protective effect of lycopene. Results showed that the mortality of dermal fibroblasts was improved by subjecting the lycopene to the culture while the cells exposure in the UVA and the activity of SOD, CAT and GSH-PX increased significantly while the the level of lipid hydroperoxide, MDA decreased. In general, lycopene can protect the dermal fibroblasts against the UVA radiation by improving the activity of enzymes which involved in the clearace of oxygen radicals.

Molecular Cloning and Analysis of the 3 ' UTR of Porcine Kobuvirus 441 by 3 ' RACE

Wang Enli, Lan Xi, Liu Wei, Yang Bin, Liu Jixing, Ma Xiaojun

2013, 0(11):  148-152. 

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The special primers of porcine kobuvirus(PKV)were designed according to the sequence logged in GenBank. The 3D gene was amplified by RT-PCR and 3' RACE. The sequence analysis showed that the amplified specific sequences were 1 064 bp, 1 046 bp and 592 bp in length respectively, including 3A，3B，3C，3D region, 3'untranslated region(3' UTR)and at least a 29-poly(A)tail. The 3D region was 1 407 bp in length, encoding 468 amino acid(aa)with a molecular weight of 53 369.29 D, pI of 6.12. The 3' UTR was 166 bp in length after the terminator codon TGA. The aligning analysis showed that the 3D gene of swKoV CH441 shared high nucleotide acid and amino acid identity with the other porcine kobuvirus strains sharing 92.2% to 93.4% and 97.9% to 99.4%, respectively. In the phylogenetic tree, swKoV CH441 was closer with Chinese strains but further with Hungarian strains.

Construction of Adenovirus Vector Containing Rat β-catenin or ΔN90 Gene and Its Expression in MSCs

Cui Hengjin, Wang Chunyan, Hu Jun, Xie Guiqin, Xu Xiaojing, Zhang Huanxiang

2013, 0(11):  153-158. 

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To construct the recombinant adenovirus vector containing β-catenin or ΔN90 gene for later research of the mechanism of β-catenin in MSCs migration. β-catenin or ΔN90 cDNA was inserted into a pAdTrack-CMV transfer vector, linearized with PmeⅠdigestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination adenoviruse Ad-β-catenin or Ad-ΔN90. Then they were linearized and transfected into QBI-293A cells by Lipofectamine^TM2000. Through PCR, endonuclease cutting and gene sequencing, the target gene was verifed to be correctly cloned in adenovirus vector. The high expression of green fluorescence protein in QBI-293A cell line was found under fluorescent microscope. Compared with control group, the β-catenin expression was obviously increased 48 h after infection with Ad-β-catenin or Ad-ΔN90. TOPFlash assay confirmed that Ad-β-catenin and Ad-ΔN90 results in a dramatic increase in signaling activity. The recombinant adenovirus vectors of β-catenin and ΔN90 were successfully constructed and the adenovirus was packaged in QBI-293A cell line.

Cloning，Identification and Eukaryotic Expression of ORF6 Gene from Highly Pathogenic PRRSV Strain TA-12

Wang Tongtong, Wang Xiaofei, Li Xin'an, Tian Guoning, Liu Litao, Wang Guangwen, Li Ning, Wang Fangkun, Li Hongmei, Zhao Xiaomin, Xiao Yihong

2013, 0(11):  159-163. 

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To obtain the efficient expression of ORF6 proteins of HP-PRRSV strain TA-12, the eukaryotic expression plasmid PfastBac HTB-ORF6 was constructed and transformed into DH10Bac^TM to get Bacmid-ORF6. The recombined plasmid was screened and identified by blue-white color selection and PCR using specific primers. Then the Bacmid-ORF6 was transfected into sf9 cells for expression. The results showed that the protein was expressed and the highest expression amount of 0.05 μg/μL was found at four days post infection using Western blot, and the expressed protein was mainly located in cytoplasm. The successful expression of the ORF6 protein has laid good foundation for the study of its potential function in the process of PRRSV infection.

Study of Changes in Abundance and Degradation Crude Oil Capacity of Endogenous Bacteria and Andexogenous Bacteria Activation in Oilfield Under Injecting Activation Agent Conditions

Wang Dawei, Zhang Jian, Ma Ting, Qi Yibin

2013, 0(11):  164-169. 

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It was study the structure change of exogenous and indigenous microbial population during activation of stratal microflora under injecting activation agent conditions, and the effect on the microbial degradation crude oil capacity. The water sample was from A18、B16 well produced water in NB35-2 Oilfield. The bacteria in the water sample under injecting activation agent conditions was activated , and samples at different time intervals were collected. These samples were analyzed by denature gradient gel electrophoresis(DGGE), and the effect of addition of activation agent on microbial degradation crude oil capacity were studied through determination of viscosity and four fractions contents analysis of crude oil. Based on the analysis of the number and lightness of DGGE bands, we studied the change of the microbial community diversity activation of stratal bacteria. The species number and total quantity of dominant microbial population were not abundant under oil reservoir conditions. After injecting activation agent, some bacteria grew and reproduced quickly along with the enhancement of nutrition condition, and the structure of dominant microbial population changed. Exogenous bacteria alone can not effectively activate endogenous bacteria, and after adding nutrients, the species number and total quantity of endogenous bacteria significantly increased, while the performance of degradation to crude oil of exogenous bacteria and endogenous bacteria was significantly enhanced.

Activities of an Oil Degrading Bacterial Strain HB-2

You Jing, Li Qing, Liu Yang, Wu Gang, Wang Guan, Lai Fengli, Li Yongbin, Duan Lisha, Cao Yanhua

2013, 0(11):  170-175. 

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An oil degrading strain HB-2 was isolated from the produced liquid of oil wells in Huabei Oilfield, which used crude oil as sole carbon. The strain was identified as a new species of the genus Luteimonas by the method of polyphasic identification. Through the reaction with crude oil, we find the viscosity-reducing rate is 47.3% and the oil-water interfacial tension decrease by 38.7%, in addition, the strain can also produce organic acid and surfactant. According to analyzing components of crude oil, we find the strain can improve the fluidity of crude oil by increasing light component of oil. According to the result of indoor model test, we can find the recovery ratio is increased by 15.4%.

Screening and Identification of an Actinomycete Strain with Nematicidal Activity

Huang Huiqin, Yuan Weidao, Wei Hua, Wang Ying, Zhu Jun, Sun Qianguang, Bao Shixiang

2013, 0(11):  176-180. 

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Root-knot nematode antagonistic strain HA11097 was isolated from mud samples collected from Dongzhaigang mangroves in Hainan province. The mortality rates of the fermentation broth against root-knot nematode were 54.2% and 46.7% while diluted 20-fold and 40-fold, respectively. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain HA11097 should belong to a member of the genus Streptomyces, and closely related to S. aculeolatus, with the highest 16S rRNA gene similarity of 98.9%. In the phylogenetic tree, HA11097 was the closest relationship with S. aculeolatus, which both located on the same branch. Their DNA-DNA hybridization value was 94.4%. The morphological, cultural characteristics and the results of physiological and biochemical properties of the strain were in well agreement with S. aculeolatus. On the basis of these data, strain HA11097 was identified as Streptomyces aculeolatus. The article first reported that the strain of anti-nematode activity.

Optimization Conditions of Fermentation and Separation and Purification of Reduced-Glutathione

Wang Shuo, Xie Yuewu, Zhang Huiwen, Yan Haolin, Xu Mingkai

2013, 0(11):  181-185. 

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The aim of the study is to provide theoretical basis in GSH large-scale production, by optimizing the conditions of its fermentation and purification. Firstly, a GSH high-yield Saccharomyces cerevisiae Y18 was obtained, with the production of 9.05 mg GSH per gram dry cells. Secondly, our results indicated that adding L-cysteine and Valine to the medium, or additional glucose during fermentation process could increase GSH production by 62.0% and 146.1%, respectively. Moreover, the weak acidity condition(pH4.5)and nitrogen input protected GSH from oxidation, and improved its stability. Finally, we obtained high-purity(98.1%)GSH and a promising recovery(73.2%)after optimizing the conditions for separation and purification GSH in Macroporous resin D001×7 and Sephadex G-10 gel chromatography.

An Analysis of Knowledge Structure Evolution on Biological Breeding Field

Hao Xinning, Sun Wei, Zhang Xuefu

2013, 0(11):  186-192. 

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Biological breeding technology has undergone a long development. From the earliest traditional cross breeding method develop to modern biological techniques, which consist of genetics, cell biology and bio-engineering basic theories. Biological breeding research field includes crop breeding and plant breeding research area. The evolution process of knowledge structure between 2008 and 2012 in biological breeding field was analyzed in this research. Described and displayed in details on the changes of research topic, co-author and country cooperation networks. Finally, development foreground and research directions were discussed.