Table of Content

20 October 2014, Volume 30 Issue 10

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Progress in Aphid-resistant Genes and Transgenic Crop Research

Chen Jin, Liu Zhi, Zhu Shengwei

2014, 30(10):  1-7. 

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Aphids damage, commonly and frequently occurring, caused serious losses to agricultural production. In the paper, we reviewed the progress in genetic engineering for aphid resistance, the genes resistant to aphids and their transgenic research, as well as problems existing in aphid resistance at present, and discussed employment of the methods such as cloning of direct toxin genes, site-directed mutagenesis, increasing of expressed regulation elements, lectin proteins as transport carrier to enhance aphid resistance for the aphid-resistant crops breeding program in the future.

The Roles of Non-cell-autonomous Transcription Factors in the Regulation of Plant Meristem Development

Gu Huiying, Jiang Wei, Li Jing, Wang Zhimin, Tang Qinglin, Song Ming

2014, 30(10):  8-15. 

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In order to adapt to the changes of environment, plants have evolved unique signaling mechanisms, almost each organ and tissue can form efficient signal transduction system. Intercellular movement refers to mechanisms that are specifically implemented during pattern formation in organs, tissues or between neighboring cells, in which transcription factors, peptides, small RNAs（sRNAs）and hormones have involved. The four types of mobile molecules mediate different signal transduction pathways, but they can interact with each other and constitute the entire intercellular signaling networks. As a kind of particular protein, transcription factors especially non-cell-autonomous transcription factors, play important roles in processes related to the formation and development of plant organs. This paper mainly summarizes the non-cell-autonomous transcription factors in plant and the mechanisms that transcription factors and other mobile molecules co-regulate plant meristem development.

Gibberellins Biosynthesis and Signaling Transduction Pathway in Higher Plant

Li Qiang, Wu Jianming, Liang He, Huang Xing, Qiu Lihang

2014, 30(10):  16-22. 

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The hormone gibberellins（GA）plays an essential role in many aspects of plant growth and development, such as promoting seed germination, stem elongation, occurrence of flower, fruit and seed development. This review elaborated the GA biosynthesis and signaling transduction pathway, as well as interactions with other hormones, response to the environment signals, and the recent advances in understanding of degradation of DELLA proteins by polyubiquitination of the DELLAs via the E3 ubiquitin-ligase SCF ^SLY1/GID2 in the 26S proteasome. This helps not only people understanding of gibberellin physiology and its molecular regulating mechanism, but also to every aspect of gibberellin mechanism are studied deeply.

Thyroid Hormone Signaling Pathway and Its Research Advance in Marine Invertebrate

Xu Jianbo, Zhang Lili, Wang Yilei, Wang Guodong

2014, 30(10):  23-32. 

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In vertebrates, thyroid hormones（THs）signaling pathway is essential for the animal’s growth, development and energy metabolism. In addition, it also involved in metamorphosis process of the amphibian and fish. In recent, increasing evidence supports the existence of signal molecules of THs signaling pathway in marine invertebrates, such as endogenous THs and thyroid hormone receptors（TRs）and so on. And, these signal molecules involved in the development and metamorphosis process of marine invertebrates. It suggests that the THs signaling pathway exists in marine invertebrates, and it shows similarity with the vertebrates. This paper aims at a summary of thyroid hormone signaling pathway and its research advance in marine invertebrate, so as to provide basic data for the study of biological function of THs signaling pathway in marine invertebrates.

Insulin-like Peptides in Invertebrates and Their Signaling Pathways—Take Insects,for Example

Zhang Longhui, Wang Guodong

2014, 30(10):  33-42. 

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Insulin-like/related peptides（ILPs）of invertebrate are homologous of vertebrate insulin. This article rewiewed the structure, expression, signalling pathway of ILPs. The functions of ILPs were also summarized in regulating growth, development, metabolism, reproduction and immunity.

Progress of Antimicrobial Peptides as Feed Additive

Qiao Wei, Hao Hua, Peng Hui, Liu Jie, Chen Huiyun

2014, 30(10):  43-48. 

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Antibacterial peptides or Antimicrobial peptides（AMPs）, a class of small-molecule active peptides, are an important component of the innate defense system of organism to resist against invasion of foreign pathogens. Antibiotics pollution is one of the most important issues that would affect the sustainable development of stock farming and aquaculture industry in our country. Antibiotic residues in agricultural（including aquaculture）products involved in this issue is the bottleneck of our food safety and exports, and excessive antibiotics addition to feedstuff is one of the main reasons for antibiotic residues. Thus, finding and developing alternatives for antibiotics is the urgent requirement of healthy development of animal husbandry and aquaculture industry in China. In this article,the sources, function and its application as feed additives in livestock were reviewed..

Applications of Environmental DNA Approaches to Ecological Researches

Xu Hao, Luo Xi, Li Yun, Xue Yang, Ye Qin

2014, 30(10):  49-55. 

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Environmental DNA（eDNA）refers to DNA that can be extracted from environmental samples, without first isolating any target organisms, which includes DNA of environmental microorganisms, alive cellular fallen off from organisms, and extracellular DNA resulting from natural death organisms and subsequent destruction of cell structure. According to metagenomics concept, eDNA technologies mainly refers to the methods of sequencing analysis with genomic DNA from environmental samples. Comparing with the traditional way, the significant advantage of eDNA technologies is more efficient in solving the classification problem of mass organisms in given environmental sample, which exert shorter time-consuming, lower cost, higher accuracy. The next generation of high-throughput sequencing technology（NGS）further expands the application fields of eDNA technologies, from microbiological field to zoological and botanic fields, and results in an innovation in research methods and ideas in traditional ecology. The present review summarized the eDNA technologies and applications aspects in analysis of biological diversity, animal diets, aquatic biomass estimation, etc. Finally, the trends and prospects regarding eDNA technologies development are presented.

The Biological Remediation Technology for the Contaminated Soil

Zhang Qiang, Liu Bin, Liu Wei, Ren Jin, Xu Sheng, Zhang Bin

2014, 30(10):  56-63. 

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Biological remediation of contaminated soil is a potential contamination control technology by its characteristic of high efficiency, low cost and no pollution. The paper analyzed several biological remediation technologies such as animal remediation, phytoremediation, microbial remediation and combined remediation, discussed the technical features of these technologies for treating heavy metals, organic compounds, etc. and provided reference for the remediation of contaminated soil in China by suitable technology.

Research Advance in Genome-wide Identification of Transcription Regulatory Elements

Li Wenmao, Jia Dongfang, Xu Rong

2014, 30(10):  64-70. 

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Expression of eukaryotic genes is a complex process, achieved through the coordinated action of transcription regulatory elements. The screening and identification of transcriptional regulatory elements in genome scale have crucial significance for understanding the mechanism and biological function of gene expression regulation. However, the effectively screening and function verification is still the major challenges for researchers. Herein, based on the latest research findings, we reviewed the methods of prediction, screening, function analysis of transcription regulatory elements in genome.

Development of A Monoclonal Antibody Sandwich ELISA for Detection of bar Gene Transgenic Plant and Element

Long Likun, Li Congcong, Zhang Ming, Li Feiwu

2014, 30(10):  71-75. 

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This study induced prokaryotic express Bar protein, by protein Immunity of BALB/c mice, cell fusion and screening clones, we obtained a hybridoma cell line. In this study, using rabbit polyclonal IgG and hybridoma cells secreting monoclonal antibodies we established sandwich ELISA method for detection of bar gene intransgenic plant which showed good specificity and sensitivity . This study provides rapid and accurate.technical tool for a secure monitoring of GMO.

Cloning and Functional Analysis of MeHDS1 from Cassava Responsing Drought

Yu Xiaoling, Ruan Mengbin, Wang Shuchang, Peng Ming

2014, 30(10):  76-81. 

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The HD-Zip transcription factors are unique to the plant kingdom. They play important roles in plant responsing to the environmental factors. Based on the cDNA of cassava, using RT-PCR technology, one gene sequence was obtained, named it as MeHDS1. The sequence was analyzed, we got a 2 469 bp gene which have integrated ORF, MeHDS1 encoded a protein which contained 822 amino acids（89.07 kD）with an isoelectric point of 5.79. MeHDS1 have typical HD-domain, START domain. It has high homology to GL2 gene of cotton and Arabidopsis, so it might belong to HD-Zip IV subfamily. There have a nuclear localization signal in MeHDS1 protein, and which was localized in the nucleus and cell wall by subcellular localization assay in tobacco epidermal cells. Real-time PCR results showed that, MeHDS1 was upregulated under drought, and its variation of expression was more highly in root cells than that in leaves cells. Through its bioinformatics and expression analysis, we concluded that MeHDS1 may be involved in cassava abiotic stress responsed as a transcription factor.

Cloning and Expression Analysis of a Cotton Microtubule Associated Protein Gene of GhSP1L5

Li Rong, Kou Wei, Tan Lan, He Yi, Liang Zhuo, Li Hongbin

2014, 30(10):  82-87. 

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A cotton Spiral1-Like5（GhSP1L5）full-length open reading frame（ORF）cDNA was cloned from fast elongating fiber tissue by RT-PCR method, The GhSP1L5 gene contains 315 bp necleotides and codes a protein of 105 amino acids. Through sequence function analysis and homology sequence alignment, GhSP1L5 protein includes several microtubule associated protein binding sites and SCOP domains. Phylogenetic tree analysis showed that GhSP1L5 has a similar relationship with SP1L5 protein family. Prokaryotic expression vector pET28a-GhSP1L5 was constructed and transformated into E.coli BL21（DE3）. Recombinant GhSP1L5 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of ~12 kD. Quantitative RT-PCR analysis showed that the GhSP1L5 gene expresses highly at 15 dpa and 20 dpa fiber tissues compared with the expressions in root, stem, leaf and fiber tissuse of other development stages. GhSP1L5 cloning and expression analysis establish a basis for its further functions in cotton fiber development.

Cloning of Chalcone Isomerase Gene from Potato Onion and Plant Expression Vector Construction

Liu Dan, Wu Fengzhi

2014, 30(10):  88-93. 

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To clone and analyse the function of the potato onion chalcone isomerase gene, we generated CHI over-expression construct and RNAi construct. Full-length CHI gene is 743 bp, and the GenBank accession number is KJ489062. Results showed that, CHI gene open reading frame contain 633 bp, translating into 210 amino acids. Full-length CHI gene and the fragment of CHI gene for silencing were amplified by PCR on complementary DNA（cDNA）, then PCR fragment was transferred to the binary vector under the control of 35S.

Research of Transferred Synthetic Nattokinase Gene in Tobacco Genome

Liu Jinglei, Zhang Yan, Sun Xiaolei, Han Lan, Kh.Mandakhtsetsen, Hasi Agula

2014, 30(10):  94-100. 

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The nattokinase gene sNK which was artificially synthesized according to plant preferential codons, and the nattokinase gene sNK-E8i which has been got by inserting the first intron of tomato specifically expressed gene E8 into sNK, were transformed into tobacco NC89 through Agrobacterium-mediated method. 12 strains of sNK transgenic tobacco plants and 10 strains of sNK-E8i transgenic tobacco plants were acquired through PCR test. It preliminarily demonstrated that the target genes were integrated into tobacco genomes. It was found that 9 strains of sNK transgenic tobacco plants and 10 strains of sNK-E8i transgenic tobacco plants were positive by using RT-PCR. The result showed that the expression level of sNK-E8i transgenic tobacco plants was higher compared with that of sNK transgenic tobacco plants by RT-qPCR. Besides, the enzyme activity was tested through agarose-fibrin plate analysis, and the dissolving circles of 5 strains of sNK transgenic tobacco plants and 3 strains of sNK-E8i transgenic tobacco plants were detected. The result showed that target genes could be transcribed and translated normally in a part of transgenic tobacco and presented the activity of thrombolysis. The T₁ transgenic straints were obtained by generation-adding cultivation.

Cloning and Functional Prediction of the Global Regulator FocVeA Gene from Fusarium oxysporum f. sp. cubense

Qi Yanxiang, Zhang Xin, Lu Ying, Zhang He, Pu Jinji, Yu Qunfan, g Xie Yixian

2014, 30(10):  101-106. 

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Based on the conserved amino acid sequence of veA homologs from F. verticillioides and F. fujikuroi, FocVeA gene and its open reading frame of F. oxysporum f. sp. cubense race 1 and race 4（Foc1 and Foc4）were cloned by PCR and RT-PCR. Sequences characterization, phylogenetic clustering and conserved domains on the two predicted proteins of FocVeA gene were also analyzed, respectively. The results showed that the complete DNA sequences of FocVeA from Foc1 and Foc4（named as F1VeA and F4VeA）were 1 693 bp and 1 690 bp, respectively. The cDNA of F1VeA and F4VeA were 1 599 bp and 1 596 bp, and encoded a deduced protein of 532 amino acid and 531 amino acid, respectively. The deduced amino acid sequences of F1VeA and F4VeA shared 99% similar to each other, and had 78%-99% similarity with the sequences of veA factor from isolates of Fusarium spp., respectively. Phylogenetic clustering suggested that the amino acid sequences of F1VeA and F4VeA showed high similarity to veA homologs of F. verticillioides and F. fujikuroi deposited in GenBank. Conservative structure domain analysis showed that F1VeA and F4VeA had the sequence characteristics of veA gene family in filamentous fungi, which meant that FocVeA might involve in morphological development, toxin biosynthesis and pathogenicity of F. oxysporum f. sp. cubense.

Studies on the Resistance of Transgenic Arabidopsis Overexpressing PcRS to Colletotrichum higginsianum

Liu Zhongyu, Zhao Shujin

2014, 30(10):  107-112. 

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In order to reveal the effectiveness of Polygonum cuspidatum resveratrol synthase（PcRS）gene in increasing tolerance to pathogenic microorganisms in foreign species, the PcRS transgenic Arabidopsis was used as material to identify its molecular characterization and study its resistance to Colletotrichum higginsianum. Integration and expression of transgene in the genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Independent homozygous transgenic Arabidopsis lines with genetical stability were obtained after the selection of T₃ progenies and tested in vitro for disease resistance to C. higginsianum. Agar-plate bioassays were used to test the extracts of transgenic Arabidopsis for antifungal activity against C. higginsianum. Results showed that the mycelial growth of C. higginsianum was greatly inhibited. Then the detached leaves of 4-week-old Arabidopsis plants were used to inoculate. The lesion diameter and spore production significantly reduced on transgenic Arabidopsis. Overexpression of PcRS in transgenic Arabidopsis could restrict colonization of C. higginsianum by inhibition of spore production, resulting in enhanced resistance against C. higginsianum.

Screening of Interacting Proteins with Fungal Elicitor PevD1 by Yeast Two Hybrid System and High Expression of Recombinant in E. coli

Tang Xiaoli, Wu Wenxian, Han Lei, Yang Xiufen

2014, 30(10):  113-118. 

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PevD1, a fungal protein elicitor, was secreted from Verticillium dahliae and could induce hypersensitive responses（HR）and systemic acquired resistances（SAR）in tobacco plant. In this study, we screened its interaction partners using yeast two hybrid system with Arabidopsis thaliana cDNA library. Three potential interacting proteins were obtained. Homologous genes NR1, NR2 and NR4 in Nicotiana tobacum were cloned. Yeast two-hybrid binding assays confirmed NR2 protein strangely binding with PevD1. The entire coding region of NR2 gene was cloned into the bacterial expression vectors pMAL-C2X. After IPTG induction and SDS-PAGE analysis showed that the target protein was expressed at a high level in bacterial cells.

Effects of Crotonaldehyde on the Protein Expression of Rice Suspension Cells

Liu Yueping, Guo Bei, Hu Hao, Zhang Guoqing

2014, 30(10):  119-127. 

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In order to study the important proteins related to cell cycle of rice, the effect of crotonaldehyde on the protein expression of suspension cell line of rice was investigated. The proteins were separated by the method of two dimensional electrophoresis, the different protein spots were analyzed by PDQuest software, and then the different protein spots were identified by LCQ-Fleet ion trap and database searching. The results showed that there were forty-five protein spots expressed differently, and twenty-six of them were identified by mass spectrometry and database analysis. The predicted function of these proteins was related to stress response, protein synthesis and processing, signal transduction, metabolism membrane function. Some of the proteins, including small molecular proteins, Skp1, 26S proteasome, cell division cycle protein 48, glucosamine-6-phosphate acetyltransferase and sucrose synthase, were related with the cell cycle process. Other identified proteins should be study further to explore its function in cell cycle of rice. The normal cell cycle activity was effected by crotonaldehyde, some of the identified proteins were related with regulation of cell cycle. Other proteins related to cell differentiation had no reports about their function to cell cycle;these proteins should be study further to explore the mechanism of cell cycle regulation.

Effect of Adjustment for Inorganic Salt Contents on Adventitious Buds Proliferation and Growth of Opisthopappus taihangensis in vitro

Zhao Yuanzeng, Shan Changjuan

2014, 30(10):  128-133. 

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The effect of different content of MS medium inorganic salts, including macro-element, micro-element, ferric salts, Ca²⁺ and K⁺, on Opisthopappus taihangensis adventitious buds proliferation and growth were investigated using adventitious buds of Opisthopappus taihangensis as test materials. The results showed that the medium containing a relatively higher content of MS macro-element was more suitable for adventitious buds proliferation and growth of Opisthopappus taihangensis. The proliferation and growth of Opisthopappus taihangensis adventitious buds, which were tiny and weak, and many of adventitious buds leaves became yellow or brown, were inhibited on the medium with reduced content of MS macro-element. The adjusted medium with doubled content of KNO₃ and KH₂PO₄ could facilitate the growth and proliferation of Opisthopappus taihangensis adventitious buds;it showed the best performance on the proliferation and growth of adventitious buds among all treatments in the study. The adjustment, whether the Ca²⁺ content increased or decreased in media, had little effect on the proliferation and growth of Opisthopappus taihangensis adventitious buds. The proliferation and growth of Opisthopappus taihangensis adventitious buds was inhibited on the proliferation medium without any micro-element. The proliferation and growth of adventitious buds was strongly inhibited on the proliferation medium containing a twofold content of ferric salts, but slightly affected on the medium with a half content of ferric salts.

Effects of Salt Stress on Telomerase Activity in Relation to DNA Stability of Ammopiptanthus mongolicus Cells

Zhang Xuyu, Wang Jinyu, Zheng Guangshun, Zhang Junqi, Lu Cunfu

2014, 30(10):  134-138. 

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Ammopiptanthus mongolicus, the evergreen broadleaf shrub indigenous to the northwest desert of China, is a residual plant of the ancient subtropical area in the Tertiary Period and has been identified as the national tertiary protection species. This research was conducted to explore the relationship between DNA damage and telomerase activity of Ammopiptanthus mongolicus cells under salt stress. The results showed that telomerase activity was increased during the first 3 d of low salt（100 mmol/L NaCl）treatment. However, when culture cells were treated with 500 mmol/L NaCl, telomerase activity increased rapidly at the initial stage, while declined after 2 day salt stress. Telomerase activity increased 1.4-fold in the recovery phase when 500 mmol/L NaCl was removed from the growth medium. DNA damage was not obvious during the NaCl treatment time or in the phase when NaCl was removed from the growth medium. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient salt stress, and telomerase may play an important role in it.

Cloning,Sequence and Tissue Expression Pattern Analysis of HOXC10 Gene in Multi-costa Properties Yaks of Jinchuan

Zhang Yanan, Xiong Xianrong, Lan Daoliang, Fu Mei, Zhang Yan, Hou Dingchao, Ma Dingmei, Li Shanrong, Li Jian

2014, 30(10):  139-143. 

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The present study aimed at cloning yak HOXC10 gene, and further analysis of the gene structure and function, and reveals the tissue specificity expression pattern. The Jinchuan multi-costa properties yak as materials, this study based on the GenBank published Ovis aries HOXC10 gene sequences for primier designing, using RT-PCR technology to amplification the cDNA full length of HOXC10 gene, and its bioinformatic analysis and expression profiling in various tissues. The sequencing result showed that the length of yak HOXC10 gene is 1 272 bp and contains a 1 029 bp open reading frame（ORF）, encoding 342 amino acids. The results of homology analysis showed that yak and bos taurus has the higher similarity（93.2%）;Followed by predicting HOXC10 protein molecular weight 38.2 kD, the theory of isoelectric point is
8.45. Evolutionary tree analysis results showed that yak was closest relative with bos taurus, and in the same branch. Tissue expression profile analysis showed that HOXC10 gene is expressed in different degrees in various organizations, which had the highest expression in the liver, the lowest expression in the stomach.

Molecular Cloning and Prokaryotic Expression of Prothoracicotropic Hormone cDNA Sequence from Heliothis viriplaca

Zhou Xia, Guo Bozhi, Gao Yanling, Zhao Kuijun, Fan Dong

2014, 30(10):  144-150. 

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Prothoracicotropic hormone（PTTH）is a group of important insect hormones. They have vital effect on regulation of growth, ecdysis, metamorphosis and diapause of insects. Total RNA was isolated from the fifth instar larvae of Heliothis viriplaca. The cDNA sequence was cloned by RT-PCR and rapid amplification of cDNA ends（RACE）. The cDNA sequence was 823 base pairs in length and contained an open reading frame of 681 base pairs, encoding for a polypeptide of 226 amino acid residues, with a predicted molecular weight of 26.3 kD and pI 8.51. Sequence analysis showed that the predicted amino acid shared extensive similarities with those from other insects, especially the lepidopteran noctuid insects. The cDNA sequence has been deposited in GenBank with accession No. JF731347. The cDNA sequence was successfully expressed by the recombinant prokaryotic expression vector pET21b in E. coli. The recombinant protein with His-tag was purified by Ni-NTA affinity chromatography.

Sequence Analysis,Cloning and Induced Expression of Chaperonin Gene in Housefly（Musca domesitca）

Zhao Xuejun, Guo Guo, Wu Qinyi, Tao Ruyu, Wu Jianwei

2014, 30(10):  151-155. 

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The aim of this study is to analyze and predict the structural and characteristics of genes and encoding proteins of MD-TCP Ⅰ（Musca domesitca Chaperonin TCP- 1（MD-TCPⅠ））, with the methods of cloning and expressing that gene. Sequence analysis indicated that the open reading frame was 753 bp, encoding a putative protein consisting of 250-amino acids, which no signal sequence and NCBI-BLAST showed acid sequence identify with other insect TCP-1 were 89%. The protein, with a predicted molecular weight of 27.07 kD, and pI of 5.92, which acid sequence as tcp-1 belong to HSP60 family. The gene coding for MD-TCP Ⅰwas amplified by polymerase chain reaction（PCR）, and then was ligated into vector pET-28a（+）and transformed into Escherichia coli BL21（DE3）competent cell, induced with IPTG. The fusion protein in the expression vector was analyzed by SDA-PAGE. The results indicated that the recombinant plasmid with the correct target gene was constructed, and the fusion protein was expressed in E. coli BL21（DE3）.

Effects of PFOS on Expression of HSP70 in Planarian Dugesia japonica

Gong Xiaoning, Yuan Zuoqing, Bai Yun

2014, 30(10):  156-160. 

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Perfluorooctane sulfonate（PFOS）is a persistent organic pollutant and has been found to be the predominant perfluorinated chemicals（PFCs）in the environment. In this study, we used planarians Dugesia japonica, which belong to the phylum Platyhelminthes, class Turbellaria, as an animal assay to evaluate the toxicological effects of PFOS on protein and mRNA expression of HSP70. The results indicated that the expression of hsp70 mRNA of planarians increased exposed to PFOS in short time and decreased when planarians regenerated of 10-days. However, protein expression of HSP70 increased significantly after regenerating planarians exposed to PFOS for 4- and 7-days, and other groups were not influenced significantly. The trend of mRNA expression were inconsistent with protern. It may be that little PFOS would inhibit the translation of HSP70, and the number of HSP70 increased to protect from toxicity of PFOS as that was accumulated in planarians.

Construction and Characterization of a Mutant of Vibrio alginolyticus ZJ03 Δdldh Strain

Pang Huanying, Chen Liming, Huang Yucong, Jian Jichang, Lu Yishan, Wu Zaohe

2014, 30(10):  161-167. 

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Vibrio alginolyticus, a gram-negative bacterium has been described to be among the most common and economically important aquatic pathogen of fish and shellfish. Dihydrolipoamide dehydrogenase （DLDH）is homodimeric flavoproteins that catalyse the NAD⁺ dependent reoxidation of dihydrolipoamide in a number of multienzyme complexes. This enzyme classically involved in energy metabolism. To identify DLDH’s role during infection, dldh gene deletion strain （ZJ03Δdldh）were constructed using the insertion mutation method. With the pRE112 suicide plasmid as the carrier, the Overlap PCR and homologous recombination technology were used to construct the mutant strains. Furthermore, the changes of the physiology and pathogenicity of ZJ03Δdldh mutant strains compared with the strain ZJ03, such as growth, swarming ability, biofilm and LD50 were investigated. The growth curve showed that the mutant grew slowly in lag phase compared to the wild-type strain, and the swarming ability, enzyme activity and biofilm formation of ZJ03Δdldh mutant strains showed significant reduced（P<0.05）. The fish lethal test showed that the virulence of the ZJ03Δdldh mutant strains was 10² times lower than the ZJ03 strain. In conclusion, the ZJ03Δdldh mutant strains were constructed successfully, and this study showed that enzymes classically involved in energy metabolism may play an important role in pathogenic mechanism of V. alginolyticus.

A Preliminary Study on the Biological Function of a Gene p17 Located on a Linear Plasmid pBSSB1 of Salmonella enterica serovar Typhi

Zhu Yunxia, Wu Jia, Gong Mingyu, Hou Shuning, Zhang Qisi, Chen Minjie, Zhang Haifang

2014, 30(10):  168-173. 

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pBSSB1 is a linear plasmid which mediates the flagellar phase variation in H：z66 positive Salmonella enterica serovar Typhi（S. Typhi）. The gene named p17 is located on pBSSB1 and it encodes the protein P17 whose function is unknown. The p17 deleted mutant of S. Typhi was prepared through the λ-Red recombination system. The growth curves of wild type and p17 mutant strain were detected to compare their growth ability. The motility of wild type and p17 mutant strains was compared through the experiments on the semi-solid LB plates. The specific primers were designed to amplify the gene p17 by PCR. The amplicon was inserted into the expression vector pET28a（+）to construct recombinant vector pET28a（+）-p17, which was then transferred to E. coli BL21（DE3）to be expressed. The recombinant protein P17-His₆ was purified with Ni-TED packed column and was used as immunogen to prepare the rabbit anti-P17 polyclonal antibody. The results showed that p17 deleted mutant of S. Typhi was constructed successfully. It was showed that the growth of p17 mutant strain was significantly slower compared with the wild type strain（P<0.05）. The motility of p17 mutant strain was decreased obviously compared to the wild type strain. The gene p17 of S. Typhi was inserted into the vector pET-28a（+）and was expressed in E. coli BL21（DE3）. The rabbit anti-P17 polyclonal antibody was prepared.

Isolation and Identification of an Actinomycetes Strain Against Root-knot Nematode from Mangrove Soil

Chen Yuqing, Huang Huiqin, Liu Min, Bao, Shixiang

2014, 30(10):  174-178. 

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110 Actinomycetes strains were isolated by plate dilution method and anti-root-knot nematode strain HA10401 was screened by 24-well plate fluid model from mangrove soil in Dongzhaigang National Nature Reserve, Hainan Province. While the fermentation broth was diluted by 20 times and 40 times, the mortality rates against nematode were 58.2% and 52.6%, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain HA10401 was closely related to Streptomyces variabilis with the highest similarity of 99.8%, and the two strains formed a cluster in the phylogenetic tree. The morphological, cultural and biochemical characteristics were basically accorded with Streptomyces variabilis. As a result, strain HA10401 was identified as Streptomyces variabilis, and its anti-root-knot nematode activity was first reported in this study and worth further research.

Isolation and Primary Identification of Salt-tolerant Bacillus in Northeast of China

Du Chuanying, Li Haitao, Liu Rongmei, Xie Binjiao, Zhang Jinbo, Gao Jiguo

2014, 30(10):  179-187. 

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To analyze the biodiversity of salt-tolerant Bacillus in the northeast of China, the temperature screening and gradient plate methods were used. 137 Bacillus strains were obtained, in which has 74 salt-tolerant Bacillus strains, 54% of total Bacillus strains. The optimum salt concentrations of these salt-tolerant Bacillus strains were all 1% and the range were 4%-14%. 16S rRNA sequences were amplified and analyzed for the determination of phylogenetic relationships. The 36 salt-tolerant Bacillus strains were different from each other in their 16S rRNA sequences or the salt tolerance, classified into 7 species of Bacillus genera. Among them, Bacillus thuringiensis was majority（14 strains, 38.9% of all, highest salt tolerance were 4%-9%NaCl）, second were Bacillus cereus（7, 19.4%, 4%-8%）and Bacillus subtilis（7, 19.4%, 8%-11%）, then Bacillus anthracis（4, 11.1%, 5%-7%）, Bacillus flexus（2, 5.6%, 9%-14%）, Bacillus sphaericus（1, 2.8%, 5%）and Bacillus aryabhattai（1, 2.8%, 6%）. Bacillus flexus and Bacillus subtilis were shown better salt tolerance. Cultural and morphology characteristics, biochemical indexes and phylogenetic analysis of 3 typical strains were given. Provide an effective data support for the discovery of salt-tolerant Bacillus.

Diversity and Phylogeny of Rhizobium Isolated from Root Nodules of Legumes in Jiuquan and Other Regions,Gansu

Li Zheng, Shan Huihui, Qi Yalin, Liu Lei, Han Suzhen

2014, 30(10):  188-195. 

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The 16S rDNA PCR-RFLP and 16S rDNA sequence analysis were used on genetic and phylogeny of 53 strains isolated from Glycine max, Vicia faba, Pisum sativum and Phaseolus vulgaris of Jiuquan and other regions, Gansu and 9 known strains. Results of 16S rDNA PCR-RFLP indicated that all isolates included 9 reference strains were clustered into 5 branches at 77.5% similarity. Branch I was Sinorhizhobium-Rhizobium, branch II Agrorhizobium-Bradyrhizobium, branch III unknown bacteria branch, branch IV Mesorhizobium, and branch V also unknown branch. Branch I included 2 sub branches at 89.8% similarity：Sub branch 1 included strains CNU1008 and 14 strains witch clustered with Sinorhizobium meliloti LISPA1002^T, Subbranch 2 included 6 strains which clustered with Rhizobium leguminosarum USDA 2370 ^T. Branch II included 3 sub branches：Sub branch 1 included CNU 1041 and other 3 strains witch clustered with 2 strains of Agrobacterium tumefaciens IAM 13129^T and IAM 13569^T together, sub branch 2 included 5 strains which clustered with Bradyrhizobium japonicum USDA 6^T, sub branch 3 included 3 unknown strains. Representative strains were selected in the analysis of 16S rDNA sequencing, and the results show that 16S rDNA PCR-RFLP and16S rDNA sequencing analysis were in good agreement. Strains of Sinorhizobium sub branch of branch I were clustered with S.fredii and S.meliloti at 99% similarity, and their exact system status should be determined by DNA-DNA hybridization. Strains of Rhizobium sub branch of branch I were clustered with R.giardinii at 99% similarity and with R.etli at 98% similarity. So the exact position of the sub branch waited for DNA-DNA hybridization to determine. Strains of Agrobacterium sub branch of the branch II clustered with A.rubi at the similarity of 99%. Strains of Bradyrhizobium sub branch of the branch II were clustered with B.liaoningense at the similarity of 99%.

Analysis of Bacterial Community Structure and Diversity in Wild Ophicordyceps sinensis

Li Hui, Liu Yang, Bai Feirong, Yao Su, Zahi Nagi, Cheng Chi

2014, 30(10):  196-200. 

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The bacterial community structure of eight wild Ophicordyceps sinensis were investigated using PCR-DGGE.DNA sequencing was proceeded to obtain dominant bacterial population information in different samples. The result of DGGE profile showed that all the samlples contained abundant bacterial community, different samples have some differences among the Shannon-winner index, Richess and evenness, significant differences were observed between samples from distinct locations. Flavihumibacter petaseus, Sphingomonas aestuarii, Geobacillus pallidus and Methylovirgula ligni were commonly detected in all samples, which laid a foundation for further studying on the roles of bacteria during the formation process of Ophicordyceps sinensis.

The Construction of E.coli Engineering Strain for Sialyllactose Production

Jin Wenbin, Zhang Xiaoxiao, Li Yu, Lu Fuping

2014, 30(10):  201-206. 

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Free sialylated oligosaccharides are known to have anti-infective and immunostimulating properties and also known to promote bifidobacterium proliferation, thus investigating the microbial synthetic route of sialylated oligosaccharides is of great value. Biosynthesis of sialyllactose involves N-acetylglucosamine-6-phosphate-epimerase（neuC）, sialic acid synthase（neuB）, CMP-Neu5Ac synthetase（neuA）and α-2, 3-sialyltransferase（nst）. We engineered a biosynthetic pathway sialyllactose production in E.coli JM109, using the expression vector pSTV29, by coexpressing the α-2, 3-sialyltransferase gene from Neisseria meningitidis with the neuA, neuB and neuC Campylobacter jejuni genes encoding CMP-NeuAc synthetase, sialic acid synthase and N-acetylglucosamine-6-phosphate-epimerase, respectively. Under the optimized fermentation conditions（inoculum volume 2%, 10g/L lactose, fermentation volume 50 mL/250 mL, temperature 34℃, rotation speed 180 r/min, fermentation time 30 h）, sialyllactose of 2.45 g/L was obtained. The above results provide a platform for exploring commercial production of sialylated oligosaccharides and its functional analogues in heterogeneous microbial hosts.

Study on Fermentation and Conversion of L-cysteine by Pseudomonas sp. F-12

Zhao Jingnan, Li Zhimin, Ye Qin

2014, 30(10):  207-214. 

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L-cysteine has been widely applied in food and pharmaceuticals. In this study, different fermentation strategies of Pseudomonas sp. F-12 transforming DL-ATC to L-cysteine were investigated. The results showed that the highest enzyme activity of 15 g/L DL-ATC batch fermentation was 149.1 U/mL, specific activity was 70.9 U/mg DCW. The highest enzyme activity of two phase fermentation using 15 g/L DL-ATC was 313.3 U/mL, 2.1 times higher than the batch fermentation. The amount of DL-ATC was reduced to 5 g/L. In this study, the major factors on the biotransformation of L-cysteine by Pseudomonas sp. F-12 were investigated. The optimal reaction conditions for cells were 30℃, pH 8.0. The addition of organic solvents could change cellular permeability and enhance the production of L-cysteine by 7.16 times. The maximum conversion of L-cysteine was 93.3%. 0.1 mmol/L Fe²⁺ benefitted the reaction, while 0.1 mmol/L Co²⁺, Zn²⁺, Ni²⁺ and other heavy metal ions inhibited transformation.

Polymorphisms and Expression of Macrophage Migration Inhibitory Factor Gene from Channel Catfish,Ictalurus punctatus

Gao Lei, Du Xiaoxi, Li Le, Gao Xianggang, Bao Xiangbo, He Chongbo

2014, 30(10):  215-222. 

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Macrophage migration inhibitory factor（MIF）was considered as a unique cytokine produced by T-cell and involved in immune response to inflammation and stress. In this study, the genomic sequence of MIF was identified, and the polymorphisms and expression level were studied in channel catfish Ictalurus punctatus. The genomic sequence of MIF was identified to be 3 918 bp, consisting of a 70 bp 5'-untranslated region（UTR）, a 160 bp 3'-UTR, a 348 bp open reading frame（ORF）, two introns as 243 and 1 441 bp, and a 1382 bp promoter. Four repetitive sequences were found in promoter and introns. Sixty sites of single nucleotide polymorphisms（SNPs）were identified in MIF genomic sequence, among which 6 sites were found to locate in the binding sites for transcription factor. MIF was found to be most abundantly expressed in intestine, and weaker in other tissues. These results could give more information and greater understanding of the degree of MIF genetic variation.

Toxicity of Beta-cypermethrin for Zebrafish Embryos

Wang Jian, Liu Lili, Yu Kaimin, Li Guochao, Wu Wei, Yan Yanchun

2014, 30(10):  223-229. 

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Beta-cypermethrin, an insecticide of type II pyrethroids, is highly toxic to fish and other aquatic organism and the residues can be detected in many foods. The impact of beta-cypermethrin for human health can not be neglected. To better define the toxicity mechanism of beta-cypermethrin for fish and human, the embryos of zebrafish were exposed to different sub-lethal concentrations of 0.05, 0.1, 0.2, 0.6 and 1 mg/L of beta-CYP solutions. With the help of a microscope, we observed the morphological changes. The results displayed that exposed to beta-CYP caused the embryonic toxicity increased in a dose-dependent manner. We chose zebrafish embryos exposure to beta-CYP（0.1 mol/L）as a model system to investigate the toxic effects on early development. A two-dimensional difference gel electrophoresis（2-D DIGE）coupled with MALDI-TOF-MS/MS analysis was employed to detect and identify the differential expressed proteins. In the present study, a total of 11 different proteins were successfully identified, which were mostly associated with cytoskeletal, stress response and metabolism. This study brings useful information and contributes to a better understanding about the embryo toxicity organisms of beta-CYP.

Research Progress of Transgenic Maize in China Based on Bibliometrics

Wang Ning

2014, 30(10):  230-234. 

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Based on the data of China Series Full-Text Database, the number of articles per year, mainly discipline, mainly level, distribution of journals, funds, core authors and affiliations, download times, and cited times of articles related to transgenic maize during 2004-2013 were statistically studied by the bibliometrics. The results revealed that the number of articles per year was increased while the download and cited times were decreased annually, and the transgenic maize articles focused on crops disciplines, the basic and applied basic research（natural）level has the largest proportion, they mainly published in professional journals, the funds which in national level is dominated, and the study from core authors and affiliations have good continuity.