Biotechnology Bulletin

Progress in Induced Pluripotent Stem Cells

Song Weihua, Liu Kun, Zhao Tongbiao

2014, 30(5): 1-7.

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Enforced ectopic expression of transcription factors Oct4、Sox2、Klf4 and c-Myc can reprogram matured somatic cells into pluripotent state, leading to the generation of induced pluripotent stem cells(iPSCs). Similar as embryonic stem cells(ESCs), iPSCs have the characters of self-renewal and pluripotency. Comparing to ESCs, iPSCs can not only provide unlimited cell source for regenerative medicine, but also hold promise to override the ethical concerns and immune rejection problems correlated with clinic development of ESCs. Here we gave a brief review on the development of iPSC technology, reprogramming mechanisms and clinic development of iPSCs.

Advances of ABA Related Genes on Plant Drought Tolerance Gene Engineering Studies

Tian Xiaowei, Peng Shouhua, Wei Jiqiang, Jiang Yong, Wang Zhiqiang, Wang Huaqi

2014, 30(5): 8-14.

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Drought is one of the most important factors which affect plant growth and development. ABA plays an essential role on plant development and growth, and response to stresses. With the fast development of molecular biology and related disciplines, people obtain much knowledge toward genes on ABA synthesis and signal transduction, and have done many transgenic studies. This article reviewed the research advances of ABA related genes on plant drought tolerance gene engineering.

Developments in Mammalian Orthoreovirus Reverse Genetics Research

Ke Fei, Wang Yun, Hou Lifan,g Hu Xing'an, Li Peipei

2014, 30(5): 15-19.

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One of the most widely distributed virus groups is Reoviridae. Mammalian orthoreovirus(MRV)is belonged to the orthoreovirus, which mainly infected the respiratory and gastrointestinal tracts of virtually all mammals, including humans. The MRV reverse genetics have improved significantly in recent years. There are several progresses in MRV genome packaging, gene function and pathogenesis based on this technology. Recent advances on the establishment of MRV reverse genetics and its application are discussed in the text.

Advances of Glycoprotein of Spring Viremia of Carp Virus(SVCV)

Zhang Jialin, Li Yang, Li Qiang

2014, 30(5): 20-24.

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Spring Viremia of Carp Virus(SVCV)is the causative agent of Spring Viremia of Carp(SVC), which results in heavy economic losses in the Cyprinid fishes farming. Glycoprotein is not only considered to have a close relationship with mediating virion endocytosis, but also posseses the main antigenic determinants of SVCV, therefore studies on Glycoprotein draw more and more attentions around the world. The progresses of Glycoprotein on relative aspects including the protein expression technology, virus detecting and vaccine development were reviewed, in order to provide valuable advices for the further studies.

Research Progress on Expression Vector Stability in E. coli System

Jiang Weihua, Liu Yili, Jiang Mingfeng

2014, 30(5): 25-31.

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The construction of stable, feasible and high-yielding plasmid vector has become one of research focuses on recombinant technology. The optimization of plasmid vector is still hindered by the lack of information on plasmid instability as well as information on the host metabolic responses. Therefore, this review highlights the stability of plasmid vector when expressing foreign proteins in E. coli and the possible influence factor for its instability, such as the inherent characteristics of the plasmid and the exogenous gene, influence on the host triggered by recombinant plasmid and other factors. Meanwhile, related solutions were briefly introduced, providing a reference for high-level expression of heterologous proteins in E. coli expression system.

Research Progress of Replication of Linear Plasmid in Bacteria

Zhu Yunxia, Zhao Xin, Zhang Qisi, Zhang Haifang

2014, 30(5): 32-36.

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Linear plasmid is a new focus of basic microbiology research in recent years. The replication of linear plasmid, compared to the circular plasmid, has its own distinct characteristics—5'end patching mechanism by which bacteria can fill the single-stranded gaps caused by the removal of RNA primers at 5'end of the strand. According to the terminal structure of linear plasmid, linear plasmids can be divided into two categories：one kind of terminal structure is covalently closed hairpin, the other one is the end capped with the terminal protein covalently. This review introduced the progress of study on the replication mechanism of linear plasmid and focused on the 5'end patching mechanism of the two kinds of linear plasmid terminal structures.

Advances of Heterogeneous Expression of Antimicrobial Peptides in Bacteria

Lü Xiaomeng, Hu Tong, Yu Ting, Cui Yanhua

2014, 30(5): 37-44.

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Antimicrobial peptides(AMPs)are a kind of cationic peptides which consist of short amino acid sequences and perform the defense system toward bacteria, virus. Different kinds of AMPs have been isolated from animals, plants, and microorganism. AMPs have important clinical application value due to their typically broad-spectrum activity against multiple pathogens. Heterogeneous expression has been demonstrated to be the most suitable tools to product antimicrobial peptides. Escherichia coli has a lot of advantages as a classic expression host, including its fast growth, clear genetic background, with a lot of available commercial expression vectors, easy operation and so on. E. coli become the preferred antimicrobial peptide expression host. Lactic acid bacteria have the advantage of being GRAS(generally recognized as safe), and have emerged in recent years as one of the most important heterogeneous expression strains of antimicrobial peptides. Developments of heterologous production of antimicrobial peptides in Escherichia coli and lactic acid bacteria were reviewed.

Research Progress of Improving the Actinomycetes Secondary Metabolite Production Method

Lei Xiuqing, Li Li, Huang Jianzhong

2014, 30(5): 45-51.

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Actinomycetes secondary metabolites have been the important sources of developing antibiotics and other drugs. Gene technology has been widely practiced for improving the yield of Actinomycetes secondary metabolites. This review summarized a series of technical methods used for improving Actinomycetes secondary metabolites，consisting of over-expressing the key structural genes and/or positive regulation genes，knocking out the negative regulatory genes，doubling the clusters，mutating ribosomal protein genes，and expressing the foreign cluster heterologously. These methods could be used for new drug discovery and strains improvement.

Research Progress of Cell-SELEX

Zhao Zhonglin, Li Yan, Yuan Chao

2014, 30(5): 52-56.

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Aptamers have high affinity to target molecules, which are selected by an in vitro process known as cell systematic evolution of ligands by exponential enrichment(Cell-SELEX). SELEX is carried out using purified target molecules, but whole live cells could be used as selection targets in Cell-SELEX progress. The advantage of this technology is that aptamers could be functional with a native conformation of the target molecule on live cells. Cell surface transmembrane proteins would also be the targets. In addition, cell-specific aptamers can be obtained without any knowledge about cell surface molecules on the target cells. In this paper, the progress of Cell-SELEX technology was reviewed.

Genetic Characteristics of 16 Kinds of Sweet Corn in China by SSR Analysis

Huang Huaru, Liang Kaiguang, Yi Hengxin, Liang Xuelian

2014, 30(5): 57-61.

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SSR markers were used to study the genetic variation of 16 varieties of sweet corn. First of all, 10 primers were screened with stable polymorphism from the 200 pairs of SSR primers；By the 10 primers, 254 allelic variations were detected in the material, each primer pair with 5 to 38 allele average. The average information quantity of polymorphism was 0.920 8. Accordingly, 16 varieties were divided into three broad categories. The study showed that SSR markers can be used to genetic analysis and heterosis breeding.

Molecular Detection and Agronomic Traits Analysis of Insect-resistant Transgenic Maize Harboring Bt cry1Ah Gene

Dai Jun, Li Xiuying, Zhu Li, Wang Hai, Zhang Jie, He Kanglai, Lang Zhihong, Huang Dafang

2014, 30(5): 62-68.

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Bt cry1Ah gene which is a novel gene cloned by Chinese scientist was introduced into inbred line zong31 via agrobacterium-mediated transformation. An insect-resistant transgenic event was acquired by population screening of 1 764 transgenic plants. In this report we evaluated the transgenic event's insect resistance through laboratory and field bioassay and analyzed the foreign gene expression and the agronomic traits of hybrids generations. The results indicated that the transgenic event had high resistance to Asian corn boner(Ostrinia furnacalis), and it had no significant difference in the plant height, ear position, ear length and ear width between the transgenic event and non-transgenic plant. However, there were significantly shorter bald in transgenic event and thousand kernels weight and yield per plant were significantly higher than the control. It was extremely significantly difference in transgenic plant and non-transgenic plant under conditions of infestation by artificial simulation.

Identification of ZmCI-1B Promoter and Its Seven Deletions in Transgenic Arabidopsis thaliana

Li Ye, Liu Xiaoqing, Li Suzhen, Zhou Xiaojin, Yang Wenzhu, Chen Rumei

2014, 30(5): 69-75.

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The expression vectors of ZmCI-1B promoter and its seven 5' truncated fragments were transformed into Agrobacterium tumefaciens strain GV3101. After identified by PCR assay, with Arabidopsis thaliana as genetic transformation, the expression vectors were transformed into Arabidopsis thaliana by floral-dip method. The transformed Arabidopsis thaliana plants were identified by PCR assay, then the seedings, flowers and siliques from positive plants were conducted to GUS histochemical staining. The results showed that the characterization of ZmCI-1B promoter in Arabidopsis thaliana is different from miaze. The ZmCI-1B promoter and its seven 5' truncated promoter-GUS constructs had different GUS staining in transformed Arabidopsis thaliana plants, revealing that different-length promoter had different promoting activity. The cis-acting elements on the promoter may contribute to this.

Transcriptional Activation Identification of the Transcription Factor HcSCL13 from Halostachys caspica and the Construction of Plant Expression Vector

Zhou Lianjie, Zhang Fuchun, Wang Yan

2014, 30(5): 76-82.

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HcSCL13 gene of the GRAS family from Halostachys caspica(GenBank accession number：KC68640)was amplified and ligated to the pGBKT7 vector by homologous recombination to construct the bait vector pGBKT7-HcSCL13, and then transformed into the competent yeast Y2HGold. Self-activation of recombinant bait vector in yeast two-hybrid system was measured. The results showed that the bait protein had not toxic to Y2HGold and could activate all the reporter genes. Therefore, we proposed HcSCL13 was a transcription factor with a transcriptional activation domain. The plant over-expression vector and subcellular localization expression vector were successfully constructed for further studying its biological function.

Expression, Purification and Characterization of Populus tomentosa PtoeIF5A4 in Prokaryote

Zhang Jiewei, Guan Yang, Zhu Dan, Chen Yajuan, Ding Liping, Wang Hongzhi, Wei Jianhua

2014, 30(5): 83-87.

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To obtain the PtoeIF5A4 protein of poplar with high purity in a large scale, its full length CDS was amplified by PCR using pGEM-T Easy-PtoeIF5A4 as template and verified by sequencing, and then was inserted into pET28a vector containing histidine. The recombinant pET28-PtoeIF5A4 plasmid was obtained and transformed into E. coli BL21(DE3). The recombinant fusion protein was produced by culturing the transformed E. coli BL21(DE3)with subsequent IPTG induction, and was observed predominantly in the supernatant of cellular extracts when cells were cultured at 28℃ and induced with 0.1 mmol/L IPTG for 4 h. The fusion protein was purified from the soluble fraction using a column of Ni²⁺ Chelating Sepharose Fast Flow, and proofed molecular mass was 18 kD. The anti-His monoclonal antibody recognized the protein, which indicated that it was PtoeIF5A4 recombinant protein.

Cloning and Bioinformatics Analysis of DFR Gene from Brown Cotton(Gossypium hirsutum L.)Fiber

Xiao Xiangwen, Zhu Qilang, Liu Haifeng, Wang Junduo, Luo Cheng, Zeng Wen, Liang Yajun, Gong Zhaolong, Li Xiaobo

2014, 30(5): 88-95.

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The main pigment that affects flower colors is the anthocyanin, and dihydroflavonol 4-reductase(DFR)is a key enzyme involved in anthocyanin biosynthesis pathway. A full-length gene from brown cotton(Cultivar Xincaimian 6)fiber at the 16 day post anthesis(16 DPA)encoding dihydroflavonol 4-reductase(GhDFR)was isolated, through homology cloning techniques. A full length coding sequence(CDS)and genomic sequence including introns were obtained. Bioinformatics approach was used to analyze and predict the function and structure domain of GhDFR. According to sequence analysis, the genomic sequence of GhDFR contained six exons and five introns. The full-length CDS of the GhDFR ortholog consisted of a 1 500 bp open reading frame(ORF)encoding a polypeptide composed of 355 amino acid residues. The molecular weight of deduced GhDFR polypeptide was 39.65 kD, with an isoelectric point(pI)of 5.67. The deduced amino acid sequence of the GhDFR ortholog had a highly conserved NADP(H)-binding site and substrate specificity site. The deduced DFR amino acid sequence showed high homology with DFRs from other plants, such as Populus trichocarpa, Vitis vinifera and Pelargonium zonale. Phylogenetic analysis showed the closest relationship with DFRs of Pelargonium zonale and Paeonia lactiflora.

Single Nucleotide Polymorphisms in A-FABP Associated with Carcass Quality and Meat Quality Traits in Five Zaosheng Cattle Groups

Tang Hongjie, Zhao Shengguo, Lei Zhaomin, Wang Xinrong, Wang Jianfu, Cai Yuan, Wu Jianping

2014, 30(5): 96-101.

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It was to study the polymorphism of A-FABP gene and its correlation with carcass quality and meat quality traits in Zaosheng cattle groups. Using PCR-SSCP to analyze the polymorphism of A-FABP gene and its correlation with carcass quality and meat quality traits in five Zaosheng cattle groups(Qingyang group, QZ and its hybrid with South Devon group, QZ×N；Pingliang group, PZ and its hybrid with Qinchuan cattle group, PZ×Q；PZ and its hybrid with Simmental group, PZ×S). Result showed that there has a base mutation at c.408G>C of A-FABP gene exon 3 and exist three genotypes GG, GC and CC. Statistical analysis of carcass traits shows that carcass weight with GG genotype was significantly lower than CC genotype(P<0.05)；dressing percentage with GG genotype was significantly lower than GC genotype(P<0.01), and significantly lower than CC genotype(P<0.05)；net meat percentage with GG genotype was significantly lower than GC genotype(P<0.05)；loin muscle area with GG genotype was significantly lower than GC and CC genotypes(P<0.05). Statistical analysis of meat quality traits showed that pressing loss with GG genotype was significantly higher than CC genotype(P<0.05)；shear force with GG genotype was significantly higher than GC and CC genotype(P<0.01)；cooking loss and pH with GG genotype was significantly higher than GC genotype(P<0.05), and significantly higher than CC genotype(P<0.01). This mutation of A-FABP gene could be considered as a locus to carcass and meat quality.

Cloning of IGF-2 Gene cDNA in Tianzhu White Yak

Li Yalan, Zhang Quanwei, Wang Xueying, Ma Youji, Zhang Yong, Zhao Xingxu

2014, 30(5): 102-106.

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It was to clone insulin-like growth factor2(IGF-2)full length cDNA in Tianzhu white Yak, thus provide a theoretical basis tostudyits mechanisms. IGF-2 gene full length cDNA were cloned by rapid amplification of cDNA ends(RACE). The result showed that the full length of IGF-2 cDNA was 1 060 bp(Gene Bank accession No：KF682139), with an ORF 540 bp in length, which encoded 179 amino acids. The amino sequence analysis indicated that IGF-2 gene in Tianzhou white yak is highly homologous in most mammals. The full length cDNA of 1 060 bp IGF-2 gene was successfully cloned, which laid a basis for further studying of its roles in whit yak.

Polymorphisms of TLR9 Gene in Sheep in Gansu Province

Lü Weili, Zhang Xiaoli, Ma Xiaojun, Zhang Guohua

2014, 30(5): 107-111.

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There is a acute and chronic respiratory disease in population of sheep，which leads to productive reduces even death. Gene TLR9 is associated with respiratory disease. The Polymorphism and variation of gene TLR9 was investigated in order to find the best alleles for respiratory disease resistance in the further observation by means of phenotype and genotype，reveal the genetic characteristics of sheep and provide data for future search. Fragments of DNA containing TLR9 were amplified using PCR. Single-strand conformational polymorphism(SSCP)analysis and DNA sequence analysis were employed to detect genetic variation in 427 healthy sheep and 58 with respiratory disease. Results showed that four alleles were identified，seven nucleotide polymorphisms were in the analysis of gene sequences，which mainly came from point mutation，including transition of 4(57.14%)and transversion of 3(42.86%). Sheep TLR9 gene in Gansu has high level of sequence polymorphism. TLR9 gene polymorphisms with respiratory disease in sheep has some relevance.

Comparison of Metallothionein-3 Gene Expression Levels in Different Brain Parts of Tibetan-sheep Between Different Altitudes Areas

Wang Jiali, Wang Xinrong, Zhao Shengguo, Wang Jianfu, Wu Jianping

2014, 30(5): 112-116.

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The study was set out to find the differences of Metallothionein-3(Metallothionein-3, MT-III)gene of Tibetan sheep in different altitudes areas and its expression differences in different parts of the brain tissue. Randomly selected 6 Tibetan rams from high altitude areas(Henan county, Qinghai)and 3 Tibetan rams form low altitude areas(Lintao county, Gansu), sampled various parts of the temporal lobe, parietal lobe, medulla, cerebellum and pineal gland in the brain tissue after slaughter, respectively, and analyzed the relative expression levels of MT-III gene at the brain tissue and other different parts of Tibetan sheep in different altitude areas by real-time PCR method. MT-III gene were expressed in various parts of brain tissue of Tibetan sheep, but there are differences of the relative expression levels in different parts；the relative expression of MT-III gene in pineal gland was significantly higher than that of other parts(P <0.01)；at high altitude, the relative expression of MT-III gene in the temporal lobe, parietal lobe and medulla were significantly higher than those of Tibetan sheep at low altitude areas(P <0.01). MT-III is a protein that expressed in different parts of brain tissue of Tibetan sheep, its high expression in the temporal lobe, parietal lobe and medulla will cause the brain cells in this region enhanced tolerance to hypoxia and high radiation.

Construction and Screening of Recombinant Interference Vector of Pluripotency Associated Genes cNanog and cPouV in Chicken Embryonic Stem Cells

Peng Te, Wang Bingyun, Li Dongsheng, Chen Zhisheng, Chen Shengfeng, Ji Huiqin, Chen Jinding

2014, 30(5): 117-121.

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The study was to construct the siRNA(small interfering RNA)expression vectors targeting cNanog and cPouV of Chicken Embryonic Stem cells(CES cells), and evaluate its inhibitory effect on cNanog and cPouV in 293T cells. Three regions of cNanog and cPouV gene were respectively selected to synthesize oligonucleotides, and construct interference vectors. These vectors were respectively transfected into 293T cells as whole-gene expression vectors were transduced simultaneously. Western blotting analysis showed target genes expression were downregulated after RNAi and the oligonucleotide sequence S3 was found to be the most efficient one in interfering effect. The results of this experiment indicate siRNA or shRNA(short hairping RNA)were translated into cells and suppress the expression of target genes.

Construction of Transgenic Goldfish Based on Tol2 Transposable Element

Dong Ying, Hu Hongxia, Wang Wei, Tian Zhaohui

2014, 30(5): 122-128.

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Medaka Tol2 transposon(Transposable element of Oryzias latipes, number 2)was the first natural active transposon in vertebrate, which had been successfully applied in transgenic research of variety model animals. The sequence containing five exons of grass carp growth hormone(GH), carp beta-actin promoter and multiple restriction enzyme cutting sites was amplified based on pCAgcGH plasmid, and was inserted into pTol2-MCS-EGFP plasmid to construct recombinant pTol2-GH-EGFP plasmid. The recombinant pTol2-GH-EGFP plasmid and the transposase mRNA synthesized in vitro were coinjected into fertilized eggs of goldfish by microinjection. The transgenic goldfish were selected by observing green fluorescence and PCR, and expression rate was 17.3%. The construction of transgenic goldfish based on Tol2 transposable element, not only expanded the range of Tol2 transposon application, but also would be useful for studies of transgene and gene expression in ornamental fish.

DNA Barcodes Reveal Intra-and Inter-Individual Sequence Variation in the Bush Cricket Mecopoda niponensis(Orthoptera：Tettigoniidae)

Zhou Zhijun, Shang Na, Chang Yanlin, Shi Fuming

2014, 30(5): 129-136.

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The mitochondrial COI barcoding region of 14 individuals from two Mecopoda species with Folmer primers were amplified. All M. elongata COI fragments were 658 bp in length, without stop codons or indels. Due to the serious sequence ambiguities caused by direct sequencing, 85 clones were sequenced from eight M. niponensis individuals, with sequence lengths ranging from 580 to 662 bp. Among these, 20 of the clones were distinct nuclear sequences of mitochondrial origin, numts(four clones with stop codons, 15 clones with indels, one clone was frameshift), and 38 clones contained point mutations that caused two or more amino acid sequence differences as compared with the orthologs. The Kimura 2-parameter(K2P)model was adopted to calculate genetic distance, and the results indicated that the genetic distances among M. elongata individuals varied from 0 to 0.008. Among M. niponensis, intra-individual clones ranged from 0 to 0.143(inclusive of numts)or 0 to 0.083(exclusive of numts), and the inter-individual distance ranged from 0.030 to 0.051(exclusive of numts). The genetic distance between the two Mecopoda species was 0.077. Moreover, the neighbor-joining(NJ)phylogenetic tree based on the Kimura 2-parameter distances indicated that all the M. elongata individuals grouped together with high confidence(99%), and the real COI sequences and numts of M. niponensis clustered into several groups. These results indicate that numerous independent nuclear integration events in M. niponensis were scattered at various time points, which suggests a continuous process of numt generation.

Cloning of DGAT1 Gene from Perilla frutescens and Construction of Scenedesmus quadricanda Expression Vector

Wang Yingying, Liu Yu, Ji Yanru, Zhang Zhenghai, Zhou Guangqi, Liu Yufeng

2014, 30(5): 137-141.

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Total RNA of Perilla frutescens was extracted by RNAiso Plus kit. It was used as a template for RT-PCR to clone cDNA coding region of DGAT1 gene of Perilla frutescens, and cloning vector pMD-DGAT1 was constructed. Cloning vector pMD-DGAT1 and empty vector pBI121 were double-enzyme digested with Xba I and BamH I. Then expression vector pBI121-DGAT1 was constructed by linking the fragments. Results showed that 1 657 bp fragment was obtained by cloning and has 97% base similarity to DGAT1 gene of GenBank. Expression vector pBI121-DGAT1 was double-enzyme digested into two fragments. The length of them was as long as the ones before linking. It was transformed and expressed in the Scenedesmus quadricanda successfully. The transformed Scenedesmus quadricanda approximately double the oil content as before.

Differential Protein Expression Analysis of Hypsizygus marmoreus Under High Temperature Stress

Liu Lin, Jia Peipei, Lu Weidong, Guo Qilin, Guo Lizhong

2014, 30(5): 142-147.

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Mycelial total proteins in Hypsizygus marmoreus from Tris-saturated phenol isolation method was analyzed by two-dimensional electrophoresis(2-DE), then the 2-DE maps obtained were analyzed with software PDQuest8.0 to compare the protein expression difference between the optimum temperature(25℃)and high temperature stress conditions(42℃), and screen the high temperature stress-related proteins. The differential expression proteins were examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and seven protein spots were identified. They are the Spa2 homology domain, DEHA2G15532p, peptidase M76 family, HSP70、SNF2 family of DNA repair proteins, cytochrome P450 and mixture of SAM domain family and zinc protein. The point 2 is up-regulated and the other six points were down-regulated. These high temperature stress-related proteins were involved in signal transduction, protein process, DNA repair and other general stress tolerance.

The Discovery and Identification of Yeast Can Produce Cutinase, and Optimization of Fermentation Conditions

Ran Qinqin, Zhang Xiaoning, Zhang Wenkun, Zhang Xuejun, Zhang Mengmeng

2014, 30(5): 148-154.

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To get effective enzyme hydrolyze cutin of Eucommia ulmoides corneous layer, the extraction of the biological active compositions within Eucommia ulmoides was done using enzymatic hydrolysis. This article from the lesion Eucommia leaves isolated bacteria capable of producing cutinase and identify the bacteria, confirmed that the strain belongs to Rhodotorula mucilaginosa, and the strain producing cutinase fermentation conditions were optimized. This finding is significant, because the yeast more secure than plant pathogens is more suitable for mass production.

Screening and Identification of Streptomyces fungicidicus Strain AL-04

Li Zengbo, Sun Hongyan, Zhao Jingxian, Zhou Jianwu, Liang Dong, Gao Ye

2014, 30(5): 155-161.

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13 actinomycetes strains were screened from soil samples of Qinghai-Tibet Plateau. Strain AL-04 which has high and stable inhibition effects on soilborne pathogens was selected by methods of agar diffusion, relative growth rate, spore germination inhibition and vegetable leaves. Results of antagonistic activity showed that AL-04 had strong antagonistic activity against 4 species of soilborne pathogens including Phytophthora capsici, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. niveum and Botrytis cinerea. The inhibition rates were beyond 70.0% for all pathogens, of which 93.0% was observed for Phytophthora capsici. AL-04 was identified as Streptomyces fungicidicus by the observation of the morphological features, cytochemical characters and 16S rDNA sequence.

A Study on Identification and Enzyme Characteristics of a Xylanase Producing Strain Isolated and Selected from the Soil in Alpine Meadow

Lu Guangxin, Chen Xiurong, Wang Junbang, Wu Chu⁴

2014, 30(5): 162-178.

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A fungus strain has been isolated and selected from the soil in alpine meadow. Using gene sequences of rDNA-ITS analysis, flask liquid fermentation and traditional culture methods. Taxonomic position of strains were identificate, and xylanase characteristics and its main biological characteristics were also been investigated. The results showed that by rDNA-ITS sequence analysis, the fungus was identified as Saccharicola bicolor. S. bicolor can secrete xylanase, its enzyme activity is relatively stable under the condition, i.e., pH3.0-9.5, showed that xylanase secreted by S. bicolor have certain tolerance ability of alkaline conditions. The fungus grew rapidly under the condition, i.e., 35℃, showing different requirements for carbon and nitrogen resources, i.e., the order of carbon resources is maltose, starch, sucrose, dextrin, and glucose；the order of nitrogen resources is proteins, amine phosphate, amine sulfate, sodium nitrate, and urea.

Effect of Biomass and Resveratrol Content of Peanut Hairy Root Strains in Different Media

Yao Qingshou, Jiang Jigang, Wu Yuyong, Liang Chengbang

2014, 30(5): 174-178.

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It was to study the effect of biomass and resveratrol content of peanut hairy root strains in different media. To induce hairy roots by using A.rhizogenes R1601 infect peanut leaves, and to detect hairy roots by PCR. Peanut hairy root strains were cultured with five kinds of media(1/2MS, MS, MS +500 mg/L carbenicillin sodium, B5, and N6), the biomass and resveratrol content of hairy root strains were measured by HPLC after four weeks. Results showed that the accumulation of biomass and content of resveratrol of peanut hairy root strains had a significant effect in different kinds media, 1/2MS medium and MS medium were conducive for the biomass accumulation of peanut hairy root strains. B5 medium and N6 medium were conducive for the resveratrol accumulation of peanut hairy root strains.

Transposon Mutagenesis of Pseudomonas sp. BS1 and Preliminary Screening of High-producing Biosurfactant Mutant

Hou Jumei, Han Wei, Guo Liling, Liu Tong, Wang Yanjie

2014, 30(5): 179-183.

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The protoplast of Pseudomonas sp. BS1 was transformed by temperature-sensitive plasmid pTV1-OK that harbor Tn917. 3 stable transformants were obtained from the LB plates with Kan and Erm. Lots of mutants were obtained by transposon mutagenesis of Tn917, sequencely a mutant library was constructed. A predicted fragment was amplified by PCR from 5 randomly selected mutants, which showed that Tn917 successful inserted into the genome of Pseudomonas sp. BS1. E24 of a mutant from 24 selected randomly reach up 67% by the measurement of the emulsification index, significantly higher than wild-type strain. These results indicated that the transposon mutagenesis is an effective method for obtaining high-producing biosurfactant.

Biocontrol Efficacy and Phylogenetic Tree Analysis of a New Bionectria ochroleuca Strain

Chen Xiuling, Li Jingfu, Zhang Lili, Zhang Junfeng, Wang Aoxue

2014, 30(5): 184-189.

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The fruit and vegetable disease controls are more difficult because the increase of protected cultivation area. Biological control means became more and more popular in disease control process. By dual culture on PDA plates, WY-1 inhibited the growth of Botrytis cinerea, Cladosporium fulvum, Phytophthora parasitica, Vertillium dahliae, Fusarium wilt, Valsa sordida Nit., Alternaria and Pseudopeziza ribis Kleb. Scanning electron microscopy revealed that WY-1 inhibited the Botrytis cinerea growth significantly. 5.8S rDNA-ITS sequence alignment and neighbor-joining phylogenetic tree reveal that WY-1 belongs to Bionectria ochroleuca, which may provide a new choice in disease control.

Study the Effect of Tachyplesin I on Proteus vulgaris

Jin Yuanbao, Wang Ying, Liu Liping, Wang Qian, Jin Gang, Zhang Lijun, Dai Jianguo,

2014, 30(5): 190-196.

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It was to investigate the effect mechanism of tachyplesin I on Proteus vulgaris. The study on the inhibition concentration of tachyplesin I, the plasma membrane permeability and electric charge of Proteus vulgaris, the activity of proteases were secreted by Proteus vulgaris and the stability of tachyplesin I in the nutrient broth medium where cultured Proteus vulgaris. Results indicated that the MIC and MBC were 40-80 and 80 μg/mL, respectively. The membrane permeability and electric charge of Proteus vulgaris would be increased with the concentration of tachyplesin I increasing. The proteinase activity which secreted by Proteus vulgaris would be increased when the concentration of tachyplesin I less than 40 μg/mL, but it presented downward trend when more than 60 μg/mL. The concentration of tachyplesin I in the medium that trained the bacterial would reduce. Tachyplesin I could inhibit growth of the Proteus vulgaris, the effect mechanism related to damage the membrane permeability of them.

The Preparation and Application of Mouse DEAF1 Polyclonal Antibody

Zhu Xiaohui, Sheng Deqiao, Kang Le, Wu Xianfeng, Shao Wen, Liu Xiaoyan

2014, 30(5): 197-201.

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To study endogenous expression of DEAF1 in mouse pancreatic lymph nodes and the mechanism of DEAF1 regulated expression of peripheral tissue antigen genes. The prokaryotic expression plasmid contain mouse DEAF1 cDNA was transformed into E. coli BL21, the induced recombinant DEAF1 protein was purified by urea gradient cleavage, and was used to immunize Balb/C mouse. The anti-DEAF1 polyclonal antibody can get from mouse serum, and the specificity, sensibility, and affinity of antibody were test by ELISA, Western blot, Immunofluorescence and Immunoprecipitation. The results showed that anti-sera antibody can reacted with antigen and detected a specific band of 60 kD, and also can be used for immunofluorescence localization. Immunoprecipitation experiments showed that anti-sera antibody can bind with the antigen with high affinity. These results suggested that the DEAF1 polyclonal antiserum revealed high specificity, sensibility and affinity.

The Construction and Interference Effects to HSV-2 of UL27, UL29 shRNA Expression Vectors

Lü Yancheng, Pan Xiaoyu, Huang Chang, Ding Juan

2014, 30(5): 202-209.

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To investigate the effect of siRNA targeting herpes simplex virus type 2(HSV-2)UL27 and UL29 simultaneously on HSV-2 replication. siRNA recombinant expression vectors targeting UL27 and UL29 simultaneously were constructed and the expression of UL27 mRNA and UL29 mRNA were tested by FQ-PCR 48 hours after transfecting in 293 cells. The viral titer are estimated by using titration end-point assay. The expression and changes(gray scale value)of protein was assessed by western-blot. MTT was used to examine the proliferative activity of cell. The results showed that：(1)The shRNA expression vectors which were constructed by pGPU6/GFP/Neo-UL27、pGPU6/GFP/Neo-UL29.(2)At 48 hours after transfection, the results of FQ-PCR showed that compared to blank group, UL27 shRNA75 the inhibition rate are 75.17%, there are significant differences(P<0.05). UL29 shRNA1461 the inhibition rate are 66.08%, there are significant differences(P<0.05). UL27 shRNA75 +UL29 shRNA1461 joint group of UL27 gene inhibition rate are about 91.28%, UL29 gene inhibition rate are about 80.40%, comparing with blank group, with significant differences(P<0.05).(3)The result of end-point assay showed each interference group could reduce the virus titers of filial generation in the supernatant in different degree, comparing with blank group, with significant differences(P<0.01).(4)Using Western blot to detect the objective gene protein has revealed that single interference group and joint group can reduce the protein expression of target genes in different degree, the joint interference group of UL27 shRNA75 + UL29 shRNA1461 can significantly inhibit the expression level of target genes protein, protein expression of joint interference group is obviously on the decline, compared with the single interference group, there is significant differences(P<0.05).(5)MTT assay showed that, UL27 shRNA75, UL29 shRNA1461, UL27 shRNA75 + UL29 shRNA1461 group cell survival rate was significantly improved, there are significant differences(P<0.05).The construction of pGPU6/GFP/Neo-UL27, pGPU6/GFP/Neo-UL29 recombinant expression vector shRNA can interfere HSV-2 UL27, UL29 gene expression from different cell level in vitro. The joint interference has more effect, which can inhibit the the replication of HSV-2 in HEK293 cells.

Construction and Activity Assay of Transcription Activator-like Effector Nuclease(TALEN)Plasmids for ALK4 Gene Knock-out

Zeng Fancai, Gu Hong, Wang Ke, Zhou Hong

2014, 30(5): 210-216.

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The plasmids of transcription activator-like effector nuclease(TALEN)to knock out Activin receptor-like kinase 4(ALK4)was obtained. Firstly, using a TALEN design tool online, the target sites of gene knock-out, TALEN recognition sequence and the restriction site for evaluating TALEN activity were defined according to the design rule and the common sequence of ALK4 variants. Secondly, the TALEN plasmids were constructed using a TALEN construction kit, and then confirmed by enzyme cutting, sequencing and BLAST analysis. Thirdly, the plasmids were transfected into HEK293T cells by lipofection and the transfection efficiency was assessed by observing the expression of pEGFP-N1 plasmid. After positive screening by puromycin, the genomic DNA of cells was extracted as the template, and the PCR products containing target sequence were digested by Hha I. The results showed that the cleavage efficiency of this enzyme for PCR products from genomic DNA of cells tranfected with TALEN plasmids was significantly lower than that from the cells without TALEN, suggesting that ALK4 genes may undergo mutation due to TALEN activity. These TAELN plasmids provide a valuable basis for constructing various cell lines with ALK4 knock-out and understanding the function role of ALK4.

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