Table of Content

25 December 2016, Volume 32 Issue 12

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Research Advances on Transcriptomics of Plant Cytoplasmic Male Sterility Lines?

YANG Peng

2016, 32(12):  1-7. 

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Cytoplasmic male sterility（CMS）is the basis of utilizing the heterosis of plants，and also favorable material for studying the interaction between mitochondrial genes and nuclear genes. Transcriptomics analysis using DNA microarray and RNA-Seq technology enabled the analysis and comparison of thousands of genes in one experiment. This allows it feasible to better understand fundamental aspects of growth and development，as well as genetic variation in plants on a genomic scale. Based on the mechanism of CMS lines，transcriptomics study may acquire the detailed relevant molecular analysis，understand the interaction mode between mitochondrial and nuclear genes，and be conducive to dissect the male sterility.

Application Advances of Quantitative Proteomics in the Studies of Animal Testis Protein

LU Zeng-kui,MA You-ji，

2016, 32(12):  8-12. 

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Proteomics is one of the most important research areas at post-genomics era and quantitative proteomics is one of the main methods to study proteomics，which may reflect the dynamic nature of proteins. As a key male reproductive organ，testis produces sperms and secrets androgens. Thus it is of great significance to investigate the protein compositions of testis by the method of proteomics. In this review，we mainly focus on the concept of proteomics and the technological means of quantitative proteomics，as well as its application in the studies of testicular development，and responses to diseases and various stress conditions, stress conditions which provids insight into the further research on protein in testis.

Research Advances on Biosorption and Detoxification Mechanisms of Heavy Metals by Bacteria

WANG Ze-huang,WANG Meng，CAI Kun-zheng，CAI Yi-xia,HUANG Fei,

2016, 32(12):  13-18. 

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With the continuous development of mining，smelting and electroplating industries，heavy metals pollutions to the environment are increasingly serious. In recent years，microbial remediation arouses the extensive attentions because of its advantages of low cost and no secondary pollution. The main microorganisms used to remove heavy metals include fungi，algae，actinomyces and bacteria；among them，the biosorption and detoxification mechanisms of bacteria are more widely studied. This paper summaries the research advances on biosorption and detoxification mechanisms of heavy metals by bacteria from the aspects of bacterial cell’s structure，i.e.，the extracellular part，the cell surface and intracellular part，which mainly includes extracellular precipitation mechanisms，biosorption mechanisms on cell surface and intracellular detoxification mechanisms. Furthermore，the paper puts forward the feasible direction of future research，and provides theoretical basis and practical references for the microbial remediation of heavy metals pollution.

Research Progress on Quorum Sensing System of Bacterial Biofilm

ZHANG Shu-mei,XU Xiang-rong,XU Hao

2016, 32(12):  19-22. 

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The quorum sensing system of bacterial biofilm is the one that the bacteria secrete signaling molecules and sense their concentration of the surrounding environment so that to regulate the expressions of certain specific genes，and to change some physiological functions and life habits of bacteria. It is one of the main regulatory mechanisms in bacteria. Based on the research of the bacterial biofilm’s quorum sensing，we can understand its internal mechanism and features，and find the best way to inhibit harmful effects of biofilms. This review focus on bacterial biofilm’s quorum sensing，such as the types，the characteristics and the related applications.

Research Progress on the Application of Bioinformatics in Prediction and Molecular Design of Antimicrobial Peptide

XIAO Yi-chen,XIONG Hao,XIE Chuan,Lü Nong-hua

2016, 32(12):  23-28. 

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Antimicrobial peptides（AMPs）are being increasingly recognized as novel bioactive peptide with critical research value owing to their activities of anti-bacterium，antivirus and antitumor as well as properties of rapidness，broad-spectrum，and high efficiency. Whereas a crucial issue in AMP drug development is how to seek appropriate approaches for maximally improving the bioactivity and decreasing the toxicity and production cost. Applying bioinformatics tools to predict novel AMP and to carry out the molecular design of natural AMP is the key to solve this problem. The new progress on bioinformatics involved in AMP prediction and optimal design is reviewed. in order to expand the sources of new AMPs and elevate the bioactivities of known AMPs.

The Introduction of Open Metabolomics Database Used for Looking for Novel Natural Active Substances

ZHANG Xiao-meng,SUN Ming-hui,LI Yan-ping,LI Feng,ZHUO Wei-wei,ZHOU Min

2016, 32(12):  29-33. 

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A lot of metabolomics database have been established after a long exploration and accumulation，they all have their own pros and cons though these databases are different on technical methods as well as collected and published data. This paper introduces several open metabolomics databases used for looking for novel natural active substances，mainly focusing on the technologies for establishing databases，the amount of data in databases，and how to access databases and seek information，etc. Concurrently，we also give some improvement suggestions for perfecting these databases in the future.

Research Progress on Methods for Isolating the Gene of Plant Glycosyltransferase，and Its Biological Functions

LUO Yan,LIU Xiao-gang,ZHOU Zhi-qin

2016, 32(12):  34-39. 

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Plant glycosyltransferases widely exist in plant for glycosylation reaction，and they could modify receptor chemicals，such as sugar and proteins，by glycosylation to change their physical and chemical properties，which is of significance for the growth and development of plant secondary metabolism and hormonal homeostasis maintenance，as well as responses to biotic and abiotic stresses. Here，we reviewed the research progress on the methods and biological functions of plant glycosyltransferase，and predicted the research focus of it in the future；aiming at providing some references for the identification and isolation of more plant glycosyltransferase genes，and assisting the further functional analysis of this gene family.

The Progress of Affinity Ligand in Affinity Separation Technology

ZENG Rong,JIN Bu-Kun,RUAN Tao,WU You,HOU Ya-Li,GONG Zhi-Wei,YANG Zhong-Hua

2016, 32(12):  40-46. 

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Affinity separation technology plays an increasingly important role in the proteins separation and purification field. Development of an appropriate ligand is the critical factor for affinity separation technology. In this work，the current research progress of the affinity ligand is reviewed according to three categories i.e. biological macromolecules affinity ligands，small molecule affinity ligands and metal iron affinity ligand. The current progress of each kind of ligand，its action mechanism and its deficiency are highlighted. Also，the prospect for development of affinity ligands is also given.

Enhanced Cordycepin Production of Cordyceps militaris by Genomic DNA Demethylation and the Evaluation of Its Stability

ZHU Wei-wei，LI Li,CHEN Fei,LI Yang,WANG Yan-hua,BAO Yong-ming

2016, 32(12):  47-52. 

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Genomic DNA demethylation may activate silenced gene，which alters secondary metabolite spectrum，and therefore it is a completely novel approach for strain improvement. Using different concentrations of DNA methyltransferase inhibitor 5-azacytidine（5-azaC）to treat the Cordyceps militaris CM-L1，then the genomic DNA methylation level reduced. Further，the improved strains LB-C3 and LD-A7 were acquired by screening with high performance liquid chromatography，and in which the contents of cordycepin in mycelium increased by 127% and 144.3% respectively. LD-A7 could not form a fruiting body，which was speculated to be related to the role of DNA methylation changed. The stability was investigated after 5 times subculture using the content of cordycepin as an indicator. With the subculture times increased，the methylated DNA in the genome increased，and the cordycepin in mycelium of liquid fermentation significantly decreased；however，the content of cordycepin in fruiting bodies was very stable，indicating that improved strains were better suited to the cultivation rather than production in industrial fermentation.

Establishment of Mutagenesis Cell Line of Macaca mulatta Gene TRIM5α by TALEN

WANG Xiao-li,YU Qing,YUAN Ya-hong,TENG Zhi-ping,LI Dong-sheng,ZENG Yi

2016, 32(12):  53-57. 

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Using transcription activator-like effector nuclease（TALEN）to establish the mutagenesis cell line of Macaca mulatta gene TRIM5α is to lay the foundation for further studying the function of TRIM5α gene. The TALEN plasmids targeting TRIM5α was constructed. The site-directed gene mutagenesis donor vector was obtained by site-directed gene mutagenesis technology and the sequence contained the seventh exon of TRIM5α at the position from 1 211 to 1 224. The TALEN plasmids and donor vector were co-transfected into the M. mulatta’s kidney cell line（LLC-MK2）by electroporation. The single cell line was obtained by the infinite dilution method. By extracting the genomic DNA and amplifying the target sequence by PCR，the cell line with base deletion（1 215-1 216）and site mutations（1 213-1 215，1 217）of TRIM5α was screened. Conclusively，the LLC-MK2 single cell line with knockout and site-directed gene mutagenesis of gene TRIM5α was obtained.

Application of Low-coverage Whole Genome Sequencing in Detecting Chromosome Micro Variations of Single Cell

CHEN Da-yang,ZHEN He-fu,LIU Ping,QIU Yong,XIE Lin,LIU Hong-tai,CHEN Fang

2016, 32(12):  58-64. 

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The paper aims to study the application of low-coverage whole genome sequencing in detecting chromosome micro variations of single cell. Using 5 cell lines with positive signal and validated by aCGH as research object，the complete genome of single cell amplified by two commercial kits was sequenced by Hiseq2000；then，comparing the results by bioinformatics with those validated by aCGH. Results showed that in preliminary tests，the whole genome amplification（WGA）was successful in the 5 single cells. Following the low-coverage whole genome sequencing，they all showed the expected karyotype. While using the GenomePlex? Single Cell WGA Kit，the overlapping rate of the detection results and aCGH results was more than 80%，but there was 1 false positive signal of about 8 Mb（Megabase）in 1 sample. While using PicoPLEX? WGA Kit，the overlapping rate of the detection results and aCGH results also was more than 80%，and there was no false positive signal in detection results. The results confirmed that the method would detect the copy number variations larger than 7 Mb via the low-coverage whole genome sequencing（0.1 X）.

Cloning and Characterization of ZmPht3；1，a Phosphate Transporter Gene in Maize

WU Shan,LIN Dan

2016, 32(12):  65-71. 

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Pht3（phosphate transporter 3）family belonging to low-affinity phosphate transporters play an important role in the regulation of phosphorus homeostasis in plants. In order to explore the characterization of gene structure and response mechanism of ZmPht3；1 to phosphorus（P）starvation，the gene ZmPht3；1 was isolated from low-phosphorus（low-P）tolerance maize inbred line Mo17 by homology cloning. Then the quantitative real-time PCR and subcellular localization of ZmPht3；1 were performed for further research in this study. The results showed that the complete CDS of ZmPht3；1 was 1 101 bp encoding 366 putative amino acids in length，and contained 6 hydrophobic transmembrane domains and had a typical conservation structure domain of mitochondrial carrier family. The PCR results exhibited that the gene expressed in roots and leaves，and the expression patterns were significantly different in the two extreme materials，respectively. While gene expression pattern in low-P tolerance maize inbred line Mo17 both roots and leaves revealed general and special response to P deficiency stress in early and late stages，respectively. Subcellular localization in the transformed tobacco（Nicotiana benthamiana）showed that ZmPht3；1 mainly expressed in cytoplasm membrane and it was a phosphate transporter in both low- and high-P environment，and played a key role in the process of adapting to P starvation in maize.

Clone and Expression Analysis of MePIL1 in Cassava

DING Ze-hong,TIE Wei-wei,FU Li-li,YAN Yan,XIA Zhi-qiang,WANG Wen-quan,HU Wei

2016, 32(12):  72-78. 

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This work aims to clone MePIL1 gene from cassava and to reveal its functional roles in abiotic stresses（e.g.，shade），which will provide theoretical bases for optimizing the planting density of cassava. MePIL1 gene was cloned from leaf of cassava cultivar ‘Ku50’ by RT-PCR method，and the sequences of MePIL1 and its homology genes with other species were aligned and their phylogenetic tree was constructed. Subsequently，structural variations of MePIL1 were revealed between wild and cultivated cassava，and their expression patterns were investigated by quantitative RT-PCR. Results showed that MePIL1，which had a 1 578 bp open reading frame and encoded 525 amino acids and contained a HLH conserved domain，was cloned from cassava. Phylogenetic analysis showed that MePIL1 and homologous genes from Populus trichocarpa and Salix purpurea were clustered together，indicating that they had close genetic relationships. Analysis of gene structural variation revealed that the region of HLH domain was highly conserved，while most variations were derived from the differences between the wild and cultivated cassava species，and the distribution overall was relatively even. Expression pattern analysis showed that MePIL1 was highly expressed in leaf，and its expression was induced by osmotic stress and shade treatments. In conclusion，MePIL1 of cassava was successfully cloned and it was involved in the responses to shade and osmotic stresses at the transcriptional level in cassava.

Analysis of the DNA Methylation of SlGLD1 Regulated by JMJ524 in Tomato

YANG Wei,PAN Yu,SU Cheng-gang,ZHANG Xing-guo,LI Jin-hua

2016, 32(12):  79-85. 

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This work is to reveal the mechanism of JMJ524 modulatinges the SlGLD1 expression by affecting the DNA methylation of SlGLD1，and leading to the dwarf of tomato insensitive to gibberellin. Bioinformatics and BSP（Bisulfite sequencing PCR）were employed to analyze the DNA methylation of SlGLD1 in inhibitory line and wild line of JMJ524. Results showed that the SlGLD1 promoter sequence was isolated，which contained 3 GA response cis-elements. The DNAs of SlGLD1 promoter and coding sequence were highly methylated，moreover，the methylation pattern varied at the different tissues and mutants. Two GC islands were found in this coding region of the gene，thus it was inferred that the methylation of SlGLD1 might happen in the region. Further，the analysis by BSP of the 2 GC islands indicated that the DNA CpG in the 2 regions of SlGLD1 was highly methylated. The whole methylation of DNA in the inhibitory line of JMJ524，especially CpG methylation was significantly lower than wild-type M82. The inhibited expression by JMJ524 resulted in the decrease of gene methylation of SlGLD1，therefore the expression of the gene was activated，which might be the one of factors causing the dwarf of tomato plant.

Revealing Deep Phylogeny of Brassicaceae Using Composition Analysis of Low-copy Nuclear Genes

ZHANG Zhe,HUANG Chien-hsun,QI Ji

2016, 32(12):  86-95. 

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Brassicaceae，as many vegetable crops and important model plants，is one of the most successful and economically valuable angiosperm families. Recent phylogenetic studies revealed that plants of Brassicaceae were classified into 3 major lineages（I，II，and III），however，detailed evolutionary relationships among them and intra-lineage still remain unknown. In order to quickly and accurately understand the phylogeny of Brassicaceae species，39 Brassicaceae species and two species of other family were chosen as research materials，and a set of low copy orthologous genes covering all the selected species was acquired via phylogenetic genomics. Further，the composition characteristics of low copy nuclear genes were analyzed by CVTree，the phylogeny of Brassicaceae in highly supported and stable relationship was obtained. The results revealed that Brassicaceae could be classified into 6 major lineages，and 3 of which agreed well with the classification by the priors，and 2 new major lineages were defined. Moreover，lineage II that was in dispute in previous studies was confirmed as the single lineage with stable supports. This indicated that a large number of low copy orthologous genes set combined with the analysis of composition vector may more accurately reflect phylogeny of Brassicaceae species. Therefore，CVTree not only is suitable for studying the phylogeny of microorganisms such as prokaryotic organisms and fungi，but also for exploring the genetic relationship of higher organisms such as Brassicaceae plants

Phylogeography of Gymnadenia conopsea from the Qinghai-Tibet Plateau

BAO Wu-yin,ZHANG Yang,LIN Peng-cheng,NAN Peng,HUANG Yan-yan,JIN Hao-fei,ZHONG Yang

2016, 32(12):  96-102. 

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Chloroplast DNA（rbcl and psbA-trnH）of Gymnadenia conopsea（G. conopsea）from the Qinghai-Tibet Plateau was used for phylogeography research，aiming at revealing the distribution pattern and evolutional process of genetic lineage of G. conopsea. Eleven haplotypes were identified in 199 samples of 10 G. conopsea populations. The analysis of molecular variance（AMOVA）showed 65.93% of the genetic variance was from inter-populations and FST value of 0.659（P<0.01）also indicated that there was significant genetic differentiation between different populations. The genetic differentiation coefficients NST（0.398）< GST（0.626）implied no significant phylogeographic structure for G. conopsea populations in the Qinghai-Tibet Plateau. Mismatch analysis of multi-peak and Tajima’s D neutral test（-1.455，P>0.1）demonstrated G. conopsea populations had not experienced abrupt expansion recently. Phylogenetic analysis and divergence time estimation showed that G. conopsea in the Qinghai-Tibet Plateau originated indigenously，haplotypes started to differentiate 19.08 million years ago（Mya）in Miocene，indicating that the genetic differentiation of G. conopsea was related to the uplift of the Qinghai-Tibetan Plateau，and the genetic distribution pattern of G. conopsea was mostly formed before the Quaternary glaciation.

Cloning，Sequence and Phylogenetic Analysis of CATSPER3 Gene in Banna Mini-pig Inbred Line

XIAO Jing,ZHA Xing-qin,CHENG Wen-min，HUO Jin-long,PAN Wei-rong，WANG Shu-yan，WANG Pei,LIU Hai-jing,ZHANG Xiu-qiong,ZENG Yang-zhi

2016, 32(12):  103-107. 

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Our purpose of this work is to obtain the partial coding region sequences of CATSPER3 gene from Banna Mini-pig Inbred Line（BMIL），and to analyze the amino acid sequence and homology comparison among different species by bioinformatics. We used boar testis material of BMIL to extract RNA，RT-PCR to amplify the coding sequence of CATSPER3 gene，and online analysis software to analyze the bioinformatics. Bioinformatics analysis showed that the protein molecular weight of deduced amino acid sequence was 19.397 3 kD and theoretical isoelectric point was 5.05. Regarding the composition of amino acid，there were 24 acidic amino acids（Asp and Glu）；and there were 16 alkaline amino acid（Lys+Arg）；among them the leucine（Leu）accounted for 13.3%，valine（Val）9.6%，threonine（Thr）9.0%，phenylalanine（Phe）8.4%，glutamate（Glu）7.2%，aspartic acid（Asp）7.2%，and their contents were high. Compared with ordinary pig，the nucleotide similarity was the highest，however，lower with that of goat. Molecular phylogenetic tree showed that inbred pig and non-inbred pig clustered in the same branch，i.e.，their genetic relationship was close，while far from goat and sheep.

Adjuvanticity of Cordyceps militaris stroma Polysaccharides in Inactivated Vaccine to Avian Infectious Bronchitis

ZHOU Mei-xian,ZHOU Ye-fei

2016, 32(12):  108-112. 

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It aimed to use Cordyceps militaris stroma polysaccharides as adjuvants that mix with inactivated vaccine to infectious bronchitis，and to investigate its effects on the immune functions of broilers. Total 150 1-day Huangyu broilers were selected and randomly divided into 5 groups. Then the broilers of 21 d after immunized（35-day）were infected with Mass 41 infectious bronchitis virus（IBV M41）strain by the ocular-nasal route. The proliferation of peripheral blood mononuclear cells（PBMC），serum antibody titer，the protection of IBV virulent strain，and histopathological changes in each group were evaluated respectively by MTT method and hemagglutination inhibition assay. The results showed that proliferation index of PBMC and serum antibody titer in the broilers with dosage C. militaris stroma polysaccharides as adjuvants significantly raised（P<0.05）. Following challenge test with IBV M41 strain，broilers inoculated with C. militaris stroma polysaccharides showed significantly lighter clinical symptoms，there were no histopathological changes in lung and kidney，and immune protection reached 96.7%. This indicated that C. militaris stroma polysaccharides as adjuvants increased the immunity of broilers to infectious bronchitis.

Molecular Cloning and Expression Analysis of Two Types of perlucin Gene from Small Abalone Haliotis diversicolor

LIN Shi,ZHANG Li-li,WANG Guo-dong

2016, 32(12):  113-123. 

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SMART RACE technology was employed to clone the full-length cDNA sequences of 2 Manna-binding lectin（MBL）members of perlucin1 and perlucin4 in Haliotis diversicolor. Real-time PCR were used to analyze their expression patterns in the different tissues of H. diversicolor. As results，the full length cDNA of perlucin1 was 668 bp，and the deduced protein was composed of 165 amino acids，with two disulfide bonds，two glycosylation sites and 13 phosphorylation sites；its predicted molecular weight was about 18.8 kD，and pI was about 4.57. The full length cDNA of perlucin4 was 587 bp and the deduced protein was composed of 156 amino acids，with two disulfide bonds，one glycosylation sites and 7 phosphorylation sites；its predicted molecular weight was about 17.8 kD，and pI was about 4.43. Gene perlucin1 and perlucin4 exclusively expressed only in hepatopancreas among 7 tissues of gill，hepatopancreas，blood lymph，up foot，mantle，mucous gland，and kidney. The above results indicate that perlucin genes involved in innate immune defense mechanisms and play a key role in immune recognition of H. diversicolor.

Identification of Critical Residues in c-di-GMP Receptor Clpxoo from Xanthomonas oryzae pv. oryzae

LI Jian-yu,LI Bo,CHEN Hua-min,YANG Feng-huan,HE Chen-yang,TIAN Fang

2016, 32(12):  124-129. 

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The purpose of this study is to identify the critical residues in Clpxoo of a c-di-GMP signal receptor in Xanthomonas oryzae pv. oryzae（Xoo）. By the gene mutation of amino acid site of Clpxoo protein，the construction of expressing vector，induced protein expressed，and analysis of Ni-NTA Resin affinity chromatography，the prokaryotic expression of Clpxoo and point mutants was conducted and the protein was purified.. The binding affinities of c-di-GMP with native and variant Clpxoo were measured by isothermal titration calorimetry（ITC）experiments. Under optimized conditions for protein expression and purification，the proteins of Clpxoo point mutants and point mutants of Clpxoo that does not bind with c-di-GMP were acquired successfully while using gene site-directed mutagenesis and bridging PCR. Results showed that site 70 of aspartic acids and site 99 of glutamic acid were found to be the key residues for their binding to c-di-GMP.

Construction of Controlled Inducible Expression System in Streptococcus zooepidemicus ATCC39920 and Its Application

ZHENG Xiao-e,WANG Zhen,LIU Hao

2016, 32(12):  130-136. 

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This work aimed at establishing an inducible gene expression system in Streptococcus zooepidemicus，and investigating the application of it in controllable two-stage fermentation. Induced expression vectors by sucrose，lactose，xylose and nisin were constructed，then using hasA of hyaluronic acid（HA）synthase as reporter gene，and ?hasA with capsule-deletion resulted from the function deletion of gene hasA as the host bacterium，the feasibility of these systems was verified by observing the generation of capsule. The results showed that：The expression of hasA was not induced in the xylose and nisin vector，at low-efficiency in lactose vector，and efficient and rigorous in sucrose vector. A controllable two-stage fermentation mode was conducted using ΔhasA/pLH201 as strain and glucose only as sole sugar source for the growth of bacteria，and added sucrose induced the generation of HA，and HA production reached at 3.4 g/L. In conclusion，this study successfully constructed a sucrose-induced gene expression system that can be used for two-stage fermentation of S. zooepidemicus.

Isolation，Identification and Heavy Metal-resistance Characteristics of a Zn-resistant Bacterium，Pseudochrobactrum sp. ZS2

QU jia,ZHAO Ling-xia,SHEN Ne-min,SUN Xiao-yu,CHEN Rui,LU Peng-peng,QIAO Xue-li,SHEN Wei-rong

2016, 32(12):  137-142. 

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Screening of resistant bacterium is beneficial to degradation of heavy metal and bioremediation of soil. A zinc-resistant strain，named ZS2，was isolated from metal-polluted soil in a lead-zinc mining area in Shangluo，Shaanxi province and identified as Pseudochrobactrum asaccharolyticum based on the analysis of its physical and biochemical characteristics and its 16S rRNA gene sequence. Studying the resistance of heavy metals and Zn2+adsorption，the results showed that ZS2 has high Zinc-resistance and maximum tolerable is 20 mmol/L. The ZS2 also could grow in a variety of single and combined heavy metals（Pb2+、Cu2+、Cr6+、Cd2+）and have obvious removal effects of low Zn2+ concentration. When the concentrations of zinc was 2.0 mmol/L，the removal efficiency could reach 55.25%. The results indicated that ZS2 is a rare strain with heavy metal resistance from Pseudochrobactrum genus and have excellent application value to remediate heavy metal contaminated soil.

Screening and Identification of Extracellular Protein Genes Relevant to Sulfur Activation of Extremely Thermoacidophilic Archaea Acidianus manzaensis

MA Ya-long,LIU Hong-chang,XIA Jin-lan，YANG Yun ,NIE Zhen-yuan,

2016, 32(12):  143-151. 

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This study focused on extremely thermoacidophilic archaea Acidianus manzaensis，13 extracellular protein genes relevant to sulfur activation were screened and identified based on comparative proteomics，and their transcription levels were verified. First of all，extracellular proteins of A. manzaensis growing on elemental sulfur（S0）and ferrous（Fe2+）were extracted by slowly shaking water bath at 80℃ for 30 min，respectively. The proteins were separated by two-dimensional electrophoresis（2-DE）and differentially expressed spots under S0 substrate were selected. The selected spots were identified by tandem time of flight mass spectrometry（MALDI-TOF/TOF），and then analyzed using bioinformatics and functional prediction methods. Finally, the genes of the screened extracellular proteins associated with S0 activation were verified by real-time quantitative PCR（RT-qPCR）at transcription levels，and 13 relevant genes were acquired. More than half of the screened proteins contained plenty of cysteine residues（Cys），meaning thiol group（-SH）rich extracellular proteins participated in the process of S0 activation. Especially，both glutaredoxin and FAD-linked oxidase contained -CXXC- domain，and their corresponding genes were highly expressed，indicating that the proteins with -CXXC- domain play an important role during the S0 activation process of extremely thermoacidophilic archaea A. manzaensis.

Expressions of Calmodulin Gene in Hyriopsis cumingii Mantle and Visceral Tissues Under the Culture Environment with Different Ca2+ Concentrations

SHANG Chao,SHI Yang,LI Wen-juan,ZHOU Zi-rui,SHI Zhi-yi

2016, 32(12):  152-159. 

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In order to explore the role of calmodulin（CaM）gene in the formation of freshwater mussel，Hyriopsis cumingii was cultured in water environment at 5 different Ca2+ concentrations of 0 mmol/L，0.5 mmol/L，1.25 mmol/L，2 mmol/L，and 3 mmol/L. At 0 d，20 d，50 d，90 d，and 120 d after nucleus inserted，mantle and visceral were sampled，then the CaM expressions in the tissues were detected using fluorescent quantitative PCR. The results showed that：1）The expression level of CaM gene in mantle and visceral mass were always low at 0 mmol/L Ca2+ concentration，decreased and then increased and remained stable at 0.5 mmol/L，decreased at the 20 d in visceral mass，then rose to level at 0 d and remain stable at 1.25 mmol/L，significantly increased at 20 d，reached the highest which was maintained at 2 mmol/L among all 5concentrations，increased at 20 d after nucleus inserted，and decreased in the later period（P < 0.05）at 3 mmol/L. 2）There was no obvious difference among different concentrations at 0 d，and the expressions differentiated along different days after 0 d. 3）Under the same culture conditions，the expression of CaM gene in visceral mass was higher than that in mantle. It was found that the concentration of 0.5 mmol/L and 2 mmol/L promoted the expression of CaM gene，but the concentration of 0 mmol/L and 3 mmol/L inhibited the expression of CaM gene. Visceral mass was more conducive to the formation of pearl.

Research on an Escherichia coli Strain Capable of Transferring Acetol to 1，2-Propanediol Aerobically

DENG Jing-jing,HU Hong-bo,ZHANG Xue-hong

2016, 32(12):  160-165. 

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We constructed an engineered Escherichia coli strain that can efficiently convert glycerol to acetol in our lab，the productivity of 1，2- Propanediol（1，2-PDO）may increase significantly if the strain can convert acetol to 1，2-PDO under aerobic condition. A mutant strain named PDO1 was screened from original strain E. coli BW25113ΔglpK，which metabolized glycerol aerobically. The further experiments showed that the strain PDO1 converted acetol to 1，2-PDO with the yield of 0.73 g/L and conversion rate of 0.493 g/g acetol under aerobic condition. The activity of glycerol dehydrogenase and the proportion of NADH were 75.6 and 1.64 times higher than that of control respectively. It is inferred that the increase of glycerol dehydrogenase activity and NADH level resulted in the mutant strain PDO1 metabolizing glycerol by glycerol dehydrogenase under aerobic condition and the conversion of acetol to 1，2-PDO .

Isolation and Identification of Marine Vibrio Producing Extracellular Polysaccharide（EPS）and the Preliminary Study on Anti-tumor Activity of the EPS

LONG Han,CHEN Sheng-feng,CHEN Jia,YANG Di,LI Xiao-yan,HUANG Yu-you,HE Xiu-miao，XUAN Jin-cai

2016, 32(12):  166-171. 

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In order to investigate the bio-activities of the extracellular polysaccharide（EPS）from the marine bacteria in the Guangxi Beibu Gulf，the marine bacteria of EPS-producing were isolated from water/mud samples by LB-aniline blue medium. Phenol-sulfuric acid method was used for measuring the yield of the EPS. The marine bacterial strains with high-yield of ESP were further identified by the sequence analysis of 16S rDNA. The molecular weight and the preliminary structure of the EPS were tested by Gel permeation chromatography（GPC）and infrared spectrometry，respectively. Finally，the cytotoxicity of the EPS on Vero cells and the inhibitory effect of the EPS on Hela cells were tested by the MTT method. The results showed that totally 167 marine bacteria producing EPS were isolated from sea water/mud of mangrove，one of which（named BHc-09）yielded ESP as high as 0.306 mg/mL and was identified to be Vibrio by the analysis of its 16S rDNA sequence. The molecular weight of the EPS from BHc-09 was 184.5 kD，and there was uronic acid in the EPS structure. The EPS produced by BHc-09 presented no cytotoxicity on Vero cell，but significantly inhibitory effects on tumor cells. The results suggested that an EPS-producing marine Vibrio was successfully isolated and identified，and its extracellular polysaccharide has anti-tumor activity.

Study on the Localization and Function of Shy1 in Schizosaccharomyces pombe

ZHANG Qing-zhen,HUANG Ying

2016, 32(12):  172-179. 

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Mitochondria are the main place for cell respiration，and mitochondrial dysfunction causes various human diseases. Here we aimed to explore the function of the Schizosaccharomyces cerevisiae mitochondrial protein SHY1（YGR112W）in homologous protein Shy1 of Schizosaccharomyces pombe. Firstly，we generated the strain Δshy1 of shy1 gene deletion by gene knockout，and examined its phenotype in the non-fermentation medium with glycerol as the sole carbon source. Then，bioinformatics analyses revealed that Shy1 of S. pombe contained the conserved MTS（mitochondrial targeting sequence）of about 30 amino acids at the N-terminus. To further study the localization of Shy1，the location of green fluorescent protein（GFP）was observed by labeling GFP at the C-terminus of Shy1 protein regulated by the nmt1 promoter. Lastly，we detected the influence of the deletion of shy1 on the expression of mitochondrial protein by Western blotting. The results demonstrated that the strain of shy1 gene deletion presented its growing deletion in the non-fermentation medium with glycerol as the sole carbon source，and was one with mitochondrial respiratory defect. When GFP was labeled at the C terminus of Shy1 protein regulated by the nmt1 promoter，the location of Shy1 protein was in the mitochondria as the green fluorescence was observed. Moreover，the results of Western blotting revealed that the expression of protein related to mitochondria resulted from the shy1 gene deletion was dramatically declined. Therefore，these findings indicate that Shy1 is localized in mitochondria and is obligatory for mitochondrial respiratory chain to play ordinary function in S. pombe.

PCR Amplification Characteristics of DNA with Inverted Repeat Sequence

LI Chun-chuan,PAN Xiao-ming,WANG Jing,DONG Ping,LI Jing,LIANG Xing-guo

2016, 32(12):  180-188. 

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This work is to explore that effect of hairpin structure formed by inverted repeats on template amplification. We investigated the factors affecting PCR amplification efficiency of DNA with hairpin structure，including ring size，stem length and the stem position as to the primer’s hybridization，and discussed how to achieve efficient amplification of DNA with hairpin structure by designing primers and changing the annealing temperature. The results showed that if the position of the primers completely or partly complemented to the 5'-sequence of the hairpin stem，the formation of hairpin structure hindered the binding of primer and template，therefore the inhibition to PCR amplification occurred；moreover，the inhibition increased with the stem length increasing. The inhibition decreased significantly when，the loop portion was longer than 50 nt. Higher annealing temperature and primer concentration were favorable for primers to compete with the formation of hairpin structure within molecule，therefore leading PCR amplification to be more efficient.

Effects of Lysophosphatidic Acid on the Migrations of Hepatoma Cells with Different Metastasis

LI Duo,LI Jia-wen,YUAN Qun-chen,LIN Chuan-chuan,SONG Guan-bin

2016, 32(12):  189-194. 

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The aim of this study is to investigate the effect of lysophosphatidic acid（LPA）on cell migration of two kinds of hepatocellular carcinoma cells（MHCC97H and HepG2）with different metastasis，and to evaluate the underlying mechanism. Transwell assay was employed to investigate the impact of LPA on the migration of the hepatocellular carcinoma cells，and RT-PCR to detect the mRNA expression of LPA receptor（LPAR）in the hepatocellular carcinoma cells. Results showed that the LPA presented no obvious effect on the migration of HepG2 cells in low metastasis，but significantly promoted the migration of MHCC97H cells in high metastasis. LPA caused insignificant impact to the proliferation of both cells. RT-PCR demonstrated that expressions of LPAR at mRNA level in MHCC97H and HepG2 cells were different，LPAR1 expressed in MHCC97H cells，however，not in HepG2 cells. Importantly，the promotion effect of LPA on the migration of MHCC97H cells disappeared after Ki16425（a specific inhibitor of LPAR1/3）was used to block the role of LPAR1 in MHCC97H cells. This indicated that LPA promoted MHCC97H cell migration via interaction with LPAR1. The effects of LPA on the migration of hepatoma cells in different metastasis varied，which resulted from the different expressions of LPAR in these two cell types.

Screening of Stem Genes Regulated by H3K9me2 in Tumor Stem Cells

TAO Hong,ZHANG Shao-ping,ZHANG Lin-na,ZHANG Man,HU Qi-kuan,

2016, 32(12):  195-202. 

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In order to analyze and compare the modification differences of the methyltransferase G9a and histone（H3K9me2）between in glioma stem cells and non-stem cells，the related genes maintaining the stem of glioma stem cells were screened. By using G9a inhibitor to facilitate U87 cells into spheres，and G9a overexpression to promote the cell differentiation，the sphered glioma stem cells and monolayer non-stem cells were cultured，and there were significant differences in CD133 expression between the two cell models. Then we used H3K9me2 antibody to compare the differences of H3K9me2 modification between glioma stem cells and non-stem cells via ChIP-seq assay. Genes in the range of TSS ± 2 000 bp among the genes with differences were selected for GO analysis. Moreover，10 transcription factors were randomly selected to confirm the expression level by QPCR，and the result of which was almost consistent with that by ChIP-seq assay.

Research Situation of Transgenic Corn Based on Bibliometrics and Content Mining

ZHENG Ying,LIU Jia-yi,ZUO Xuan,LI Sheng-yan,WU Sheng,NIE Feng-ying

2016, 32(12):  203-213. 

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Corn is the most widely cultivated cereal crop in the world，thus it possesses critical niche in agricultural production. Since the 80's of last century，the research of genetically modified corn（GMC）has been being a hotspot in the field of agricultural science. In order to understand the research situation of transgenic corn in the world，we analyzed the items that have great influence in this field，such as countries，research institutions，key journals，outstanding authors，and high cited papers，based on the papers of 10 years（2005 - 2014）on GMC collected in the Web of Science. With the content mining method，we extracted 10 core sentences in this topic，and analyzed the research hotspot of transgenic corn. The results showed that the United States and China led the world research level of GMC. China's scientific research institutions issued larger amount papers，but the papers issued from United States were in higher quality and caused larger impact in GMC field. The key journals publishing transgenic corn were concentrated in United States，Britain，Germany，Australia，Kenya，Ireland and Holland. Among the top 20 high cited papers，10 were from the United States. The research hotspot of the transgenic corn focused on the 4 aspects：the pest resistance，the anti-reverse，the risk assessment of environment，and the improvement of crop performance. Some studies，such as the use of RNAi technology against insects，shade-tolerant corn（resistant to high density），male sterility，etc.，could become the next hot topics on GMC in the future.