Biotechnology Bulletin

Analysis，Comparison and Classification of Metagenomic Samples

CHENG Fu-dong,DING Xiao,LI Sheng,SUN Xiao

2016, 32(5): 1-10. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.001

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Metagenomics attempts to understand the diversity of the environmental microbial community and the interaction between microorganisms and environment by analyzing the sequence data of metagenomic samples. Microbiology has been revolutionized by metagenomics，which makes it feasible to research the microbial communities in complex biological systems without cultivating the microbes. The high-throughput metagenomic study is promoted by the rapid development of next-generation sequencing technology and bioinformatics. As a mass of high-quality metagenomic sequencing data are produced，also are accessible to the scientific community，the role of metagenomics has been recognized by various scientific areas. On the other sides，huge metagenomic data for individuals with different health status，or for different habitats of the human body makes the comparison and classification of metagenomic samples more important，leading the comparative metagenomics to become an important branch of metagenomics. This review mainly introduces the related researches and algorithms in the analysis，comparison and classification of metagenomic sequencing data.

Research Progress on Methanotrophic Bacteria in Landfills and the Reduction of Methane Emission

WANG Xiao-lin,CAO Ai-xin,ZHOU Chuan-bin,ZHAO Kai-ning,ZHAO Guo-zhu

2016, 32(5): 16-25. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.003

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As the main source of anthropogenic methane emission，landfills globally produce 35-69 Tg methane per year. The technology of reducing the methane emission in landfills has become a hot topic at present. Methanotrophic bacteria decomposing methane are the important biological collection to reduce atmospheric methane emissions，which is of significance in keeping the balance of the methane concentration in the atmosphere. Starting from the taxonomy and characteristics of methanotrophic bacteria，and the mechanism of its oxidizing methane，we summarized the latest research progress on the methods of studying diversity，factors affecting the activities of methanotrophic bacteria in landfills，and applications of them in the biological reduction of methane emission. Based on the prior researches，the issues in current studies of methanotrophic bacteria are also discussed. We propose comprehensive measures of utilizing the complex microbial agents of methanotrophic bacteria in landfills，providing a new thought in the research and application of reducing methane emission in landfills.

Research Progress on the Biosynthetic and Regulatory Mechanisms of Natamycin in Streptomyces natalensis

LU Shi-yao,SUN Li-jie,YUAN Li-xia,ZHU Wei-wei,YAO Jian-ming

2016, 32(5): 26-33. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.004

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Natamycin，a polyene-macrolide antibiotic，is a secondary metabolite produced by Streptomyces natalensis. It can not only effectively inhibit the growth of fungi，but also depress the generation of aflatoxin. Thus，it is widely used in the fields such as food preservatives，remedial reagent of fungal keratitis，etc. Skeleton ring of natamycin is firstly synthesized by the catalyst of acetic acid-activated polyketone synthase，and then modified by some other correlated synthases，involving the regulation and expression mechanisms of natamycin biosynthesis-related genes（i.e.，transcriptional regulator’s regulation，pathway-specific regulation，global regulation，and cross-talk regulation，as well as ROS regulation，etc）. A matured molecule of natamycin can be exported out of cells through intracellular transporters. Finally，perspective in the future has been proposed based on the research progress of latest reports.

Research Progress on the Strategies of Preventing Drug-resistance in Fishery

PAN Hao,WANG Di,LU Tong-yan

2016, 32(5): 34-39. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.005

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Currently，the drug treatment is still the main way of preventing and controlling the aquatic animal diseases，but abuse of drugs has also led to a large number of drug-resistant bacteria. How to formulate a reasonable strategies of preventing drug-resistance while applying common drugs in fishery has become the focus on the current study. This paper systematically reviewed the window theory of drug-resistant mutation selection，and the research progress on the strategies of preventing drug-resistance such as applying Chinese herbs and the effects of culture conditions.

Research Progress on Protein Microcapsules

HE Xiao-lin,TENG Kai,GUO Shu-yuan

2016, 32(5): 40-46. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.006

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Protein drugs are easy to be degraded and inactivated. Thus how to sustain the activity of protein to make them function effectively has been a key problem in the application of protein drugs. Microencapsulation is to load a protein into a capsule，therefore，the target protein is isolated from the environment and could be protected. This paper reviews several common preparation methods of protein microcapsules，including layer-by-layer self-assembly，emulsification process，electrostatic droplet generation，etc.，which lays a foundation for the wide application of protein microcapsule in the future.

The Development of CRISPR/Cas9 Technique and Its Applications in Genome Editing

ZHANG Kai-li,LI Rui,HU Tong-tong,XU Yong-jie

2016, 32(5): 47-60. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.007

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CRISPR/Cas9 is one of novel genome editing technologies developed from the unique prokaryotic acquired immune system in bacteria and archaea，and it can be guided to specific locations within complex genomes by a short RNA，and thus cleavage of the target can be recognized. Using this technology，DNA sequences within the endogenous genome and their expressed products are now easily edited or regulated in virtually any organism of choice. The technology has become the most popular genome editing tool，promoting the development of basic biology，biotechnology and medicine. This paper introduced the research progress on CRISPR/Cas9 from the aspects of development history，structure and function，and the mechanism of precise recognition，also reviewed the application of this new technology in genome editing in detailed，aiming at providing a reference for related researchers.

The Transformation of Drought-resistant Gene into Maize by Microprojectile Bombardment

LI Zhi-liang,WU Zhong-yi,YANG Qing,ZHANG Xi-tai,YE Jia,XING Hao-chun,CHEN Jian-zhong,HUANG Cong-lin,

2016, 32(5): 61-68. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.008

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A drought-resistance function gene，VaP5CS，was cloned from mothbean（Vigna aconitifolia）. Plant expression vector pBPC-P5CS-F129A was constructed using gene engineering strategy，then the genetic transformation of young embryo callus in maize inbred line Jing 501 was performed by microprojectile bombardment. Further，the transformed explants were directly selected on the medium containing 10 mg/L glufosinate，and the glufosinate-resistant plantlets were confirmed by PCR amplification and Northern blot identification. Finally，the different drought resistance indexes of P5CS-transgenic maize plants were analyzed. The results showed that the transgenic plants presented the improved drought resistance comparing to control plants. Under drought stress，the proline content in the P5CS-transgenic maize plant was higher than that in the control，while the MDA content was lower than that in the control.

Cloning，Expression and Bioinformatics Analysis of IQM5.2 from Arabidopsis

GONG Lu-ping,XIAO Wen-hui,ZHOU Yu-ping,HUANG Xiao-ling,TIAN Chang-en

2016, 32(5): 69-74. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.009

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The Arabidopsis IQM5，the fifth member of IQM family，is encoded by At5g57010. So far，there is only one splicing mode found for the IQM5 gene in the biological literatures and databases，i.e.，At5g57010.1（IQM5.1）. In this study，a new splicing mode for IQM5 gene was found by RT-PCR and T-A cloning method，designated as At5g57010.2（IQM5.2）. Bioinformatics methods and semi-quantitative and quantitative RT-PCR were employed to analyze the properties of proteins encoded by new CDS sequence，and the ratio of IQM5.1 and IQM5.2 in different organs，respectively. The results revealed that IQM5.2 encoded a polypeptide of 300 amino-acid residues，and its N-terminus shared sequence homology with the pea heavy metal-induced protein 6A（HMIP6A）. The protein retained an IQ motif and was a calmodulin-binding protein of the calcium independent. There were transcripts of IQM5.1 and IQM5.2 in the seedling，rosette leaf，cauline leaf，stem，and flower bud，moreover，the ratios of them in the different organs varied.

Eukaryotic Expression，Purification and Activity Testing of Human C-Src Protein Tyrosine Kinase

XU Lan,XIAO Bin,CHEN Hui-qin,LI Xiao-rong,HAO Wen-bo

2016, 32(5): 75-81. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.010

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The objective of this work is to construct a eukaryotic expression vector for the C-Src tyrosine kinase（Csk）gene from human. Total RNA was extracted from HeLa cells. The full-length cDNA sequence of Csk gene was isolated and amplified via RT-PCR，and cloned into a eukaryotic expression vector pENTER，the recombinant pENTER-Csk-his plasmid was constructed. The recombinant plasmid was transfected into 293T cells，after 48 hours，the expression levels of Csk protein were determined by SDS-PAGE and Western blot. The localization of Csk protein was detected via indirect immunofluorescence，and the protein Csk was purified by nickel chelate beads；in addition，the activity of the protein was measured via his-pulldown and CO-IP. Results showed that：Double digestion and sequencing reveled that recombinant plasmid pENTER-Csk-his was constructed properly without mutation. Both SDS-PAGE and Western blot detected a 51 kD protein，indicating that Csk protein was expressed successfully in the 239T cells. The indirect immunofluorescence confirmed the expression of Csk protein in cytoplasm. Finally，the purified protein Csk by nickel chelate beads interacted with the IGF1R and SHC1 by his-pulldown and CO-IP. Conclusively，this study successfully acquired the full-length sequence of Csk，the eukaryotic expression vector pENTER-Csk-his was constructed，and the gene was expressed efficiently in 293T cells，moreover，the expressed protein presented bioactivity.

Eukaryotic Expression of MSTN Gene from Hetian Sheep，Carla Kul Sheep，and Duolang Sheep in the Southern Xinjiang

WANG Hai-tao,JIN Bo,LI Shu-wei

2016, 32(5): 82-90. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.011

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This work aims to construct the fusion expression plasmids of myostatin gene（MSTN）from 4 local varieties（lines）sheep，namely plain Hetian sheep（HP），mountain Hetian sheep（HS），Carla Kul sheep（KL），and Duolang sheep（DL）in the southern Xinjiang with enhanced green fluorescent protein（EGFP），and the expression of MSTN in eukaryotic cells. Total RNA was extracted from the skeletal muscle in 4 varieties of sheep，cDNA was acquired by reverse transcription，and MSTN amplified by PCR was ligated with pMD18-T vector to construct recombinant plasmid and sequenced. Then MSTN（HP，HS，DL，and KL）gene was inserted into the fusion expression vector pEGFP-N1，the recombinant plasmid of pEGFP-N1-MSTN in a eukaryotic fusion expression was constructed. The plasmid with over 80% cells covered was transfected into CHO by liposome，the green fluorescent of fusion protein of EGFP and MSTN in CHO cells was observed. The results showed that the ORF sequence of MSTN gene in any of HP，HS，KL，and DL was 1 125 bp（excluding stop codon），encoding 375 amino acids. Comparing MSTN sequences in GenBank，there was differences in HS and HP；and 2 nucleotide variation in KL caused 2 amino acid mutations；and 6 nucleotide differences in DL resulted in 4 amino acid variations. In conclusion，the recombinant plasmid for EFGP and MSTN co-expression was constructed and expressed in eukaryotic CHO cells.

The Genetic Diversity of Six Chinese Sheep Breeds by Six Microsatellite Markers

CHEN Li-peng,HUANG Yong-fu,ZHAO Yong-ju,E Guang-xin,NA Ri-su

2016, 32(5): 91-98. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.012

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Six microsatellite loci were used to analyze the genetic diversity of 280 sheep from 6 Chinese breeds of Ujumqin sheep，Small-tailed Han sheep，Tan sheep，Zhaotong sheep，Hulun Buir sheep，and Gansu Alpine Merino. We calculated the polymorphism information content，heterozygosity and genetic distance，analyzed the principal components and clustered UPGMA from these 6 sheep breeds. Altogether，101 alleles were found，the mean heterozygosity was 0.599-0.691，and mean polymorphism information content was 0.609-0.680. The Gansu Alpine Merino sheep had the most genetic divergence compared to other sheep breeds，while the Hulun Buir and Ujumqin ones were the least in genetic divergence. The above results showed that these 6 breeds had relatively high genetic diversity，and their genetic relationships were consistent with their formation history，differentiation and geographical distribution.

Cloning and Analysis of DNA and cDNA Sequence in NPY Gene of Northern and Florida Subspecies of Largemouth Bass

LIU Hao,BAI Jun-jie,LI Sheng-jie

2016, 32(5): 99-106. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.013

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Neuropeptide Y（NPY）plays an important role in the activity of body feeding，and is the most potent endogenic orexigenic factor in mammals. In order to understand the structure and the function of NPY gene in the largemouth bass（Micropterus salmoide），the NPY’s cDNA of northern and Florida subspecies of largemouth bass were cloned by RT-PCR and RACE techniques. The result revealed that the NPY’s cDNA in two subspecies all contained an ORF encoding 99 amino acids and 5′-UTR of 52 bp. Using PCR and genomewalker technology，a 3 561 bp and a 3 565 bp DNA sequence of of NPY gene were acquired from northern and Florida subspecies of largemouth bass respectively. Sequence analysis indicated that gene from both subspecies contained 4 exons and 3 introns. By bioinformatics analysis with MATINSPECTOR，the basic transcriptional regulatory elements such as TATA Box，CAAT Box，CCAAT Box，and GATA Box were found in the promoters of both subspecies. Six single-site base differences between the two genomic NPY DNA sequences of 2 subspecies were discovered，4-base insert segments were identified in promoter sequence of Florida subspecies compared with the northern one. The homology of NPY genes with other species was also compared，the largemouth bass shared the highest nucleotide homology with mandarin fish and grouper，reached 90% and 88% respectively，and the amino acid homology were 93% and 95%.

Cloning and Prokaryotic Expression of Interferon Regulatory Factor 2 from Takifugu rubripes

SUN Sai-hong,MA Pu,SUN He,KONG De-rong,Li Hui,QIU Xue-mei,JIANG Zhi-qiang,LIU Hai-ying,LIU Yang

2016, 32(5): 107-113. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.014

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Interferon Regulatory Factor（IRF）is a transcription factor with multifunctions. The full length cDNA of Takifugu rubripes interferon regulatory factor 2（IRF2）was cloned by RT-PCR and rapid amplification of cDNA ends（RACE）. The full-length cDNA of the gene was 1 552 bp，including a 187 bp 5' non-coding region，a 429 bp 3' non-coding region，and 936 bp coding region，and it encoded 311 amino acids. The recombinant pET32a（+）-IRF2 was constructed by ligating code sequence of IRF2 with prokaryotic expression vector pET32a（+）. Then，the recombinant genetic engineering bacteria with expressed IRF2 were obtained by transforming pET32a（+）-IRF2 into competent cell Escherichia coli BL21（DE3）. pET32a（+）-IRF2 was expressed in BL21 under the induction of IPTG. The results from the detection of the purified products by Western blotting showed that the expression of the IRF2 gene in E. coli was high，and the target protein was accurate.

Cloning and Expression Analysis of Keratin 18 Gene in Loach（Misgurnus anguillicaudatus）

WU Wei-jun,LIU Shi-li,LI Qian,WANG Yu-chen,LIAN Qing-ping,HU Ting-jian,JIA Yong-yi,JIANG Wen-ping,LI Xi-lian

2016, 32(5): 114-123. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.015

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Keratin 18（K18），one of the keratin，is the intermediate filament proteins and an integral part of the cytoskeleton. In order to understand the sequence characteristics and expression of K18，cDNA full-length sequence of K18 gene was cloned from loach（Misgurnus anguillicaudatus）using RACE，and its expression in different tissues was explored using real-time quantitative PCR. The results showed that the cDNA full-length of cloned K18 gene was 1 694 bp，containing a 1 299 bp open reading frame encoding 432 amino acids. There were the sequence DGKVVS，and the very similar sequence DNA（R/K）LAADDF of type I keratin. Identity analysis revealed that loach had the highest identity up to 98% with large-scale loach（Paramisgurnus dabryanus）in K18，and there were various levels of identity with other species. Phylogenetic analysis demonstrated that the K18 gene of loach was in phylogenetic homology with other species，and in true orthologs with terrestrial vertebrates（tetrapods）and teleosts，moreover，closest with large-scale loach. K18 gene was expressed in the 8 tissues of intestine，muscle，heart，stomach，liver，testis，ovary，spleen，the highest in the intestine，and the lowest in the heart. Statistical analysis showed that the differences of expression among different tissues were significant.

Cloning and Analysis of Gene for Adenine Nucleotide Translocase in Portunus trituberculatus

LI Bing-yue,ZHU Dong-fa,QIU Xi-er,ZHOU Yan-qi,LIU Zhi-ye,XIE Xi

2016, 32(5): 124-130. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.016

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Adenine nucleotide translocase（ANT）is a transport protein responsible for the conduction of energy molecules in the inner membrane of mitochondria，playing a vital role in the energy metabolism. In order to study the role of ANT gene in the molting of crustacean，a full-length cDNA of ANT gene（GenBank access number：KM921660）was cloned from Portunus trituberculatus using RT-PCR and RACE. Sequence analysis showed that it was 1 414 bp long，including a 132 bp 5' non-coding region，a 352 bp 3' non-coding region，and a 930 bp coding region，and encoded 309 amino acids. The phylogenetic comparison of ANT gene sequence between P. trituberculatus and other species revealed that ANT gene from P. trituberculatus and other species clustered as one same branch，and the similarity with Scylla paramamosain was up to 96%. Amino acid alignment proved that it had 3 conserved mitochondrial transmembrane domains，forming a channel for the conduction of energy molecules，and catalyzing transmembrane exchange between ATP in mitochondria and ADP in cytoplasm. Real-time PCR results indicated that ANT gene expressed in all 10 different tissues with the highest expression in muscle，while quite low in all other tissues，and there were significant differences among the expressions of the tissues（P<0.05）. During the whole molting process，the level of ANT in muscle reached the maximum at postmolt（stage A），then began to decline until the least during intermolt（stage C），and then gradually rose until the second peak at stage D1，then declined again. The combined result indicated that ANT gene was closely correlated with the activity of muscle of P. trituberculatus，and might play an essential role in the molting.

Cloning and Expression Analysis of Spook Gene in Portunus trituberculatus

ZHOU Yanqi,ZHU Dong-fa

2016, 32(5): 131-139. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.017

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Ecdysteroids in crustaceans are mainly synthesized and secreted in the Y-organs（YO），and involve in the many essential physiological activities such as molting，reproduction，etc. The cytochrome P450（CYP）307a1 encoded by Spook is the key enzyme in early steps of ecdysteroids biosynthesis. To study the regulatory role of Spook in molting process，the full-length cDNA of gene was cloned and characterized from Portunus trituberculatus（GenBank accession number：KM030021）by reverse transcription PCR（RT-PCR）and RACE，designated as Pt-Spook. The cDNA of Pt-Spook was 2 200 bp in length and contained an opening reading frame（ORF）of 1 563 bp encoding 520 amino acid residues. Multiple alignment revealed that the Pt-Spook contained 5 conserved domains of helix-C，helix-K，helix-I，PERF and heme-binding. The analysis by phylogenetic tree discovered that the Pt-Spook clustered in the same branch with other species，however，Halloween genes were in another branch，indicating that the deduced amino acid sequence was truly the protein sequence of P. trituberculatus Spook. The tissue distribution of Pt-Spook was detected by qPCR，and the results showed that Pt-spook expression was highly specific to YO. During the molt cycle，the expression of Pt-spook gradually increased from post-molt（stage A and B），and reached the maximum in intermolt（stage C），then gradually decreased during pre-molt to the minimum in sub-stage D3 and D4. The results suggested that the Pt-spook involved in the regulation of molt cycle in P. trituberculatus.

Research on the Expression of Porcine Circovirus Capsid Protein in the Bacillus subtilis

CHEN Ling-yan,CHEN Xian-jin,JIANG Ye,Lü Tun

2016, 32(5): 140-145. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.018

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The objective is to study the expression of porcine circovirus capsid protein in the Bacillus subtilis. Based on 3 different plasmids，different types of recombinant vectors for porcine circovirus capsid protein gene（PCV2-ORF2）were constructed，and they were transformed into strain 168 and WB800 of B. subtilis. The expression of the target protein was detected by SDS-PAGE and Western blot，and the mRNA expression of the target gene in B. subtilis was detected through RT-qPCR. The original ORF2 was difficult to be translated in B. subtilis expression system. After the codon optimization，truncated ORF2 gene was successfully expressed as target protein in intracellular expression system of B. subtilis. Thus，the optimization of the codons promoted the expression of the target gene.

Construction of Eukaryotic Expression Vector for Glypican-3 and Its Expression in Cell

CHENG Hua,FANG Xiang-dong,CUI Shuo,WU Meng,ZHANG Zhao-jun,LI Ze-xia

2016, 32(5): 146-150. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.019

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This work aims to construct the eukaryotic expression vector of glypican-3（GPC3）for acquiring anti-GPC3 monoclonal antibody. The GPC3 gene was amplified by PCR and was cloned into an expression vector p3XFLAG-CMV-14，and the expression vector pCMV-gpc3 was constructed. HEK293 cells were transfected with the vector by liposome，and the final results were detected by Western blot. The stably-expressed cells were collected and ground，and the high-purity GPC3 protein was obtained by affinity column. The eukaryotic expression vector of pCMV-gpc3 was constructed successfully. After the transfection to HEK293 cells，the monoclonal cell line of stably-expressed was gained by G418 screening. Western blot analysis revealed that the target protein expressed markedly. A large number of GPC3 proteins were obtained via genetic engineering and purifying technology，which laid a foundation for the biological function and application of the protein as well as the preparation of monoclonal antibody.

The Identification of a Fungal Endophyte of Suaeda salsa and Preliminarily Research on Active Products of Promoting Growth

YU Fei,GU Yue,ZHANG Qi,BU Ning

2016, 32(5): 151-157. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.020

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The purposes of this work are to identify the endophyte fungus JP4-1 of Suaeda salsa in Red Beach of Panjin，Liaoning Province，and to research the growth promotion activity of its metabolites. Endophytic fungus JP4-1 was identified using its morphological features and 18S rDNA，and several technical means such as filtering centrifuge，ultrasonic crushing，extraction，and rotary evaporateion were used to extract its active components，and hydroponic experiments were taken to test the growth promotion activity of its fermentation broth to rice seedlings. The JP4-1 was identified as Glomerella sp. with hyphae branched，septate，and producing spores. The result of hydroponic experiment revealed that the micromolecular products extracted from fermented liquid with n-butanol promoted the plant height 10.84%，dry weight of leaves 14.67%，and chlorophyll content 44.22% of the rice seedlings，i.e.，the growth promotion effects were remarkable. In conclusion，the extracellular fermentation products by the endophyte fungus JP4-1 of S. salsa significantly promoted the growth of rice seedlings，and the micromolecular active products extracted by n-butanol had the most significant promotion effects.

Identification of a Moderately Halophilic Bacterium Halobacillus dabanensis N522 and Study of Its Antimicrobial Activity

NI Zhi-hua,ZHANG Yu-ming,ZHOU Yan-fen，

2016, 32(5): 158-164. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.021

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The aims of this study are to isolate and identify halophilic bacteria from the sediments in Xuwen Solar Salt Field of Guangdong Province，to investigate the culture conditions of the target strain，and to study the physiochemical properties of active antimicrobial products in the fermentation. Serial dilution and plating method was employed to isolate halophilic bacteria from sediment samples. Then，the isolated strain was identified by physiological and biochemical experiment as well as 16S rRNA gene sequence analysis. Further，the antimicrobial activity of the target strain fermentation broth was detected using several bacteria，yeasts and fungi by cylinder plate method，and the physicochemical property of antimicrobial materials was preliminarily studied. A halophilic bacterium was isolated and identified as Halobacillus dabanensis strain N522 based on 16S rRNA sequence comparison and physiological and biochemical tests. The strain N522 belonged to moderately halophilic bacterium due to its optimal growth concentration of NaCl was 100 g/L，and pH was 7.0. The fermentation broth of the strain inhibited the growth of several bacteria and fungi. Particularly，the strain showed the strongest antimicrobial activity to Staphylococcus aureus. The experiments of ammonium sulfate salting-out and protease degradation preliminarily revealed that the materials with antimicrobial activity in the broth of strain N522 was peptides，and its molecular weight was between 3 kD to 5 kD. Moreover，the antimicrobial peptides were resistant against pH and temperature. In conclusion，the moderately halophilic bacterium（H. dabanensis N522）isolated from the sediments of the Solar Salt Field may be developed as a potential new source for the novel antimicrobial medicines.

Screening of Exopolysaccharide-producing Strains and Structural Analysis of the Exopolysaccharides

LI Bin,CHEN Xiang-nan,ZHANG Jian-fa,WANG Shi-ming

2016, 32(5): 165-171. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.022

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This study aims to screen and identify strains producing exopolysaccharides（EPSs），to study the rheological properties of EPSs，and to analyze the structure of EPSs. The strains producing EPSs were screened from soil，and were identified based on their morphology and 16S rDNA gene sequencing. The viscosity changes of EPSs in different conditions were determined by viscometer to study its rheological properties. Then，GC，IR and NMR were used to analyze the structure of EPSs isolated and purified by ethanol precipitation，Sevag method and dialysis. As a result，a strain producing EPSs was identified as Enterobacter sp. WL113. The viscosity of the EPSs exponentially increased with the concentration of EPSs，i.e.，viscosity increased about 33 times when concentration of EPSs from 1 g/L to 10 g/L. The EPSs were sensitive to temperature，and viscosity decreased 89.8% when temperature from 25 ℃ to 100 ℃. The viscosity of EPSs remained relatively stable under the condition of acid，neutral and weak alkaline. The EPSs also well tolerated with metal ions such as Na+、K+、Mg2+ and Ca2+. The analysis of EPSs by GC，IR，and NMR indicated that the EPSs consisted of arabinose，mannose and glucose in a molar ratio of 1.63：3.16：2.38，also containing α-pyranose，β-pyranose and uronic acid.

Isolation and Utilization of a Thifensulfuron-methyl-degrading Bacterium，Bacillus subtilis LXL-7

LI Xiao-lou

2016, 32(5): 172-179. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.023

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A strain LXL-7 isolated from soil highly degrades thifensulfuron-methyl，and was identified as Bacillus subtilis. The degradation rate by LXL-7 reached 99.6% in mineral salt medium of thifensulfuron-methyl（100 mg/L）after 72 h. The optimal pH value and temperature were 7.5 and 30-35℃. The strain tolerated well to thifensulfuron-methyl and salt，at least to 400 mg/L of thifensulfuron-methyl and 4.0% NaCl. In addition，adding strain LXL-7 in the commercial preparation for soil amendment，a new multiple-function microbial preparation was developed，and it had an additional new function of degrading thifensulfuron-methyl residue in soil except original functions. Therefore，the strain LXL-7 could be considered for bioremediation of thifensulfuron-methyl pollution and development of microbial preparation.

The Effects of Heterologous Expression of groEL from Arthrobacter simplex on the Resistance Performance of Escherichia coli

WANG Yan-xia,WANG Hong-ling,WANG Min,LUO Jian-mei

2016, 32(5): 180-186. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.024

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Arthrobacter simplex，a strain with steroid C1，2 dehydrogenation ability，has been widely used in the steroid industry，usually adding ethanol in the transforming system may catalyze the dissolution of hydrophobic substrates. Our prior work by LC-MS/MS and bioinformatics analysis found that heat shock protein（HSP）GroEL was closely correlated to the cell adaptive behavior under ethanol stress condition. Based on this concept，groEL gene was cloned by PCR while using A. simplex TCCC 11037 genome as a template. The results showed that the homologies of nucleotide and amino acid sequence of GroEL in the strain were the highest with that from Nocardioides sp. JS614 by the ratio of 89% and 95%，respectively. Then its heterologous expression was conducted in Escherichia coli BL21（DE3）with the vector pET-22b（+），and the results showed that the recombinant strain presented the significant increase on the survival performance under the shock condition with high-concentration of methanol，hypertonicity and superacid，as well as the growth performance under the proper stresses of them. The study was the first report on the cloning and expression of gene groEL from A. simplex，and also provided fundamental data for exploring the biological function of HSP protein GroEL from A. simplex.

Effect of Phosphate-solubilizing Bacteria on Oxidative Response of Pleurotus eryngii Under Lead Stress

HE Nan,XU Heng

2016, 32(5): 187-193. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.025

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In this study Pleurotus eryngii was selected to enrich Pb2+，and 8 strains with capability against Pb2+ of 800 mg/L were screened from the strain library of our laboratory. Two strains CG8 and FFT1 with high phosphate-solubilizing capacity，as well as FFC6 and FFC11with low phosphate-solubilizing capacity were selected. Using 16S rDNA analysis，CG8 was identified as Klebsiella sp.，FFT1 as Pseudomonas sp.，FFC6 and FFC11 as Bacillus sp.；the similarity of their sequences reached 99%. Further，the effects of 4 strains of phosphate-solubilizing bacterium（PSB）on the mycelium growth，Pb accumulation，lipid peroxidation，the content of sulfhydryl-containing protein，and antioxidant enzyme of P. eryngii in Pb-contaminating liquid medium were investigated. The result showed that inoculation with PSB enhanced the hyphal biomass of P. eryngii and accumulation of heavy metals. Moreover，the content of MAD，and the activities of SOD and POD in mycelium decreased significantly，while the content of total sulfhydryl（TSH）increased. All results confirmed that PSB efficiently reduced the toxicity of heavy metals to P. eryngii mycelium and the oxidative stress induced by heavy metals. CG8 with the strongest capacity of solubilizing phosphate had more significant effects to resist the mycelium accumulating heavy metals and oxidative stress induced by heavy metals in P. eryngii，the accumulation of Pb increasing 96.1% and the PB-SH increasing 91.3%.

On the Activities of Two Enzymes Related to Salicylic Acid Metabolism in Gracilariopsis lemaneiformis Under Adverse Environment

ZOU Tong-lei,WANG Fang-jun,HOU Sai-nan,SUN Xue,XU Nian-jun

2016, 32(5): 194-199. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.026

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The article mainly studied two enzymes related to Salicylic acid（SA） signaling pathway：phenylalanine ammonia-lyase（PAL）and β-1，3-glucanase（β-1，3-GA）in marine algae Gracilariopsis lemaneiformis，and measured their activity change under sick，high salt，high temperature stresses and addition of SA. Results showed that the activities of PAL and β-1，3-GA in G. lemaneiformis of 3 sick groups increased 1.27-1.42 times and 1.26-1.35 times compared to healthy groups，respectively. For high salt stress，the activities of PAL had no significant difference from the control，whereas β-1，3-GA increased 1.90 times and 1.42 times at 24 h and 48 h compared to the control，respectively. The activities of PAL and β-1，3-GA under high temperature stress increased 1.25 times and 1.27 times compared to the control groups. The activities of PAL and β-1，3-GA increased after adding 100 μmol/L SA，but high concentration of SA inhibited the activities of 2 enzymes. This study indicated that the increases of activities of PAL and β-1，3-GA in G. lemaneiformis might be associated with the SA resistance to environment. In addition，100 μmol/L SA had the most significant effect on the induction of PAL and β-1，3-GA activities.

Effects of Different Temperature on Starch Metabolism in Different Growth Stages of Tobacco（Nicotiana tabacum）

ZHANG Jian-bo,JIN Yun-feng,WANG Sha-sha,YANG Hui-qin,PANG Tao,LI Jun-ying,CUI Ming-kun,GONG Ming,

2016, 32(5): 200-211. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.027

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In order to clarify the effects of different temperatures on starch metabolism in different growth stages of tobacco，using Yunyan 87 as material growing in artificial climate chamber to simulate the different climates（Jiangchuan in Yunnan，Zunyi in Guizhou，and Xiang in Henan）in the stages of transplanting to resettling，resettling to squaring，and mature，we studied the effects of growth temperature on tobacco starch content and enzyme activity，and detected the expression changes of genes related to starch metabolism by RT-PCR. The results showed that，the starch content increased with the continuous growth of tobacco，and achieved the highest in the stage of mature. The temperature of Zunyi in Guizhou was beneficial to the accumulation of starch and the AGPase activity increased in the stage of transplanting to resettling. The temperature of Jiangchuan in Yunnan was conducive to the accumulation of starch content and the activities of AGPase and SSs raised in the stages of resettling to squaring and mature. The starch accumulation under the temperature of Xiang in Henan in any stage was little. The above results indicated that the temperature ultimately determined the formation of tobacco leave style by impacting the genes expressions of genes related to starch metabolism，thereby affecting the activities of related enzymes，as well as the metabolism and transport of starch.

Effects of GC Clamps on RpoB-PCR-DGGE Fingerprint of Three Types of Food Borne Pathogens

LIAO Chao,ZHANG Zhao-huan,YAO Xin,XIE Jing,ZHANG Wei-jia,PAN Ying-jie,ZHAO Yong, ,

2016, 32(5): 212-219. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.028

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This work is to investigate the effect of different GC clamps and different positions of primers connected with GC clamps on the DGGE fingerprint of food borne pathogens. We synthesized 6 pairs of primers encoding RNA polymerase β subunit（rpoB 1-6）containing 3 different GC clamps（GC-1，GC-2 and GC-3）that linked to 5'endings of both forward and reverse primers，respectively. Then，rpoB-PCR-DGGE was used to analyze Vibrio parahaemolyticus（5 strains），Listeria monocytogenes（5 strains），and Salmonella spp.（3 strains）. The results of fingerprint analysis were compared with that of V3-PCR-DGGE and ERIC-PCR. GC-3 clamp linked with reverse primer presented the best discriminating effect，and the discriminating index of rpoB-PCR-DGGE reached 0.9 that was equal to ERIC-PCR and greater than V3-PCR-DGGE. In conclusion，both of the different GC clamp sequences and linked positions of primers with GC clamp affected the result of rpoB-PCR-DGGE fingerprint. Choosing or designing GC clamp without continuous guanine（G base）and linked with 5'ending of reverse primer will improve the detecting and typing effect of rpoB-PCR-DGGE for food borne pathogens.

Genetic Transformation of Human IL-12 Driven by Tuber-specific Promoter from Potato（Solanum tuberosum）

YANG Jia-wei,CHENG Xiao-ling,LONG Chao-kang

2016, 32(5): 220-225. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.029

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To obtain transgenic potato plants that express human interleukin 12（hIL-12）protein，the early-constructed plant expression vector driven by Ppatatin of potato tuber-specific promotor was transformed into potato by Agrobacterium-mediated transfection，followed by co-cultivation，screening and differentiation，and the transgenic plants were acquired. Techniques of PCR，RT-PCR，GUS staining，and ELISA analysis were performed for the identification of transgenic plants. The results showed that 12 transgenic lines were obtained，and in the 10 transgenic lines the target gene was in the potato genome，i.e.，positive rate was 83%. RT-PCR analysis illustrated that exogenous genes expressed successfully in 7 transgenic lines at mRNA level，moreover，ELISA data showed that 5 transgenic lines presented the high level of hIL-12 protein expression. In addition，the expression of hIL-12 was genetically stable in these 7 transgenic potato lines.

Dynamic Changes of Sheep Oocyte Meiosis from Metaphase I to Metaphase III Matured in vitro

LI Xin-xin,BAI Chun-ling,WEI Zhu-ying,LI Guang-peng

2016, 32(5): 226-233. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.030

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Meiosis is a division mode in diploid organism to produce haploid gametes，which is the special mechanism of ensuring the chromosome’s number of a species after fertilization to be constant. The key step in the meiotic division is to separate the homologous chromosome and sister chromatids correctly，the dynamic changes of the cytoskeleton，in particular microtubules（MTs）and microfilaments（MFs），play critical roles in this process. In this study，the dynamic changes of chromosomes and spindle MTs and MFs during the maturing and parthenogenetic activation of sheep oocyte cells were studied by immunohistochemistry method. The results showed that：1）The morphology of spindle changed from barrel-shaped at metaphase stage to cylinder-shaped at early anaphase，and then to long，thin cone-shaped at late anaphase and telophase. Chromatin connected with the floor of the cone became the polar bodies and expelled. 2）Chromosome morphology changed from visible individual at metaphase to condensed chromatin during anaphase and telophase stages，and then backed to visible individual chromosomes at the next metaphase. 3）The size of MI spindle was larger than that of the MII spindle and MIII. MII spindle，however，was more barrel-shaped than the MI spindle. 4）Almost all of the MTs and MFs composing the spindles were partitioned into the polar bodies.

Cloning，Expression and Identification of F Gene Deletants from Peste Des Petits Ruminants Virus

DENG Rui-xue,MENG Xue-lian,ZENG Qiao-ying,CAI Xue-peng

2016, 32(5): 234-239. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.031

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The F gene deletants were constructed and expressed in prokaryotic cell in order to clarify the fusion mechanism between the envelope of peste des petits ruminants virus（PPRV）and the host cells. Considering the key roles in the fusion process，HR1 and HR2，the two conserved fragments of F gene，were cloned into prokaryotic expression vector pET-30a，respectively. The recombinant plasmid pET30a-HR1 and pET30a-HR2 with the confirmed correct sequences were transformed into Escherichia coli BL21（DE3）and expressed by the induction of IPTG. The fusion proteins were largely expressed under the optimal condition and purified using the nickel agarose gel. The results showed that the relative molecular masses of the fusion proteins expressed in form of soluble protein were consistent with the expectation，and the purities of the expressed proteins were over 90%. The fusion proteins reacted with both anti-His tag antibody and anti-PPRV Nigeria75/1 positive serum，indicating that the fusion protein had reactionogenicity.

A Preliminary Study on Infection Model of Mongolian Gerbil by Hepatitis E Virus

ZHU Guang-ze,QU Yong-gang,LI Yi-quan,LAN Tian,FAN Yuan-yuan,HU Ning-ning,ZHANG Nuo-na,LI Xiao,JIN Ning-yi

2016, 32(5): 240-245. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.032

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To explore the feasibility of using Mongolian gerbils（Meiiones unguiculataus）to construct an animal model of hepatitis E virus（HEV），HEV RNA was extracted from the diluted suspension of swine feces，then Mongolian gerbils were experimentally inoculated with the HEV via intraperitoneal and oral. Subsequently，the samples of blood，relevant organs（heart，liver，spleen，lungs，kidneys，mesenteric lymph nodes，and spleen）and feces were collected weekly. Samples were detected by RT-PCR and immunohistochemistry. As a result，from day 7 after infection，HEV RNA was detected from the blood，kidney，spleen，mesenteric lymph nodes，liver，lung and feces. The analysis of immunohistochemistry discovered that HEV was expressed in the liver，and caused the pathological changes in liver tissue，but it was lack of obvious clinical manifestations in Mongolian gerbils after HEV infection. All above results suggest that Mongolian gerbils have susceptibility to HEV，and have pathological changes of lobular hepatitis in the liver after HEV infection，indicating that Mongolian gerbils have a potential value to be the animal model of HEV infection.

Gene Expression of AAV-ITR Mini Vector in Mice vivo

LI Tai-ming,XU Qi-lin,PAN Jun-jie,LIU Xiao-mei,ZHANG Chun

2016, 32(5): 246-254. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.033

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In order to compare the expression difference between AAV-ITR expression mini vector and its monomers in different parts of mice，the same copy number of AAV-ITR mini vector and pUC57-minivector-EGFP were separately transferred into the brain of mice. In addition，the same copy number of monomers of AAV-ITR mini vector and pUC57-minivector-EGFP were transferred into mouse skeletal muscle，and the number of residual molecules were compared in the different tissues at different periods. Analysis by florescence quantitative PCR，microscope observation，average optical density，and gray value showed that monomers of AAV-ITR mini vector in the same copy number had higher transfection efficiency and more stable than AAV-ITR mini vector and pUC57-minivector-EGFP. The comprehensive results suggest that the monomers of AAV-ITR mini vector present the feature of efficient，secure and stable expression，which may be expected to become a novel，secure，reliable，stable，and efficient vector in gene therapy.

Characteristics of ML00253764 Rescuing the MC4R Deficient Mutants

WANG Xiao-hua,CAO Xiao-hong,FAN Zhen-chuan

2016, 32(5): 255-259. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.034

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To determine the specificity and internalization kinetics of non-peptide small molecule ML00253764 rescuing the obesity-causing MC4R mutants，the cell model for the stable expression of melanocortin receptors and their mutants were established. The results by flow cytometry（FACS）showed that ML00253764 resulted in the increase of the membrane surface expression of MC4R deficient mutation receptor N62S and P78L by 43% and 34%，respectively，while almost no effects to other melanocortin receptor and their mutants，demonstrating this rescue mediated by ML00253764 was MC4R receptor- and mutant- specific. Further study showed that the internalization of rescued receptors was similar to the wild type（WT）receptor，and there was no significant difference in rate of internalization t1/2. Both endogenous ligands α-MSH and AgRP had no rescue effect on the membrane surface expression of MC4R deficient mutation receptor，proving that action way of ML00253764 was different from that of above two，and what mutant receptors were rescued resulted from the specificity of pharmacological chaperone with cell membrane permeability.

The Inhibition Effect of Recombinant Trichosanthin Combined with YH-16 on the Proliferation and Xenograft Tumor Growth of Melanoma

GUO Hui-can

2016, 32(5): 260-264. doi:10.13560/j.cnki.biotech.bull.1985.2016.05.035

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The objectives of this study are to acquire trichosanthin（rTCS）and to explore the effect of rTCS combined with YH-16 on the proliferation of melanoma B16-F10 and xenograft tumor growth in mice model. rTCS was expressed in Escherichia coli BL21 strain and purified by Ni affinity chromatography. Then the effects of rTCS，YH-16，and combination of rTCS with YH-16 on the cell growth were determined by MTT assay and in vivo trial. Results were as：pure recombinant rTCS protein was obtained. The proliferation of B16-F10 was inhibited significantly by rTCS，and inhibition rate was significantly higher than by YH-16 alone，however，no significant difference between rTCS alone and the combination of rTCS and YH-16. The xenograft tumor growth in the group treated with the combination of rTCS and YH-16 was significantly smaller than the ones by rTCS-treated alone or control. In conclusion，the combination of rTCS with YH-16 presented significant inhibitory effect on proliferation of B16-F10 as well as on xenograft tumor growth，and this strategy with the combination would provide new opportunity for treating melanoma patients.

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2016, 32(5): 300.

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