Table of Content

20 June 2013, Volume 0 Issue 6

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Genome Wide Association Study: Opportunities and Challenges in Genomic Research

Zhang Yanming, Xing Guofang, Liu Meitao, Liu Xiaodong, Han Yuanhuai

2013, 0(6):  1-6. 

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Genome wide association study(GWAS)is about screening for high-density molecular markers in certain populations in the range of the whole genome, then analyzing the correlations between the data of the molecular markers and the phenotypic traits. GWAS opened up a new chapter in genomic and genetic research, enabling linking genomics and genetics in unprecedented scale. GWAS is mainly applied in the analysis of complex traits of human diseases, leading to the identification of a number of genetic variants related to complex diseases and quantitative traits in human, hence it is to become one of the key approaches for human genomics. Application of GWAS in plant genomics just began, yet it has shown great advantages. It is becoming a research trend in plant genomics to use GWAS to discover genes related to complex quantitative traits to guide breeding programs. However, there exist some problems in GWAS, which are not as simple as expected. This review summarizes the current knowledge on GWAS with special emphasis on its applications in human and plant genomes and highlights its potential areas for future research.

Advances in Transgenic Plants for Phytoremediation

Chen Mei, Tang Yunlai

2013, 0(6):  7-11. 

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Phytoremediation, which is a green, sustainable and promising solution to problems of environmental contamination, defines the use of plants for uptake, sequestration, detoxification or volatilization of inorganic and organic contaminants from polluted soils, water, sediments and air. Phytoremediation was coined from the discovery that some plant species displayed the extraordinary ability in accumulating high quantities of toxic metals. This was shortly followed by the application of breeding techniques and artificial selection to genetically improve the remediation efficiency of some plant species, which give a high biomass, be easy to be transformed, and grow fast. Now, after years of research, transgenic plants for phytoremediation have been produced in laboratory, but none have reached commercial existence. This review aims to give main advances in the field of transgenic plants for phytoremediation.

Research Progress of Structural Characteristics and Functions of Calcium-dependent Protein Kinases in Plants

Jiang Shanshan, Zhang Dan, Kong Xiangpei, Zhou Yan, Li Dequan

2013, 0(6):  12-19. 

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The calcium ion(Ca²⁺)is known as a key second messenger in plants, intracellular Ca²⁺ signals are relayed to downstream(transcription factors, NADPH oxidases genes)via different calcium sensor proteins(CaMs, CaMLs, CBLs, CDPKs), which further causes expression of related genes and responses to abiotic and biotic stresses. Calcium-dependent protein kinases(CDPKs)which are Ser/The protein kinases found in plants and some protozoa, play crucial roles in Ca²⁺-mediated signaling pathways. CDPKs are encoded by multigene families and are divided into four subgroups. CDPKs exhibit overlapping and distinct expression patterns, sub-cellular localizations, substrate specificities and redundancy and/or diversity functions. Here we review the recent advances on the structural characteristics, expression patterns, localizations, regulations substrates both in vivo and in vitro, inhibitors and functions in response to abiotic and biotic stresses of CDPKs in order to shed light on the functions and regulatory mechanisms.

Research Progress of Nanoparticles for Immobilized Enzymes

Gao Qiyu, Xu Guangcui, Chen Hongli, Zhou Chenyan

2013, 0(6):  20-24. 

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Immobilization of protease and ribozyme by nanometer carrier are researched as a more useful means, including of the magnetic nanoparticle and nonmagnetic nanoparticles. Currently, the types of immobilized carrier and methods and results of nanoparticles are discussed. In this paper, we describe the current application of immobilized enzyme by nanocarrier, the effect of nanoparticles matrix to enzymatic properties and the prospect of application for the above mentioned technology were introduced, and the direction of the development of nanoparticles immobilization of enzyme was analyzed.

Research Progress of Structure and Function of Oligopeptide Transporter PepT2

Zhao Dongxin, Zhao Shanshan, Lu Kui

2013, 0(6):  25-31. 

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The oligopeptide transporter PepT2 of mammal is a high-affinity and low capacity transporter, it can transport small peptides and peptide-like drugs to all kinds of tissue cells . PepT2 is mainly expressed in the renal medulla cells, but also found in brain, central nervous system, lung, mammary gland and pancreas. PepT2 functionally characterized in the kidney has been considered to play a key role in efficient reabsorb small peptides generated by luminal peptidases. The structure and function of PepT2 were summarized in this paper.

Progress in the Mechanism of Host Cell Apoptosis Induced by Pathogen Virulence Factors

Hu Qingliang, Li Lingjuan, Wang Xiao

2013, 0(6):  32-38. 

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Apoptosis may occurs under pathological conditions and is sometimes accompanied by necrosis. In particular, bacterial infections play an important role in triggering apoptosis. Pathogenic bacteria can trigger apoptosis by activating some factor or suppress inhibitors of apoptosis, then change the host cell signaling pathways and induce cell apoptosis. In recent years, researchers have found that cell apoptosis is mainly related to pathogenic bacteria of virulence factor. Here, we reviewed the mechanism of host cell apoptosis induced by pathogen virulence factors, which aims at revealing the integrated effects of bacterial virulence factor triggering host apoptosis, and the same kind of bacteria can trigger to cell apoptosis through different mechanisms. Finally, with the help of current research progress and our own studies, we discussed and prospected pathogen virulence factor induced host cell apoptosis mechanism and application prospect, and discovered the pathogen virulence factor is of great potential in cancer and some bacterial resistant diseases.

Structure and Regulation Mechanism of Bacillus stearothermophilus dnaB-dnaG Complex

Lu Ting

2013, 0(6):  39-45. 

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In Bacillus stearothermophilus, helicase dnaB and primase dnaG form primosomes to unwind duplex DNA at the replication folk, and it’s important for the synthesis of Okazaki fragments. A helicase-interacting domain at the C-terminal of primase dnaG(P16)can regulate the interaction structurally and functionally. 9 amino acid residues as well as their linker regions at the N-terminal and C-terminal of helicase dnaB can affect the interaction between dnaG and dnaB. Here we summarize the regulation mechanism of dnaB-dnaG complex from the protein domain and functional aspect and the recent progresses.

Research Progression of Animal Gonad and Vitellogenin Under the Influence of Environmental Estrogens

Jia Yina, Liang Gang

2013, 0(6):  46-52. 

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Environmental estrogens(EEs)is able to disturb normal endocrine activity of human and animal, the affect reproductive functions. This paper summarized the recent researches on the effect of EEs on animal reproductive gland structure, sex ratio and vitellogenin synthesis. We recommend to determine the vitellogenin levels of abnormal expression in male or larvae of oviparous vertebrates as EEs index in environmental monitoring;mechanism research on the influence of EEs on reproductive function of human and animal, should be start from a single compound in EEs.

PI3K-Akt Signaling Pathway and Viral Infection

Liu Rongdiao, Ruan Lingwei

2013, 0(6):  53-62. 

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Many viruses have developed the ability to modulate key signaling pathways, and govern host cell functions to facilitate its proliferation. PI3K-Akt pathway is one of these key pathways which participate in host-pathogen interaction. Viruses take advantage of this pathway to create an environment favourable for its replication, resulting in dysfunction of the host cells, or even carcinogenesis. A thorough study on the roles of PI3K-Akt pathway in virus infection is of great significance to comprehensively understand pathogenesis, prevention and treatment of virus diseases. In this review, we summarize the recent advances in research on the regulation of PI3K-Akt signaling during virus infection.

Mechanism of RNA Interference and Its Application in Animal Disease Research

Fan Lu, Fan Jianming, Zhang Gaiping, Wang Aiping

2013, 0(6):  63-69. 

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RNA interference(RNAi)is a sequence-specific gene silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since it is discovered, RNAi has been developed into an useful tool for studying gene function, it has great advantage over traditional vaccine and therapy in preventing animal diseases. Additionally, introduction of virus-specific small interfering RNA(siRNA)or short hairpin RNA(shRNA)into cells, thus programming the RNAi machinery to target viruses, is an effective therapeutic approach to inhibit virus replication in vitro and in animal models. In this review, we summarize the mechanism of RNAi and its application in animal diseases.

Research Progress on Biosensors for Antibiotic Detection

Dun Wentao, Li Mian, Bi Qingsheng, Zhao Zhonglin, Yuan Chao, Li Shuying

2013, 0(6):  70-74. 

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Inadequate application of antibacterial drugs accelerated bacterial resistance phenomenon, which is a tremendous challenge for human health care and environment. Due to the merits of biosensor development and applications in the antibiotic field, this review gives an overview on the use and prospects of biosensor applications for antibacterial detection in environmental and foodstuff. The limited factors and future development trends of biosensors were also discussed.

Cloning and Sequence Analysis of PsaH Gene from Sasussured involucrata Kar. et Kir

Zhang Linhua, Qiu Honglin, Liu Chao, Wang Aiying, Deng Fujun, Zhu Jianbo

2013, 0(6):  75-79. 

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Ps I H is an intrinsic membrane protein of 10 kD that is a subunit of photosystem I(Ps I), Ps I H is one of the three PSI subunits found only in eukaryotes, plays an important role in plant photosynthesis. A cDNA clone encoding a PS I H subunit of photosystem I was cloned from Sasussured involucrata Kar. et Kir leaf cDNA library. Sequence analysis indicated that the psaH cDNA contained an open reading frame of 432 nucleotides encoding a 143 amino acid which consisted of a 47 amino acid transit peptide and a 96 amino acid mature PS I H peptide. Forecasted the protein relative molecular weight is 15.04 kD, Theoretical pI was 9.81, contains a span membrane structure. Sequence alignment indicated that the deduced amino acid sequence of Sasussured involucrata Kar. et Kir showed high identity of 83.3% to those of PsaH from Ricinus communis and Populus trichocarpa. Phylogenetic analysis further showed that the SikPsaH gene has the nearest relationship with Lycoris radiate.

Identification and Characterization of a Novel Gene, GhWRI1, Encoding an AP2-type Transcription Factor in Gossypium hirsutum

Li Xin, Wang Zhengming, Xue Wei, Chu Mingguang

2013, 0(6):  80-86. 

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AP2(APETALA2)/EREBP(ethylene-responsive element binding protein)genes comprise a family of plant-specific transcription factors, many of which have been linked to the regulation of essential biological processes. In this study, a novel AP2 gene, named GhWRI1, was isolated from Gossypium hirsutum(cotton). The GhWRI1 cDNA has a total length of 1 314 bp and encodes a protein of 437 amino acids with a molecular weight of 48 kD and a calculated pI of 5.77. Sequence analysis showed that GhWRI1 contains two AP2 domains and belongs to the AP2 subfamily. Using a green fluorescent protein fusion protein, we demonstrated that GhWRI1 accumulated specifically in the nuclei of onion epidermal cells. Real-time RT-PCR revealed that GhWRI1 was constitutively expressed in true leaves and roots, and its expression in fibers and seeds was upregulated over the course of development. In addition, GhWRI1 enhances the activity of a luciferase reporter by approximately 20-fold, by using a GAL4 activating system in tobacco cells. Our results indicate that GhWRI1 is a strong transcriptional activator. The possible role of cotton GhWRI1 in controlling the seed oil content is discussed.

Study on Characteristics of Constitutive Expression of GbLTP1 and GbLTP3 Genes in Land-cotton

Shen Haitao, Wang Aiying, Li Yuxia, Zhu Jianbo

2013, 0(6):  87-93. 

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According to the conserved sequences of LTPs gene from G. barbadense variety Xinhai 21 clone two lipid transferase gene GbLTP1 and GbLTP3. Constructing the constitutive expression vector pCAMBIA2301-GbLTP1 and pCAMBIA2301-GbLTP1 two plant expression vectors were transformed into upland cotton. By kanamycin screening, PCR and RT-PCR technique, to obtain transgenic GbLTP1 cotton and transgenic GbLTP3 cotton in all two strains. The progeny of transgenic plants disease resistance, agronomic and economic traits analysis, results showed that: the expression of GbLTP3 and GbLTP1 gene and Xinjiang upland cotton resistance to Fusarium wilt, resistance to yellow, and no significant increase of cotton. Effects of exogenous GbLTP3 gene and GbLTP1 gene into not agronomic traits, yield traits of cotton fiber quality, especially on fiber length has a certain improving effect.

The Effect of Physiological Traits of Transgenic Cotton Induced by Verticillium wilt Pathogen

Ou Xiuling, Geng Yawen, Li Feng, Li Caolong, Zhu Jianbo, Wang Aiying

2013, 0(6):  94-98. 

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To provide theoretical basis for improving resistant in transgenic cotton by researching the effect of physiological traits of transgenic cotton induced by Verticillium wilt pathogen. Chitinase and β-1, 3-glucan of bivalent resistance genes were delivered to two cotton lines of Byd, 28p by pollen-tube pathway method. The transgenic cotton lines with different resistant levels were obtained by many years screening. After the Verticillium wilt pathogen infestation to cotton seeds, the physiological and biochemical changes in transgenic cotton were determined at seedling stage. The results showed that the enzyme activity in transgenic disease-resistant cotton plants had reached the control's level, however, the level of enzyme activity in susceptible and disease-resistan cotton plants were different. The enzyme activity and changes of proline content were related to disease resistance of transgenic cotton plants.

Evolution of RNA Editing in Land Plant Mitochondria

Wan Ping

2013, 0(6):  99-103. 

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Mitochondria harbor the greatest number of RNA editing sites, why RNA editing emerges so frequently in mitochondria is unclear. In this study, we analyzed the 6 838 mitochondrial RNA editing sites from hornwort, clubmoss, fern, conifer, cycad, monocot and eudicot in features of(1)the probability of amino acid transition, (2)the probability of codon transition, (3)the probability of position of editing site occurring in the codon, (4)the probability of the occurrence of four bases at the minus 1 position near the RNA editing site, and(5)the probability of the occurrence of four bases at the plus 1 position near the RNA editing site. We find that the codon transition of mitochondrial RNA editing in angiosperms(monocot and eudicot)is significantly different from that of other plant groups.

Study on the SNP and Bioinformatics of Promoter Region of JAK2 Gene in Cattle

Gong Yu, Yang Yongqiang, Jiao Rengang, Hui Yanting, Liu Ruoyu

2013, 0(6):  104-109. 

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In order to screen the polymorphisms of JAK2 promoter in cattle and analyze the effect of SNPs on function elements of promoter. Two cattle breeds(Wuchuan black cattle and Guizhou Holstein cow)with significant difference in breeds property were selected to construct DNA pools, SNP sites were screened by direct sequencing subsequently. Results showed that 2 single nucleotide polymorphisms(SNPs)were found in the 5' flanking region and part of exon 1 which included G+5A and G+104A. Furthermore, bioinformatics tools were used to predict the core region of the promoter. It demonstrated that 1 new transcription factors binding sites emerged while 9 previous transcription factors binding sites disappeared based on the SNPs found in this study, and it also showed that SNP of G+5A could change the secondary structure of RNA, transcription factors binding sites and range of CpG island dramatically by using various softwares.

Identification on a Bacterial Strain HB-1 Separated from Stratum Water in Huabei Oilfield

Li Qing, Liu Yang, Wu Gang, Du Huijing, You Jing, Li Hong, Ke Congyu, Zhang Xin, Yu Jiliang, Zhao Ting

2013, 0(6):  110-115. 

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A bacterial strain HB-1 was isolated from stratum water in Huabei Oilfield, and identificated by polyphasic taxonomy basing on morphologic and physiologic methods, chemical composition analysis, genetic characteristics test and 16S rRNA gene phylogenetic analysis. The result showed that the strain was a member of the genus Chelatococcus, and was belong to the known species of Chelatococcus daeguensis. 37℃ was its optimum growth temperature. The major polar lipids presented in strain HB-1 were DPG, PE, PG and PC. Major fatty acids were C_{18: 0} ω7c(39.49%)and C_{19: 0} cyclo ω8c(30.39%), and ubiquinone Q-10 was the predominant quinone. HB-1 had enzyme activity of oxidase, catalase, esterase(C4), esterase lipase(C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphate hydrolase. Chelatococcus daeguensis HB-1 was isolated from stratum water in Huabei Oilfield for the first time.

Recombinant and Expression of Amidase from Nocardia farcinica in E. coli and Application on PA Modification

Guo Yingchun, Chen Sheng, Su Lingqia, Wu Jing

2013, 0(6):  116-121. 

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The amidase from Nocardia farcinica was recombined and expressed in E. coli. The recombinant vector pET24a/AMID was constructed and transferred into E. coli Origami(DE3). After 20 h induction(0.4 mmol/L IPTG, 25℃), the amidase activity was reached about 1.02 U/mL. In this study, the effect of temperature, pH, treatment time and rotation rate was investigated when PA fabric was treated with recombinant amidase, the water absorbability and wettability was detected by the methods of capillary effect and dying test. The result was demonstrated that the optimization of treatment condition were as follow: 40℃, pH7.5, 100 r/min rotation rate and treated 1 h, under this condition, the rising height of PA was increased by 8.7 cm, the dying percent was increased by 23.1%. The effect of enzymatic modification by recombinant amidase was almost the same as alkali treatment.

Cloning, Expression and Activity Analysis of Lipase Gene from Rhizomucor miehei

Miao Changlin, Luo Wen, Liu Shuna, Lü Pengmei, Li Huiwen, Yang Lingmei, Yuan Zhenhong, Jiang Jianchun

2013, 0(6):  122-127. 

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Lipase gene cDNA fragment from Rhizomucor miehei(RML)was amplified by RT-PCR method. Expression vector RML-pPIC9K containing lipase gene was constructed and the gene was expressed in His4 mutant Pichia pastoris yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression product of RML gene was analysis by SDS-PAGE, and molecular weight of the expressed protein was estimated to be 39 kD. The activity of lipase was up to 84 U/mL determined by the method of titration of NaOH. The result indicated that RML gene was functionally overexpressed in Pichia pastoris.

Cloning, Expression and Characterization of β-glucosidase from Stachybotrys chartarum

Yu Hailing, Li Shuwei, Wang Huaming

2013, 0(6):  128-132. 

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To express β-glucosidase from Stachybotrys chartarum using Aspergillus niger G1 as host. Through genome sequencing and analysis, the β-glucosidase gene(g10158)was isolated from the genome of Stachybotrys chartarum through PCR. The coding region of the gene was inserted to the vector pGm. The expression plasmid was transformed into A. niger G1 strain. An β-glucosidase producing strain(G1-pGm-g10158-13)were selected after screening several transformants using amdS selection plates and confirmed by PCR validation. Results showed that the molecular mass of the enzyme was 87.9 kD by SDS-PAGE gel analysis. Our results indicated that the recombinant enzyme exhibited optimum activity at pH4.8 and 50℃. The β-glucosidase activity was 1 856.48 U/mL under the optimized conditions. The β-glucosidase from Stachybotrys chartarum was expressed in A. niger G1 strain and it was proved to have a high activity.

Clonging and Expression of Heparinase III Gene from Flavobacterium heparinum and Characterization of the Recombinant Fusion Enzyme

Li Ye, Wu Jingjun, Ye Fengchun, Su Nan, Zhang Chong, Xing Xinhui

2013, 0(6):  133-139. 

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Heparinase III is an important enzyme for heparin structure analysis and low molecular weight heparin production. To achieve the soluble expression of HepC with high activity in recombinant E. coli, the heparinase III gene(hepC)from Flavobacterium heparinum was amplified by the PCR method, and the fusion expression vector and system of expressing fusion protein of a maltose binding protein(MBP)and HepC were constructed. The results showed that the fusion strategy by using MBP was effective to enhance solubility of the MBP-HepC when induced at low temperature of 15℃. By shake flask cultivation, the specific enzyme activity of MBP-HepC could reach about 46.41 IU/mg, which was the highest among the values reported so far. By one-step affinity purification with amylose resin, the MBP-HepC fusion protein was easily purified to more than 95% purity. The enzyme characteristic study showed that the optimum pH value of MBP-HepC was 7.3, the optimum reaction temperature was 42℃-48℃, and the thermal stability of MBP-HepC is better than Heparinase I(MBP-HepA)at 30℃.

The Influence of Spacer Peptide and C-Terminal His-tag on Activity and Secretion of MAT from Pichia pastoris

Qin Xiulin, Qian Jiangchao, Chu Ju

2013, 0(6):  140-146. 

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S-adenosylmethionine synthetase(MAT)catalyzes the synthesis of S-adenosylmethionine(SAM)from L-methionine and ATP in all living organisms. SAM is an important biochemical molecule participating in a variety of biochemical reactions, and has attracted much interest in clinical research because of its potential to cure many diseases, such as depression, liver disease, and osteoarthritis. Accordingly, to develop a simple and effective way to enzymatically synthesize and produce SAM, the MAT gene(sam2)from Streptomyces spectabilis was expressed successfully under the control of the AOX1 promoter in the recombinant yeast Pichia pastoris KM71. Introduction of a spacer peptide(G-Spacer: NTTEEGEPK)after the α-factor, creating a G-spacer of the N-terminal extension of the MAT(GM), greatly increased the MAT spectivity activity of GM to 158% and concomitantly improved the secretion level of the GM by 92%. In contrast, the MAT activity of the recombinant GM with C-terminal His-tag(GMH)was decreased by 13% as compared to that with the GM. Conclusively, the G-Spacer seem to be of crucial importance in determining the secretion efficiency and activity of MAT in P. pastoris. The C-terminal region of the MAT might also be important to the active protein structure.

Reliability Study on the Genus Bacillus Species Based on Fatty Acid Identification

Liu Guohong, Liu Bo, Lin Naiquan

2013, 0(6):  147-154. 

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In this study, 10 type Bacillus species were selected and carried out a different repeats of fatty acid respectively using Sherlock MIS system. From the identitication results, similarity index(SI), fatty acid content and clustering analysis, it was showed that all species were accurately identified as species level, SI had a good reproducibility, the fatty acid content was little changed in each pepetition, and was no effection on the taxonomic status of Bacillus species by clustering analysis. Finally, the results were determined that the Bacillus fatty acid identification had a good reliability.

Bacterial Adsorption of Heavy Metal Ions such as Cd²⁺

Zhou Guangqi, Ren Zhengyu, Yang Hongze, Li Rongjiao, Xing Yanjie, Zhao Yuqing

2013, 0(6):  155-159. 

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This paper studies the bacterial adsorption and desorption of cadmium ions and other heavy metal ions. The test results showed that the appropriate adsorption time of cadmium ions by the bacteria is 60 min. Infrared spectral analysis indicates that the amide, hydroxyl and carbonyl functional groups of polysaccharides and proteins on cell surface play a key role in the adsorption process. Further experimental results show that pretreatment of 0.1 mol/L NH₄OH solution on cell can significantly increase the bacterial adsorption capacity for Cd²⁺, Ni²⁺ and Pb²⁺;0.02 mol/L HCl solution can make more than 96% of the heavy metal ions to be desorbed from the microbial cell, and after desorption, the bacteria can be reused.

Heavy Metal Resistance and Its Related Mechanisms in Pseudomonas aeruginosa

Li Qun, Yang Hongjiang, Lin Shuxiang, Wang Wei, Ye Yujie

2013, 0(6):  160-166. 

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It was to investigate the prevalence of heavy metals resistance in Pseudomonas aeruginosa clinical strains and study the related mechanisms. Measurement of bacterial growths in the medium with various heavy metals;construction of transcription fusion reporter gene for analyzing the expression level of the RND efflux pump CzcCBA;analysis of the regulatory gene czcRS with PCR method and sequence alignment. Results showed that among 158 clinical strains, the majority was able to thrive in the presence of 0.002 mol/L Co²⁺, 1 strains didn’t grow in the presence of 0.001 mol/L Co²⁺, and control strain ATCC27853 could tolerate 0.003 mol/L Co²⁺ during the incubation. Two clinical strains and strain ATCC 27853 were selected for other heavy metals tolerance analysis and the resistance profiles were similar to that of Co²⁺. To investigate the mechanisms of heavy metal resistance, the expression level of gene czcC was determined via detecting β-galactosidase activity encoded by the transcription fusion reporter gene czcC-lacZ. The results showed that β-galactosidase activity corresponded with their respective resistance to heavy metals and heavy metals could induce β-galactosidase activity increase in each strain. Two-component regulatory genes czcR and czcS of the czcCBA were amplified and their sequences were analyzed. Comparing with the genome of strain PAO1, regulator CzcR had the same sequence in all the three strains, while each regulator CzcS had multiple amino acids substitutions in each strain. Clinical strains of P. aeruginosa were resistant to heavy metals. The expression level of RND efflux pump czcCBA contributed to heavy metals resistance. Mutations in regulator CzcS might play key role in the expression variation of operon czcCBA.

Optimization of Receptors Expressed on Xenopus laevis Oocytes

Fang Licong, Shen Lizi, Yu Jinpeng, Zhu Xiaopeng, Hu Yuanyan, Zhang Ben, Luo Sulan, Zhangsun Dongting

2013, 0(6):  167-171. 

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It was to optimize the conditions of AChRs expressed on Xenopus laevis oocytes, including the oocytes’ digestion time, frequency and the amount of collagenase. Results showed that when using two-step digestion method and 0.5 mg/mL concentration of collagenase, the isolated Xenopus laevis oocytes was beneficial to express AChRs. The conditions of receptor expression on the oocytes were also optimized. The optimized expression methods are significant to set up the expression system of AChRs on Xenopus laevis oocytes, which are the basis for screening new drugs to target AChRs with this experimental model.

Study on Infection of Pupal Ovaries Cells of Antheraea pernyi with ApNPV

Wang Linmei, Yue Dongmei, Li Shuying, Fan Qi, Ye Bo, Zhao Zhenjun, Zhang Bo

2013, 0(6):  172-176. 

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Mechamisms of interaction and morphogenesis of Antheraea pernyi somatic cells infected by Antheraea pernyi nuclear polyhedrosis virus(ApNPV)were studied for the further development of baculovirus expression system. Pupal ovaries cells from Antheraea pernyi were cultured in vitro followed by infection with Antheraea pernyi nuclear polyhedrosis virus.  Cells infecting rate and median tissue culture infective dose were determined. Cellular morphology was observed by an inverted phase contrast microscope and a light microscope. Results showed that, pupal ovaries cells were found expanding and nuclear polyhedrosis virus occurred in cells under the inverted phase contrast microscope after 5-days infection. After 7-days infection, the quality of nuclear polyhedrosis virus was found increased, cells infecting rate reached 40.2% and the titer of budded virus in cell supernatant was 6.95×10⁵ TCID_{50/mL. An early sign of virus infection was enlargement of nuclei, then virogenic stroma and nucleocapsids were observed within the nucleus, nucleocapsids were enveloped and formed to virus bundls from the region between virogenic stroma and nuclear membrane, the well-developed virus were then enveloped into a strongly polarible proteinaceous polyhedra with a diameter of 0.7-10 μm under the electron microscope. It proved that ApNPV was highly sensitive to pupal ovaries cells and replicated asynchronously to form typical baculoviruses in the cells.}

Construction of Rac1 Related Plasmids by Improved Overlap Extension

Duan Guihua, Shen Shanshan, Zhuge Yuzheng

2013, 0(6):  177-182. 

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Construct plasmids contains Rac1 and Rac1 mutant gene by improved Overlap extension and observe the expression of EGFP-Rac1 in hepatocyte LO2. Rac1 gene was cloned by Reverse Transcription-Polymerase Chain Reaction, Rac1 mutant gene was generated by site-directed mutagenesis which was carried out by improved overlap extension. Both Rac1 gene and Rac1 mutant gene were inserted into the pEGFP-C1 vector, three recombinant plasmids: pEGFP-C1-Rac1, pEGFP-C1-Rac1V12 and pEGFP-C1-Rac1N17(constitutively active mutation Rac1V12 as Gly at codon 12 of Rac1 CDS is mutated into Val, and dominant negative mutation Rac1N17 as Thr at codon 17 is mutated into Asn.)were constructed. All kinds of plasmids were transfected into LO2 cells, Expression of EGFP was examined by fluorescence microscope, and exogenous EGFP-Rac1 fusion protein was determined by Western blotting. Three recombinant plasmids were verified by double digestion and gene sequencing. Exogenous EGFP-Rac1 fusion protein was highly expressed in cells transfected by three recombinant plasmids. Three recombinant plasmids which can express EGFP-Rac1 protein in LO2 cell were successfully constructed.

Response Surface Methodology to Optimize Biodiesel Production Conditions by Jatropha curcas oil

Zhang Xuelin, Tang Xianghua, Li Junjun, Song Tuo, Mu Yuelin, Xu Bo, Yang Yunjuan, Huang Zunxi

2013, 0(6):  183-187. 

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Response surface methodology(RSM)was used to optimize the conditions for biodiesel production by Jatropha curcas oil. Based on the single factor experiment, we screened the important parameters by Plackeet-burman design, and used the path of steepest ascent to approach to the biggest methyl ester yield production subsequently. Then, we obtained the optimum values of the parameters by Box-Behnken design. Predicted values were found to be in good agreement with experimental values(R-sq=0.999 and Adj R-sq=0.998), which indicated suitability of the model employed and the success of BBD in optimizing the conditions of biodiesel production process. The optimum technical conditions were: alcohol to oil molar ratio 1.72:1, lipase concentration 112.5 U each gram of oil, n-hexane content 11.96%, water content 20 %, reaction temperature at 25 ℃, and reaction time for 24 h. The methyl ester was higher 2%-3% than that under the original condition.

Optimum Technique of Extracting Mycelium Polysaccharide from Auricularia auricular Using Box-Behnken Design-Response Surface Methodology

Zhao Chao, Zeng Feng, Huang Yifan, Liu Bin

2013, 0(6):  188-193. 

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To optimize the extraction technique for mycelium polysaccharide from A. auricular, the effects of extraction temperature, extraction time, the liquid to solid ratio and their interaction on extraction rate were studied by Box-Behnken design. The predictive model and reliability were developed by Design Expert software and response surface analysis(RSM). The optimal extraction conditions were achieved and listed as follows: extraction temperature 81.59℃, extraction time 81.40 min, liquid-to-solid ratio 36.42:. Under the optimum conditions, the extraction yield of mycelium polysaccharide from A. auricular reached 7.79 mg/g, while the predicted one was 7.92 mg/g. The results showed the suitability of RSM in optimizing the extraction of mycelium polysaccharide from A. auricular. The necessary technical support was provided by this experimental study for the industrial production of mycelium polysaccharide from A. auricular.

Optimization of Cultivation Conditions of Saccharomyces boulardii Against Diarrhea Using Response Surface Method

Hu Xiaoyuan, Teng Da, Zhang Yong, Mao Ruoyu, Wang Xiumin, Huang Jianzhong, Wang Jianhua

2013, 0(6):  194-199. 

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The fermentation medium and conditions of Saccharomyces boulardii were optimized based on single-factor experiments and response surface method. Single-factor experiments were performed to select the optimal carbon source, nitrogen source and inorganic salt. Response surface method was used to optimize the fermentation medium and conditions. A Plackett-Burman design was used to evaluate the effects of eight factors;the path of steepest ascent and the Box-Behnken design were performed for further optimization. The optimal fermentation medium contained 2% of maltose, 2.08% of yeast extract, 2% of glucose, with initial pH value of 6.0. The optimal fermentation conditions were as follows: 5% inoculums added to 250 mL erlenmeyer flask with 29 mL medium volume were cultured with shaking speed 250 r/min at 29.6℃ for 24 h. In the optimal fermentation medium and conditions, the cell wet weight of Saccharomyces boulardii increased from 20.9 g/L to 32.2 g/L, increased by 54.1%, the number of viable yeast increased from 5.5×10⁸ CFU/mL to 8.3 ×10⁸ CFU/mL, increased by 50.9%.

Construction of Recombinant E. coli JM109 Capable of Producing 3-Hydroxypropionic Acid and Its Fermentation Condition Optimization

Zhang Xiaomei, Li Xinsheng, Xu Zhenghong

2013, 0(6):  200-208. 

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The expression vector pEtac harboring aldh gene and dhaB gene was transformed into E.coli JM109 to construct recombinant E.coli JM109(pEtac-dhaB-aldh). And the response surface method(RSM)was firstly applied to determine and optimize the fermentation condition for the novel recombinant E.coli JM109(pEtac-dhaB-aldh)capable of producing 3- hydroxypropionic acid. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 3- hydroxypropionic acid. The model estimated that, a maximal yield of 3-hydroxypropionic acid(5.4 g/L)could be obtained when the concentrations of glycerol, VB₁₂, yeast extract KH₂PO₄ were set at 62 g/L, 0.05 g/L, 6.2 g/L and 7.4 g/L, respectively. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design.The yield and productivity under the optimal parameters and process can reach 5.8 g/L.

Dectection of Vibrio parahaemolyticus by Outer Membrane Protein ompK Immunomagnetic Beads

Li Yuchen, Wen Yiming, Tong Jiyu, Xiang Junjian

2013, 0(6):  209-214. 

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It was to construct recombinant gene cloning and express the membrane surface protein outer the membrane protein K(ompK)of Vibrio parahaemolyticus, developing a method to detect Vibrio parahaemolyticus via immunomagnetic separation combined with color plate method. Using Primer 5 to design primers of ompK gene, the ompK gene was amplified by PCR subsequently and cloned into pET28a(+)prokaryotic expression vector, optimally transferred into E. coli BL21 to express. The expression products were purified by Nickel column and immunized mouse to prepare polyclonal antibody. Western blotting analysis was applied to identify the recombinant protein and ELISA was used to analyze its immunogenicity. The titer and cross reactivity of the polyclonal antibody were determined using indirect ELISA, then the polyclonal antibodies we prepared were coupled with protein G immunomagnetic beads to develop a chromogenic plating detection of Vibrio parahaemolyticus. The sensitivity of immunomagnetic beads reached to 10⁴ CFU/mL. We managed to clone and express Vibrio parahaemolyticus outer membrane protein ompK, in addition, we prepared mouse anti-Vibrio parahaemolyticus polyclonal antibody to create a new detection method using immunomagnetic beads, this method which combined with color plate method could save 72 hours comparing with traditional enrichment method, all process only need one-third of the time of traditional methods. When compared to the common immune detection methods, it was two magnitudes more sensitive.

The Improvement on the Protocols of Total RNA Extraction Kits for RNA-Seq from Grape Leaves

Yang Xiaoyan, Zhang Bo, Huang Fang'ai, Yan Huan, Li Yuerong, Zheng Qiusheng

2013, 0(6):  215-220. 

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Commercial RNA extraction kits were wildly used in obtaining high quality RNA samples from plants. To seek the optimal method for the extraction of high quality RNA in RNA Expression Sequences Array(RNA-Seq), the topic of present article is to evaluate and optimize the effects of three common RNA extraction kits on leaves samples from grapevine(Vitis vinifera cv. Red Globe). The quantities of RNA were analyzed between the recommendatory and optimal usages of three kits in term of times. Results showed that the optimized procedure for RNA extraction displayed higher yield and better integrity on total RNA. There are a ratios of OD₂₆₀/OD₂₈₀ from 1.8 to 2.2 and further OD₂₆₀/OD₂₃₀ ratio no less than 2, resulted in a integrate 28S and 18S bands by electrophoresis. The extracted samples also kept their quality and gene coverage from RNA-Seq sequencing. Our research demonstrated that the prolonged incubation time for samples preparation and other procedures would significantly benefit the separation of total RNA from grape leaves(Vitis vinifera cv. Red Globe)with a higher purity and integrity.

Rapid Determination of Diarrhetic Shellfish Poisoning Toxins in Shellfish Samples by Immune Colloidal Gold Strip

Zhang Yang, Jia Rui, He Peimin

2013, 0(6):  221-225. 

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In the present study we determined the Okadaic Acid(OA), the main pathotoxin of diarrhetic shellfish poisoning toxins(DSPs), in shellfish samples in Shanghai sea food markets by the immune colloidal gold strip for the first time which is researched and developed by our lab. There are 69 samples which were collected from May 2011 to October 2011 for each month from the seafood market. The samples were tested by OA immune colloidal gold strip and enzyme-linked immunosorbnent assay(ELISA)after a series of processing. The result showed that there are 11 positive samples contain OA for OA Immune colloidal gold strip, and the positive rate is 15.9％. The 11 positive shellfish samples were Mytilus edulis(n=5), Argopecten irradians(n=4), Ostrea(n=1)and Bullacta exarata(Philippi)(n=1). The result of ELISA showed that the 11 samples which were tested positive by immune colloidal gold strip contain the OA, and the concentration of OA was quantified higher than 15 μg/100 g. The correlation between OA concentration determined via Immune colloidal gold strip and ELISA is 100%. The time of determining the shellfish samples using the OA immune colloidal gold strip is within 30 min from collecting the samples to get the result, and the result is accurate with no false positives. In the present study, immune colloidal gold strips test were shown to be an effective tool for rapid screening in marine environmental monitoring and seafood quarantine and inspection with the limit of detection meeting the safe threshold of OA in shellfish.

Gold Immunochromatography Assay for Rapid Detection of Fluoroquinolones——Being Used with a Readout Instrument of Colloidal Gold Test Paper Card Together

Ma Yinsheng, Wan Yuping, Liu Qingjun, Jia Fangfang, Zhang Yu, Du Meihong

2013, 0(6):  226-231. 

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To establish a rapid assay for detecting fluoroquinolones in milk, gold immunochromatography assay was studied, which was used with a readout instrument of colloidal gold test paper card together. The limit of detection(enrofloxacin, sarafloxaxcin, difloxacin, ofloxacin, norfloxacin, ciprofloxacin, pefloxacin, flumequine, danofloxacin)is 20 μg/L, and(enoxacin, oxolinic acid)is 40 μg/L. We can obtain the results in 10 minutes. The assay gives no false positive and false negative results. The method is accurate, simple, reliable and convenient to rapidly detect fluoroquinolones of a lot of samples on the spot.

Protection and Development of Agro-biotechnology Intellectual Property Rights

Pang Junfeng, Qi Yong, Feng Feng, Zhang Shaolei

2013, 0(6):  232-234. 

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Agro-biotechnology as one of the most representative high-tech, has been an important work on how to protect the intellectual property rights, which could promote technology development and economical development. In this paper, we present the development background of protection of intellectual property rights in agricultural biotechnology, and discuss the status and problems of China’s protection of agricultural biotechnology. Meanwhile, we provide some useful strategies and suggestions to enhance the protection of agro-biotechnology intellectual property rights and promote agriculture science and technology development.