Table of Content

19 July 2013, Volume 0 Issue 7

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Review

Functional Genomics Approaches for the Study of Low Phosphate Responses in Higher Plants

Xing Guofang, Guo Pingyi

2013, 0(7):  1-6. 

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Phosphorus（Pi）plays an important role in higher plants on the growth and development, and Pi availability is frequently a limiting factor for crop quality and productivity due to the deficiency of soluble inorganic Pi in many soils worldwide. A number of morphological, physiological, biochemical and molecular responses have evolved in plants that allow them to grow and prosper in the presence of low levels of available. This review examine and compares several recent advanced studies related to the functional genomics aspects of plant responses to Pi stress, such as the gene expression profiles of phosphorus stress induced, the differences in gene function categories, the complex network of regulatory genes, the non coding RNAs and plant hormone involved in the regulation mechanism of plant tolerance to low phosphorus were also highlighted and assessed.

The Dispute About CHLH Being an ABA Receptor

Ren Jie,Leng Ping,Sun Fushan,Xu Xiuhong

2013, 0(7):  7-11. 

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Mg-chelatase H subunit（CHLH）is a key enzyme involved in chlorophyll synthesis. Since CHLH was proved to be an ABA receptor in Arabidopsis in 2006, the relation between CHLH and ABA signal transduction has been reported in barley, sweet cherry, strawberry and peach, but whether CHLH acts as an actual ABA receptor remains controversial. This paper summarizesd the research on the relation between CHLH and ABA signal transduction, focused on the dispute about whether CHLH acts as an ABA receptor and advances of CHLH in perception and transduction of the ABA signals.

Review on High-level Aspergillus niger Glucose Oxidase Production Engineering Strains

Liu Yu, Li Piwu

2013, 0(7):  12-19. 

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Glucose oxidase, which can catalyze the oxidation of glucose to glucono-δ-lactone, has been applied in various fields. The traditional production of glucose oxidase using Aspergillus niger as the industrial production microorganism has reached the limit. Nowadays, the main expression systems which have been used in the expression of glucose oxidase are Asprgillus niger, Saccharomyces cerevisiae and Pichia pastoris. Directed protein evolution has recently come into use in the molecular modification of glucose oxidase, which is expected to improve the yield of Aspergillus niger glucose oxidase in industrial production. The progress of high-level glucose oxidase production engineering strain were reviewed.

Advance of the Classification in Ralstonia solanacearum Species

Cai Liuti,Wang Hancheng,Liu Yanxia,Cao Yi,Yuan Saifei,Zhao Zhunbei,Shi Junxiong

2013, 0(7):  20-23. 

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Ralstonia solanacearum is a soilborne Gram-negative bacterial pathogens, infecting more than 200 species belonging to more than 50 botanical families, including Solanaceae economic crops such as potatoes, tobacco and tomato, cause to serious loss of their yield and quality. R. solanacearum is a complex species, this paper reviewed the research progress of classification in the R. solanacearum, including the races, biovars and phylotypes, and the different type schemes were compared, which povide phylotype information for the analysis of the composition and genetic diversity of the tobacco R. solanacearum, for the study the interaction between tobacco and R. solanacearum.

Research on Glutathione Related Enzyme’s Response to Persistent Toxic Substances in Aquatic Organisms

Li Shaojuan, Wang Yixiao, Zhao Huan, Zhou Yibing

2013, 0(7):  24-28. 

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Glutathione related enzymes, which are the phase Ⅱ detoxification enzymes and antioxidant enzymes in vivo, have an important role in biological metabolism of persistent toxic substances. In this paper the detoxification effect of glutathione S-transferase and glutathione peroxidase on organic pollutants and heavy metals, and molecular research in these two enzymes are briefly discribed. The results of these researchs will provide us insight in the future applications of glutathione related enzymes in environmental monitoring and pollution diagnosis.

Research Progress on Regulation Mechanism of Ovary Development in Shrimps and Crabs

Wang Xiaowei,Zhang Ziping,Wang Yilei,Jia Xiwei,Lin Peng

2013, 0(7):  29-35. 

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Regulation of reproduction in shrimps and crabs is a highly complex process that requires participation of several regulative factors like neuropeptides, hormones, neurotransmitters etc. Neuropeptides are mainly secreted by X-organ-sinus gland（XO-SG）, brain and thoracic ganglia（TG）. The neuropeptides of CHH family synthesized and secreted by XO-SG play a key role in gonad development regulation process. In this article, recent studies related to gonadal regulation in shrimps and crabs are reviewed.

Advance in the Nucleotide Binding and Oligomerization Domain （NOD）-like Receptors （NLRs）

Li Yiping, Wang Xiao

2013, 0(7):  36-40. 

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The innate immune system is the first line of defense against microorganisms, recent studies identified a new kind of protein, the nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) which can recognize and respond against pathogens, play an important role in the innate immune of the plant and animal. Activation of NLR proteins results in inflammatory responses mediated either by NF-κB, MAPK or Caspase-1 activation. The paper summarized the NLR family and the inflammatory diseases associated with it. The NLR family in the treatment of inflammatory diseases were also prospected.

Phenomics: A Science of Unravelling the Genotype-Phenotype Relationship

Li Hong, Wei Xiaolan

2013, 0(7):  41-47. 

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Phenomics is a new branch of science developed in recent years, which mainly study the cell phenotypes in different environments at systems level. Phenomics is widely used in the genotype-phenotype mapping, the genetic basis research of complex trait diseases, crop genetic improvement. Meanwhile, phenomics research faces huge challenges. Extensive features of genomic data directly spawned a new scientific research based on the genome sequences. This paper introduces the scope of phenomics in simple terms. It summarizes the application of phenomics in the genotype-phenotype mapping, the genetic basis research of complex trait diseases, crop genetic improvement, and the challenges of phenomics research. Meanwhile, it discusses the application of bioinformatics in phenomics, and the future and perspective of phenomics.

Research Progress of the Mechanisms of Semipersistent Viruses Transmission by Vectors

Tian Xiao,Li Lingdi,Liu Jinxiang,

2013, 0(7):  48-53. 

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Most of plant viruses are transmitted by insect vectors. Hemipteran is the most common vector which spread viruses in nonpersistent, semipersistent and persistent manner. The complex interactions between vector and virus can be classified into two general mechanisms. One is capsid mechanism and other one is helper mechanism. Semipersistent transmitted viruses mainly belong to Closteroviridae, Caulimoviridae, Flexiviridae and Sequiviridae. In recent years, the mechanism of the virus transmission, especially of semipersistent viruses transmission, has attracted an extensive attention. This paper summarizes the research progress of the mechanisms of semipersistent viruses transmission by vectors.

Study Report

Cloning and Expression Characterization of Ethylene Receptor Gene Cm-ETR1 from Melon（Cucumis melo L.）

Chen Yujie,Chen Ming,Wulan Bateer,Hao Jinfeng,Gao Feng,Hasi Agula

2013, 0(7):  54-59. 

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Ethylene receptor is the initial composition of ethylene perception and signal transduction. In order to investigate the function of melon ethylene receptor gene Cm-ETR1 during fruit ripening in melon, a pair of gene specific primers was designed based on the cDNA nucleotide sequence of Cm-ETR1 gene from melon（accession number：AF054806）in GenBank. The full-length cDNA of Cm-ETR1 was cloned by RT-PCR from ripening fruit of melon（Cucumis melo L. cv. Hetao）, and was submitted to GenBank（accession number：EF495185）. Sequence analysis showed that the cloned cDNA was 2 256 bp long, contained an 2 223 bp ORF that encoded a polypeptide of 740 amino acids, and the cDNA sequence was consistent with that of reported melon variety cantalupenis ETR1 cDNA. Cm-ETR1 protein phylogenetic tree analysis show that the ethylene receptor protein is highly conserved in all species, and Cm-ETR1 is highest similarity with cucumber ethylene receptor protein by 99% consistency, and lowest similarity of longan ethylene receptor protein by 86% consistency. Quantitative PCR analysis indicated that the expression level of Cm-ETR1 gene was increased, which coincided with the raise of the melon fruit ethylene production and fruit ripening process, and peaked at ethylene climacteric stage. The amount of endogenous ethylene had a positive correlation with the expression of Cm-ETR1 gene. It reveals that Cm-ETR1 gene may play an important role in fruit ripening.

Molecular Cloning and Expression Analysis of ERF Transcription Factor Gene in Gardenia jasminoides During Fruit Development

Gao Lan, Lu Jingwen

2013, 0(7):  60-65. 

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Ethylene-responsive element-binding factor（ERF）genes constitute one of the DNA-binding protein gene families in plants, and are transcription factors associated with ethylene signal transduction, developmental and physiological processes. In this paper, a new member of the AP2/ERF transcription factor family, GjERF, was isolated from Gardenia jasminoides fruit cDNA library. The GjERF cDNA is 962 bp, contains a predicated 780 bp open reading frame that encodes a protein of 259 amino acids with calculated molecular mass of 28.6 kD, and with a 82 bp non-coding region at 5' end, a 100 bp of non-coding region flank at 3' end. Sequence analysis showed that GjERF contained an AP2/ERF domain of 59 amino acids and a N- terminal MC motif（MCGGAII）. It belonged to a group Ⅶ protein in the ERF subfamily. The RT-PCR analysis indicated that the transcription of GjERF gene accumulated primarily in mature leaves of G.jasminoides, and was not related with fruit developmental stages.

Cloning and Expression Analysis of Chalcone Synthase Gene from Rosa hybrida

Zhang Yunting, Yu Dingqun, Cheng Yi, Tang Haoru, Wang Qingming, Zhang Yong

2013, 0(7):  66-70. 

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It was to clone and identify chalcone synthase gene（RcCHS）from Rosa hybrida‘Fairyland’, analysis the characteristic of the sequence and expression. The total RNA was isolated from the flower petal of Rosa hybrida‘Fairyland’by improved CTAB, then reverse transcription. Using PCR degenerate primers designed based on GenBank published the conserved sequences of chalcone synthase genes from other plants to amplify cDNA fragments, a new chalcone synthase gene named RcCHS was cloned. The expression patterns of RcCHS in different developmental stages were analyzed by semi-quantitative RT-PCR. The length of RcCHS was 860 bp which encoded 287 amino acids, GenBank accession No. KC145553. Homology analysis showed that the RcCHS of Rosa hybrida‘Fairyland’had high homologies with other plants, and indicated that CHS in evolution process kept the high conservatism. RcCHS had a highest transcript level in petals at blooming stage.

Construction and Application of a Plasmid Reference Molecule for Detecting Transgenic Rice

Li Xiaofei,Tan Xiaoli,Li Jun,Wu Yuhua,Cao Yinglong

2013, 0(7):  71-77. 

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Five targets, including the CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene were selected to be detecting targets of transgenic rice screening, based on the application frequency of the regulation elements and function genes used in rice（Oryza sativa）genetic engineering research. A rice reference gene SPS, five screening targets（CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene）and three event-specific detection fragments（TT51-1, KF-6, KMD1）were fused using a overlap extension PCR technique and cloned into a plasmid vector pBluescript II SK+ to construct pBS Rice. The results showed that the plasmid molecules pBS Rice could be applied not only for the screening of genetically modified rice, but also in the event-specific detections of insect-resistant rice TT51-1, KF-6 and KMD1. This study provides a raw materials independent positive control with a coverage rate of 100% for the worldwide approved rice events, meeting the needs of the transgenic rice security supervision.

Purification and Activity Assay of Flage llin from Acidovorax avenae sub. avenae NB001

Wu Tian,Liu Ying,Yu Chao,Bian Qiang,Tian Fang,Huang Qiong,Chen Huamin,He Chenyang

2013, 0(7):  78-81. 

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To elucidate the mechanism of PAMP-triggered-immunity（PTI）of rice by bacterial flagellin, a pathogen-associated molecular pattern（PAMP）, the isolation and purification of flagellin from Acidovorax avenae sub. avenae（Aaa）strain NB001 and it’s activity in elicitation of H₂O₂ generation of rice were performed in this study. The results showed that Aaa flagellin（about MW45 kD）was effectively purified by combined using of vortex oscillation, acetone precipitation and anion exchange chromatography. Moreover, the purified flagellin significantly induced H₂O₂ production in the leaf tissues of 14-day-old rice seedling. Therefore, the purified flagellin from Aaa could be further used for function analysis in PTI of rice.

Yak HSFY Gene Cloning and Structure Anlysis

Zeng Xianbin,Wang Yong,Liu Zhongna,Ma Zhijie,Hai Ting,Zhong Jincheng

2013, 0(7):  82-88. 

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Heat shock transcription factor linked Y chromosome is one of the candidate genes for spermatogenesis obstacle. In this study, sequences of HSFY genes of yak were obtained by cloning and alignmented with 84 HSFY sequences on the Y chromosome of the cow. The results show that：The nucleotide homology of yak, HSFY genes which are composed with two exons and one intron with the corresponding sequences in the genome of the cow is high up to 99.54%. We found four insertion/deletion sequences. Between the 1 012-1 020 bp（Ⅰ）, it lacks 9 nucleotides（AAAGAAGA/TG）which is located in the intron. While between the 1 071-1 073 bp（Ⅱ）and 1 249-1 251 bp（Ⅲ）lack AAG and CCA/C respectively, which are located in the second extron and that lack lysine and histidine respectively in the amino acid sequence. Between the 1 708-1 710 bp（Ⅳ）, there is a 3 nucleotides deletion（CAA）, which is located in 3'-URT. In the 84 HSFY genes of the cow, the frequency of the insertions/deletion is 29，3，2，1 forⅠ，Ⅱ，Ⅲ and Ⅳ, respectively. It is not found one with both Ⅱ and Ⅲ deletion. But all Ⅱ or Ⅲ deletion are accompany withⅠdeletion.Ⅱ and Ⅲ deletions change three-dimensional structure of HSFY protein. Yak HSFY which is a multicopy gene prefers using codes that are ended with A or T.

Screening and Sequence Analysis of Cashmere Goat DBY

Xiao Hongmei,Xiao Xu,Liu Zhihong,Li Jinquan

2013, 0(7):  89-93. 

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Cashmere goat is a unique biological resource in China. After a long period of natural selection and artificial breeding, Cashmere goat has best fiber quality and higher cashmere yields at present in the world. Therefore, the high reproductive rate of Cashmere goat is essential for developing economy. In this experiment, 240 bp gene fragment was obtained, though screening from Cashmere goat BAC library, cloning, and identifying DBY gene controlling spermatogenesis on the Y chromosome by PCR. Then the sequence of Cashmere goat was compared with other species from GenBank. The results showed Cashmere goat has homology of 96% with Ovis aries, homology of 93% with Cervus eld and Bubalus bubalis. The homology is up to 92% and 91% with Axis porcinus, Bos indicus and Bos teurus, respectively, no homology with the human, chimpanzee and mouse. The sequence fragment encodes 52 amino acids. The clustering consequence of species NJ consistent with the taxonomic status.

Culture and Characterization of Chicken Embryonic Stem Cells in vitro

He Wenjun,Liu Hong,Ye Lingling,Li Shichong,Wang Qiwei,Chen Zhaolie

2013, 0(7):  94-98. 

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The blastoderm cells isolated from the stage X embryos of chicken were cultured on feeder layer of mouse embryonic fibroblasts（MEF）with high glucose DMEM supplemented with 10 ng/mL bFGF, 20 ng/mL hIGF-1, 2 ng/mL mSCF, 2 ng/mL hIL-11 and 1 000 U/mL LIF. Typical chicken embryonic stem cells（cESCs）colonies with high endogenous AKP activity were observed in the culture system. The cESCs derived from blastoderm cells expressed pluripotent markers SSEA-1 and Oct-4 as determined by immunocytological analysis. RT-PCR analysis further confirmed the expression of cENS-1, a gene specifically expressed in cESCs and early embryo. These results demonstrate the feasibility for using the culture system in isolating cESCs from the stage X chicken embryos and supporting the growth of cESCs with undifferentiating state in vitro.

Genetic Diversity of The MHC-UBA Gene in Rainbow Trout

Hu Dandan, Liu Zhe, Shao Shujuan, Yang Juan, Wang Jianfu

2013, 0(7):  99-106. 

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Major histocompatibility complex（MHC）is central to the vertebrate immune system. The genes of the MHC are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. In this study, the variation of MHC classⅠUBA gene exon 2 in the 184 Rainbow trout was investigated by PCR-SSCP, followed by cloning and DNA sequencing. For this research 12 alleles were identified, of which 11 were novel. A maximum of four alleles was amplified per individual in this locus, suggesting that there are at least two copies site of UBA gene in Rainbow trout. Variation percentage of nucleotide and amino acids were 6.13% and 17.44%, respectively. The polymorphism information content is 0.794 3. Sequence homology analysis showed that gene homology range from 98.08% to 99.23% among the 12 alleles. The non-synonymous substitutions were significantly higher than the synonymous substitutions in the peptide-binding regions（PBR）and non-PBR（P<0.001）, this result might suggest that the polymorphism of UBA gene seems to be maintained by a positive selection. Frequencies of synonym codons were not distributed equally. The neighbor-joining phylogram of UBA gene showed Rainbow trout and Atlantic salmon was assumed to be closely related. The study indicated that a high level of genetic diversity existed in Rainbow trout, and it might be a potential genetic marker in Rainbow trout breeding.

Genetic Diversity of Different Geographical Populations of Simulium quinquestriatum Based on ISSR Analysis

Xiu Jiangfan, Zhang Chunlin, Chen Hanbin

2013, 0(7):  107-113. 

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In this paper, the genetic diversity of geographical populations of S. quinquestriatum from China was investigated with ISSR method using eight selective primers in order to clarify thegenetic diversity of eight different geographic populations of S. quinquestriatum. Establishment of ISSR fingerprint of S. quinquestriatum from eight populations of 160 individuals in this study. The results showed that total 111 bands were generated, of which 96 bands were polymorphic bands（the percentage of polymorphic band, P= 86.47％）. The characteristic ISSR pattern for each individual was detected. S. quinquestriatum displayed the higher degree of genetic diversity at the species level（P=86.4%, A=1.991 0±0.094 9, A _E=1.553 2±0.334 5, H=0.323 1±0.157 9, I=0.484 7±0.203 7）than the population level（P=78.37%, A=1.492 1±0.499 7, A _E=1.316 6±0.370 0, H=0.180 1±0.206 6, I=0.265 5±0.295 0）. Results of AMOVA analysis demonstrated that the among-population component had a high degree which accounted for 59% of the total genetic differentiation；while the within-population component accounted for 41%. The gene flow that among-population is 0.347 5. It is indicated that the main factors for the high degree of genetic differentiation among different geographic populations of S. quinquestriatum are geographic barrier（mountains and plains）and habitat fragmentation.

Purification and Optimization of Prokaryotic Expression of Perlucin Protein in the Freshwater Pearl Mussel，Hyriopsis cumingii

Lin Jingyun, Bai Zhiyi, Li Jiale

2013, 0(7):  114-118. 

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Perlucin was originally isolated from nacre of abalone inner shell and improved to promote calcium carbonate precipitation at ambient conditions, nucleate the calcium carbonate crystallization and modify the morphology of calcium carbonate crystals. The open reading frame（ORF）of the perlucin gene was amplified from the mantle cDNA of the freshwater pearl mussel, Hyriopsis cumingii. The ORF from this species was ligated into the expression plasmid pET-28a. Following transfer of the recombinant plasmid into host bacteria, Escherichia coli BL21（DE3）cells, the recombinant pET-28a-Hcperlucin protein was expressed by induction with isopropyl-β-thiogalactopyranoside（IPTG）. SDS-PAGE analysis showed that inducing the cells at 37℃ in 0.1 mmol/L of IPTG for 6 h was the optimal conditions for expression of the recombinant pET-28a-Hcperlucin protein. Inclusion body was the main existence form of the recombinant protein. The expression products were detected by SDS-PAGE and Western blot. The results showed that the recombinant expression plasmid of pET-28a-Hcperlucin, expressing a 21 kD protein that could be recognized by mouse anti-His-tag Mab, was successfully constructed.

Screening and Characterization of a Marine Actinomycete with Antagonist Activities Against Multidrug Resistant Bacteria

Chen Meiqun，Feng Guangda,Deng Mingrong,Zhu Honghui

2013, 0(7):  119-124. 

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It was to screening the antagonist activities against multidrug resistant bacteria from 71 marine actinomycetes. The inhibitory effect of 71 marine actinomycetes were detected against multidrug resistant bacteria by disk diffusion method, the strain was identified by morphological, physiological, biochemical characteristics and 16S rDNA sequence analysis. Results showed that strain MQ-86 showed strong inhibitory effect against multidrug resistant bacteria. The fermentation crude extract of strain MQ-86 using M₂₊ medium shows strong inhibitory effect to Escherichia coli 8739, Staphylococcus aureus 6538, Candida albicans 10231, Escherichia coli 480, Escherichia coli 460 and Staphylococcus aureus 206. Strain MQ-86 has a strong inhibitory effect against multidrug resistant organisms, which was identified as Streptomyces parvus.

Screening and Identification of a Galactosamine-producing Strain and Study of Its Cultural Conditions

Zhang Rongling, Wang Ruiming, Li Piwu

2013, 0(7):  125-130. 

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A galactosamine producing-strain YR06 was isolated from the sludge where the dead fish and crab was decomposed in Dongying Yellow River estuary wetland. Based on morphology, physiological and biochemical characteristics as well as the 16S rDNA gene sequence, strain YR06 was identified as Aeromonas hydrophila. Optimization of YR06 fermentation conditions was studied via single factor and orthogonal test design. The optimum fermentations conditions were showed as followed：fructose 10 g/L, urea 15 g/L, Na₂HPO₄ 15 g/L, CaCl₂ 22.2 g/L, MgSO₄ 0.18 g/L, KH₂PO ₄ 5 g/L；initial pH6.5, fermentation holding 60 mL/250 mL, fermentation temperature 33℃. The yeild of galactosamine achieved 128 mg/L after the optimization.

Screening and Identification of a High-yield Glucoamylase Bacteria

Zhang Wenli,Yu Hansong,Piao Chunhong,Wang Shuting,Hu Yaohui

2013, 0(7):  131-135. 

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An high-yield glucoamylase bacterial strain of ZWL-1（GenBank accession number of the 18S rDNA gene：KC433410）used dilution plate method was isolated from a soy processing plant in Jilin sewage outfall at soil samples . The species identification was made on it, on the basis of morphological observation and through 18S rDNA sequence analysis further to the bacterial strain. The 18S rDNA sequence shows that the homology of this bacterial strain and the18S rDNA of Aspergillus niger was above 99.0%. As shown in the phylogenetic tree, strain ZWL-1 and Aspergillus sp. LQ21 constituted a branch and they constituted a high-order branch together with Aspergillus niger strain.CS1-1. Thus, the primary identification proved that this bacterial strain is Aspergillus niger.

Gene Cloning，Expression and Enzyme Activity Assay of UDP-glucose Dehydrogenase in Escherichia coli

Chen Yihan,Qian Yue,Hou Yongtai,Zhou Qingwei,Gan Renbao,Guan Shimin,Rong Shaofeng

2013, 0(7):  136-143. 

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UDP-glucose dehydrogenase, as the key rate-limiting enzyme in the hyaluronic acid synthesis process, can catalyze UDP-glucose to UDP-glucuronic acid which is essential for cell to produce hyaluronic acid. The ugd gene amplified from genomic DNA of Escherichia coli BL21（DE3）and YK537 by PCR were cloned into pBLMVL2 vector containing P _{L promoter. The plasmid pBLBugd and pBLYugd were constructed and transformed respectively into Escherichia coli BL21（DE3）and YK537 to get four recombinant strains. UDP-glucose dehydrogenase activity of different recombinant strains was measured by thermal induction. The differences of expression level and enzyme activity of UDP-glucose dehydrogenase were detected under different temperature induced conditions. These result demonstrate that adopting double stage of heating induction and adding some moderate amount of yeast extract to the cultivating medium can improve the expression level and enzyme activity of UDP - glucose dehydrogenase.}

Full-length Gene Cloning and Common Antigen Analysis of Outer Membrane Protein of Pathogenic Aeromonas Isolated from Diseased Eels

Guo Songlin, Wang Yu, Guan Ruizhang, Feng Jianjun, Lin Peng, Yang Qiuhua

2013, 0(7):  144-152. 

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In order to search the common antigen of out membrane protein of pathogenic Aeromonas isolated from diseased eels, 17 Aeromonas gene sequences of outer membrane protein（OMP）from the NCBI/GenBank were compared and the conserved fragments of one OMP（porin II）were determined. According to the whole genome sequences of Aeromonas hydrophila and Aeromonas salmonicida, the full-length gene sequences of porin II（ompII）were ascertained and the related up and down fragments out of ompII sequences were ascertained. One pair of specific primers was designed at the up and down fragments. Using the primers, 11 OmpII genes were successfully amplified by temperate DNA of 30 strains of pathogenic Aeromonas, and 10 of 11 products were sequenced. Protein structure, hydrophilicity and epitope were analyzed using the predicted expression products of a full-length sequence of ompII. The results showed that the predicted protein was a trans-membrane protein with tertiary structure, and it was also a hydrophilic protein with multiple epitopes. The study provided theoretical basis on gene engineering vaccine designing through common antigens of OMP of pathogenic Aeromonas isolated from farming eels.

Characterization of Intestinal Microbiota in Feces from Captive Healthy Rhesus Macaques

Zhao Na,Liu Shelan,Lu Jiqi,He Hongxuan,Zhao Baohua

2013, 0(7):  153-160. 

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With the denaturing gradient gel electrophoresis（DGGE）and real-time quantitative PCR（q-PCR）, we analyzed the predominant bacteria in fecal samples of 24 individuals divided into 6 groups. DGGE profiles showed abundant bands for the 16S rRNA gene V3 region, and the composition of the gut microbiota was apparently different in each group. Furthermore, a clustering between infant and pregnancy was shown by Principal Coordinate Analysis（PCoA）. Identified in the DGGE gels by sequencing the V3 regions were overwhelmingly affiliated with Firmicutes, Actinobacteria and Bacteroidetes. Q-PCR indicated that Clostridium and Bifidobacterium were the dominant species, and the least dominant species was Enterococcus faecali. C.clustersⅪⅤ, E.faecalis and Bacteroides-Prevotella group have the most significant diversity among individuals（P<0.01）The Bifidobacteria /Enterobacteriaceae（B/E）ratio, which indicates microbial colonization resistance（CR）in the gut appeared to be lower than the peers, subadults and adults, in pregnancy（P<0.01）. The study revealed the characterization of Rhesus Macaquesintestinal microbiota. The characteristics of infant gut microbiota are remarkably similar to those found of the pregnancy groups.

Botryosphaeria dothidea of Multiple Genes Joint Identification

Jia Guangcheng,Zhou Zengqiang,Hou Hui,Wang Li,Zhu Jianlan

2013, 0(7):  161-166. 

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In order to identify apple ring rot and make sure of the present situation of apple ring rot’s main pathogenic genes, taking the method of PCR to amplificate and sequence the ITS genes, EF-1α gene and β-tubulin gene. The results showed that the fungi of apple ring rot is Botryosphaeria dothidea. There were EF-1α gene and β-tubulin gene in the apple ring rot’s DNA. Tubulin and transcription factors may be involved in the disease process of apple ring rot, and they are important in the pathogenic process of the apple ring rot.

Establishment and Optimization of CPA-nucleic Acid Test Strip for Rapid Detection of Vibrio cholerae

Qin Qiang, Zhu Jinling

2013, 0(7):  167-171. 

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It was to estabilish a cross priming amplification and nucleic acid test strip based rapid detection method of Vibrio cholerae. Specific primers and probes according to the the MDH gene sequence of the Vibrio cholerae were designed, and via optimizing the reaction system and the reaction conditions to establish the optimal reaction system, the CPA method with the PCR method were compared and evaluated. Result showed that the method was highly specific for the detection of Vibrio cholerae, the detection limit reached 6 × 10 ² CFU/mL, higher than that of the PCR, and has satisfactorg stability. The CPA-nucleic acid test strip detection method established in this study is a specific, sensitive, rapid and easy detection method of Vibrio cholerae.

Phosphoenolpyruvate Carboxylase from Corynebacterium glutamicum Inhibited by Diverse Biochemicals

Wu Xinyang,Pei Guangsheng,Zheng Xiaomei,Cao Guoqiang,Liu Jiao,Zheng Ping,Wang Min,Sun Jibin

2013, 0(7):  172-178. 

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Phosphoenolpyruvate carboxylase（PPC）, an enzyme involved in replenishment of the four-carbon dicarboxylic acid intermediate pool of the TCA cycle, plays an important role in both biological and biotechnological synthesis pathways. To discover the effect of diverse inhibitors on the activity of PPC, the full-length sequence of the ppc gene from Corynebacterium glutamicum ATCC13032 was cloned and expressed as soluble form in Escherichia coli. The recombinant PPC was purified with Ni-Column. The purified enzyme showed high phosphoenolpyruvate carboxylase activity, and was inhibited by diverse inhibitors such as citrate, isocitrate, fumarate, glutamate, aspartate, malate etc. Aspartate and malate were the strongest inhibitors. For the first time, the inhibition of PPC activity by N-acetyl glutamine was clearly demonstrated in a glutamate-producing strain of C. glutamicum.

Constrution and Application of T7 Phage Vector with a Histidine Tag

Fan Jiangping,Zhong Li,Dong Xiaomin,Zhang Xufei,Wang Zhiqiang

2013, 0(7):  179-183. 

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A new type of histidine tag expression vector was obtained on the basis of the vector T7 Select10-3b through gene recombination technology. The recombined vector was used to construct cDNA library as an effective method of screening open reading frame（ORF）. A His-Tag was inserted into multiple clone site of vector T7. The sequence of His-Tag was obtained by renaturation. In order to get infectious T7 phage, T7 packaging extracts was utilized to package recombined DNA. CDNA library was constructed using modified vector, and ORF was purified by Ni-nitrilotriacetate affjnity chromatography. DNA sequencing revealed that His-Tag was inserted into the expression vector accurately. Chemiluminescence immunoassay displayed that inserted His-Tag sequence could express accurate protein. Screening rate of ORF rised from 5.6% to 82%. It proved that vector T7 inserted His-Tag had important significance for ORF screening.

Synthesis and Bioactivity of T-superfamily Conotoxin B8.1T Native to Hainan

Wu Yong, Wu Xiaosa, Zhangsun Dongting, Luo Sulan

2013, 0(7):  184-188. 

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A new T-superfamily conotoxin B8.1T with sequence DCCPESPPCCH was discovered from C. betulinus native to Hainan. Conotoxin B8.1T contains two disulphide bridges between Cys3-Cys9, Cys4-Cys10. The linear conotoxin B8.1T was synthesized by Fmoc solid-phase synthesis method at first. Three oxidative methods were used for folding linear peptides according to different cysteine thiol protecting groups：（1）two-steps oxidative folding；（2）one-pot folding；（3）random one-step oxidative folding. Oxidative folding parameters were optimized to improve folding efficiency. The bioactivity of conotoxin B8.1T was assayed. The results demonstrated that all the three strategies can be used to produce native conotoxin B8.1T. However, the efficiencies of the two-step and one-pot methods were higher than that of the one-step. The presence of folding additives helped to improve native isomer accumulating by parameters’ optimization. The bioassay showed that conotoxin B8.1T inhibited the movement behavior of mice by intracranial injections, while there was no effects on goldfish by intramuscular injections. The results would lay a solid foundation for follow-up synthesis and research of T-superfamily contoxins.

Cloning，Prokaryotic Expression and Purification of ANKIB1

Xie Yunfei,Yang Zishan,Ma Zhenling,Xie Bohong,Liu Shirao

2013, 0(7):  189-194. 

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Ankyrin repeat and IBR domain-containing protein 1（ANKIB1）contains RING-IBR-RING domain, ankyrin repeat（ANK）motif and ubiquitin-interacting motif（UIM）. It is likely that ANKIB1 has E3 ubiquitin ligase activity and specific catalytic mechanism. However, the function of ANKIB1 remains unclear. For the first time we cloned human ANKIB1 cDNA using nestled PCR and segmental amplication. Full length of ANKIB1 coding DNA sequence was constructed by gene recombination technology. The cDNA was ligated into prokaryotic expression vector pGEX-5X-3, and the recombinant plasmid pGEX-5X-ANKIB1 was transformed into E.coli BL21（DE3）. Using culture medium cotaining 200 mmol/L ZnCl ₂, the stabilizer for RING-type protein, expression of soluble GST-ANKIB1 fusion protein with a molecular weight of 148.5 kD was successfully induced by 0.1 mmol/L IPTG at 15℃ for 10 h. The recombinant protein was purified by affinity chromatography using Glutathione Sepharose 4B.