Biotechnology Bulletin

Chen Yangxun, Zhang Zhiguo, Lu Tiegang

2012, 0(12): 1-7.

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Selectable marker genes（SMGs）based on antibiotic or herbicide resistance have been widely used to identify ‘true’transformants. To release the public concerns for the bio-safety of SMGs，a new strategy has been developed to obtain marker-free transgenicplants to eliminate the marker gene from the nuclear or chloroplast genome after selection. Marker-free transgenic crops have a lot of uniqueadvantage such as reducing the trepidations from the SMGs and transferring exogenous genes gradually into transgenic crops. In this review，different approaches of marker-free transgenic method were discussed.

Advances in fat-1 Transgenic Animals

Yang Huiting, Wang Hongmei, Fang Yongzhi, Liu Wenhao, Wu Jianming, He Hongbin

2012, 0(12): 8-12.

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Mammals engineered by fat-1 gene from Caenorhabditis elegans can convert n-6 polyunsaturated fatty acids（PUFAs）to n-3PUFAs，and decrease the ratio of n-6/n-3 PUFAs in vivo. Although n-3 polyunsaturated fatty acids have been shown to play an important role inhealth，and can reduce the risk of many diseases，humans can not synthesize the n-3 PUFAs automatically. This review summarized the statusof research about fat-1 gene and fat-1 transgenic animals.

Research Progress on Spermatozoa-mediated Gene Transfer to Product Transgenic Animals

Du Wei, Cui Haixin, Wang Yan, Sun Changjiao, Zhao Xiang

2012, 0(12): 13-18.

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Spermatozoa-mediated gene transfer（SMGT）is a low-cost and fast method used to produce transgenic animals. Theexogenous DNA can be either integrated into the sperm chromosomal DNA or simply transferred to the egg by the spermatozoa and laterintegrated into the zygote’s genome. Although there are many transgenic animals produced by SMGT，its efficiency is still low，mainly dueto the ability of sperm to bind，internalize，and transport exogenous DNA into an oocyte during fertilization. The purpose of this review is tosummarize what has been achieved with this method，such as the binding of DNA molecules to spermatozoa，and attempts made to producetransgenic animals in this field.

Systemic RNA Interference and Its Application in Insect

Wang Huidong, Gong Liang, Hu Meiying, Hong Peng

2012, 0(12): 19-24.

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A remarkable property of RNA interference（RNAi）in the nematode and plants is its systemic nature: silencing signals cancross cellular boundaries to silence gene expression distant from the site of initiation. In the past years，it is found that some insects can alsoshow systemic RNAi. RNAi technology has significant technological advances in diverse fields including functional genomics and agriculturalpest control. Here，we collected the current status of systemic RNAi in nematode and insects，we also introduce the current applications ininsects as well as expect its potential applications.

Research Advances on Core Enzymes of Anthocyanidin Biosynthesis

Zhao Qiming, Li Fan, Li Ping

2012, 0(12): 25-32.

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Anthocyanidin is the material basis of the plant flowers showing different colors, its biosynthetic pathway is mainly controlledby Chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid3'-hydroxylase, flavonoid 3', 5'-hydroxylase dihydroflavonol4-reductase, anthocyanidin synthase and flavonoid 3-O-glucosyltransferase. The biosynthesis pathway of anthocyanidin, core enzyme’s crystalstructure, modification of flower pigment were introduced in this review, discussed the existing issues in transformation of flower pigment and putin perspective.

S-adenosyl-L-methionine Production by Microorganism

Hu Xiaoqing, Guo Wen, Han Guoqiang

2012, 0(12): 33-39.

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S-adenosyl-L-methionine（SAM）is a type of active amino acid having broad market prospects，and there have been manyreports on SAM production catalyzed by microorganisms. This paper integrated advances in genetic modification and fermentation optimizationof SAM-producing strain. From the points of elevating expression and activity of SAM synthetase，optimizing feeding mode of glycerol andmethanol，improving the generation of ATP and supplement policy of L-methionine，and blocking downstream metabolic pathway，themultiple strategies of enhancing SAM synthesis and accumulation and their mechanisms had been reviewed. Furthermore，on the basis of theauthors’ research practice，some new prospects on SAM biosynthesis by microbes were discussed.

Application of Elastin-like Polypeptide Tag for RecombinantProteins Purification

Lin Heng, Xu Chongbo, Sun Lihui, Hu Xuejun

2012, 0(12): 40-45.

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Elastin-like polypeptide（ELP）is an extremely promising recombinant protein purification tag. The artificial polypeptidesconsist of repeats of the pentapeptide（VPGXG）with a reversible phase transition at a specific temperature. ELP fusion protein retains thisbehavior when a target protein is fused to the ELP at the gene level. Once isolating a recombinant ELP fusion protein from the protein solution byrepeating inverse transition cycling（ITC）, the ELP tag can be proteolytically removed, or self-cleave mediated by intein in response to solutioncondition shift, then we can recover the pure target protein. At present, the method is used successfully in expression systems such as E.coli andplants. The highest yield of GFP-ELP fusion protein is 1.6 g/L in E.coli. This non-chromatographic purification method is simple, rapid, costeffective,and easy to scale up. This review summarizes mechanism, technical route and development direction of this method.

Advances of Metatranscriptomics Technology

Ma Shu, Liu Huhu, Tian Yun, Lu Xiangyang

2012, 0(12): 46-50.

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Metatranscriptomics is a new subject to study the transcription situation and regulation rules of entire genomes of colonialorganism under a certain specific environment and period in the overall level. The emergence, research strategies and current applications of theMetatranscriptomic were reviewed briefly and the application prospect of it was also anticipated in this paper.

Transcriptome and RNA-Seq Technology

Zhang Chunlan, Qin Zijuan, Wang Guizhi, Ji Zhibin, Wang Jianmin

2012, 0(12): 51-56.

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The transcriptome is the complete set of transcripts for certain type of cells or tissues in a specific developmental stage orphysiological condition. Transcriptome analysis can reveal the organism's gene expressing level, structural variation, discovery of new genes. Theresearch methods and platforms of transcriptome are undergoing rapid changes. And bioinformatics analysis is also improved gradually. RNASeqas a new research method, it can be more quickly and afford more accurate transcriptome’s information while using the Next-generationSequencing（NGS）technology. This article compares several main methods and platforms of transcriptome in recent years, and review theprinciple, purpose, steps, bioinformatics’ analysis and applications in related fields of RNA-Seq. This will be afford useful reference for relatedresearch.

Interference and Its Application in Fruit Trees

Yu Dingqun, Tang Haoru, Zhang Yong, Chen Qing, Zhang Xiaonan, Yu Haowei

2012, 0(12): 57-64.

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RNA interference, which widely exists in organisms, is a phenomenon of specific gene silencing by small interfering RNAinduced.RNA interference is a biological self-protective mechanism resisting to abnormal RNA. However, it also regulates gene expression in theprocess of growth and development. So it has opened up new avenues on research of plant gene function. This review summarizes the mechanismand characteristic of RNA interference. Meanwhile, it also focuses on applications of RNA interference in growth and development, diseaseresistance, and fruit quality improvement of fruit trees in recent years, which will provide more guidance for further studies on breeding andquality improvement of fruit.

The Application of Modern Biotechnology in the Research of Rhizosphere Microbial Community

Zhao Jia, Sun Yi, Liang Hong, Huang Jing, Du Jianzhong

2012, 0(12): 65-70.

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Rhizosphere microorganisms are defined as the microbial community in the area of direct impact on the soil of the root surfaceand root of the plant. Rhizosphere microorganisms interact with plant roots are the main medium for the material exchange of plant ecosystems.Studies on rhizosphere microbial community are essential for microbial ecology and plant ecology. In recent years, with the rapid developmentof modern biology techniques, studies on unculturable microbials has made a breakthrough on the natural communities in the rhizosphere. Thisarticle reviews research progresses in the field and summarizes the application of modern biotechnology in the rhizosphere microbial communityto provide a reference for the further in depth study of rhizosphere microorganisms.

Plant Height and Physiological Index Analysis of Variation Offspring ofRice Transduction Exogenous General DNA

Zhao Ying, Wang Liping, Xiao Jun, Wang Na, Gong Na, Chen Xun, Yang Zhen, Wang Hong, Yang Tao

2012, 0(12): 71-75.

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Through inspecting physiological indexes and plant height of G series salt-tolerant rice varieties of field screening, we canfurther appraisal salt resistance of new screening salt-tolerant rice varieties. We cultivate rice seedlings by sand culture method. The ricein growing period of seedling and were treated with different concentrations of NaCl（0.3%, 0.6%, 0.9%）and clear water. The former, wemeasured MDA content, Pro content, SOD activity and palnt height in the third leaf stage. The above 4 indicators were measured in third day andsixth day, which begin to run from the day of pouring salt water. With the salt concentration increasing in the two treatments, the MDA contentin H5、H6 were gradually decreased, however, in the same watering conditions are lower than the control G6 ；Pro content increased gradually,However, in the same watering conditions are higher than the control G6 ；SOD content increased first, and then decreased, however, in the samewatering conditions are higher than the control G6. There were significant change in MDA content, Pro content, SOD activity and palnt height.The study was obtained that G series salt-tolerant rice varieties of field screening were stronger than the control G6. The results indicate thevalidity of G series salt-tolerant rice varieties from physiological indexes to plant height. Experiments conducted that MDA content ；Pro content,SOD activity and palnt height can be used as physiological indexes and morphological indicators for Identification of Genetic Dissection of SaltTolerance.

Analysis of Single-copy Nuclear TPI Gene Sequences of Oryza L. and ItsApplication in Identification of Oryza granulata

Song Yun, Chen Yan, Xu Han, Li Mingfu, Liu Shiqin

2012, 0(12): 76-81.

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In this paper, single-copy nuclear TPI genes of Oryza L. and its related genera were amplified and sequenced, we analyzedtheir phylogenetic relationships and sequence differences, designed specific primers for Oryza granulata. Sequences analysis indicate thatthere were few variations between cultivar rice, but many variations between wild rice species, especially sharp variations were found for Oryzagranulata. On the basis of these variations, we designed Oryza granulata specified primers for species identification.

Screening on Recipient Material for Agrobacterium-mediatedTransformation in Maize

Jiang Huiquan, Ren Wen, Xu You, Zhang Zhifang, Chen Hao, He Haohua, Zhao Jiuran, Liu Ya

2012, 0(12): 82-87.

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At present, Agrobacterium mediated genetic transformation in maize is widely used in the world. A good recipient material isvery important for the transformation system. In this study, 14 new inbreed lines were used to choose the suitable recipient material by analysingembryogenic Type Ⅱ callus induction, plant regeneration rate and GUS transient expression. Zpm2 and zpm5 were finally screened for maizetransformation system. In the study of mature embryos callus induction, mature embryos callus induction rate can predict the growth status ofimmature embryos callus and their callus induction show a positive correlation.

Cloning and Expression Analysis of CeActin Homologous Gene from Cymbidium ensifolium

Wu Jinghua, Wu Shaohua, Yang Chao

2012, 0(12): 88-92.

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The full-length cDNA sequence of Actin gene from the bud of Cymbidium ensifolium was cloned（GenBank accessionnumber: JN613147）by using RT-PCR and RACE with degenerate primer which were synthesized based on the high homologous region amongthe sequences of several monocotyledon Actin genes. The full length cDNA of CeActin was 1 434 bp with an ORF of 1 134 bp encoding a proteinwith 377 amino acids. Homology search showed that this gene shared high identity with other Actins. Semi-quantitative RT-PCR analysis showedthat the expression of CeActin had no notable difference in different tissues and different floral development stages. The results laid basis foridentification of gene transcription level in Cymbidium ensifolium using CeActin as internal control.

Profile of Drought Resistance of Tomato Transcription FactorSlDREB2 Gene Mediated by VIGS

Yu Guohong, Ma Xuefeng, Xu Na, Xin Shichao, Cheng Xianguo

2012, 0(12): 93-100.

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SlDREB2 gene was isolated by yeast one-hybrid system using DRE element of rd29A promoter through hybridization withtomato seedlings（Lichun）cDNA library, and it is a transcription factor belonging to EREBP family. In this study, the VIGS virus vectors wereconstructed, and transformed into the GV3101 Agrobacterium strains, and the resulting recombinants carrying pBINTRB6, pTV00-SlDREB2-1and pTV00-SlDREB2-2 vectors were used to infect leaves of tomato plants respectively by injection of liquid filling respectively. All the plantsinfected after 7 days were identified by viral DNA testing and the physiological index of wild-type strains of tomato plants as well as infectedplants successfully were determined under drought stress for three weeks, and Real-time qPCR was performed to analyze expression profileof the SlDREB2 transcription factor. Data showed that the contents of proline and soluble sugar in wild tomato plants were higher than that oftomato plants infected by VIGS virus, and malondialdehyde levels in wild plants were lower than that of plants infected by VIGS virus. Whenall tomato plants stressed by drought for three weeks were re-watered for one week, the contents of proline and soluble sugar in wild tomatoplants were lower than that of tomato plants by VIGS virus infection, and the contents of malondialdehyde in wild plants were higher than that ofplants infected by VIGS virus infection. Real-time qPCR results indicates that the expression of SlDREB2 in plants by VIGS virus infection waslower significantly than that of in wild type plants under drought stress, indicating that 500 bp of specific fragment at the end of SlDREB2 geneconservative domain has better silence effect, and the expression of SlDREB2 mediated by VIGS was inhibited by drought stress, and indirectlyimplying that the SlDREB2 gene can be a valuable candidate gene for improving crop varieties with drought resistance.

Bt New Gene sip Cloning, Expression and Bioinformatics Analysis

Liu Yanjie, Li Haitao, Liu Rongmei, Gao Jiguo

2012, 0(12): 101-105.

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Bacillus thuringiensis is the most widely applied type of microbial pesticides because of its high specificity and environmentalsafety. There is a wide range of researchs about ICPs and Vip, but little was known about Sip. To further explore novel Bacillus thuringiensisstrains and the new gene resources in our country, we isolated 400 wild strains from different entironment for sip gene cloning and identification,of which amplification from QZL26 isolate got a sequence of 1 038 bp which codes 345 amino acids and the similarity of the amino acid sequencewith the Sip1A is 91.83%. Sequencing result was submitted to GenBank, and the registration number is JQ965994.

Bioinformatis Analysis of PHGPx Gene in Goat Capra hircus

Shi Liguang, Xun Wenjuan, Zhou Hanlin, Hou Guanyu, Yue Wenbin, Zhang Chunxiang, Yang Rujie

2012, 0(12): 106-113.

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This study aimed to clone the full length cDNA of goat PHGPx gene. Total RNA was extracted from goat testis and the fulllength cDNA was obtained by the reverse transcription PCR and RACE method. The results demonstrated that the full length of goat PHGPxcDNA was 844 bp, which encode 199 amino acids. The multiple sequence alignments revealed that the identity of goat PHGPx amino acidssequence were above 90% with other species. Bioinformatics analysis indicated that goat PHGPx belonged to GSH-Px family in the secondarystructure, and predicted the signal peptide at the position of 23-24 or 28-29. The molecular phylogenetic trees among species according theUPGMA method indicated that goat and cow assembled, and then assembled with pig, rat, human and chicken, respectively and assembled withbee last. This result of phylogenetic clustering was identical to the zoological classification. In summary, Capra hircus PHGPx gene has beencloned, and it’s concluded that PHGPx has typical characteristics with the homologous genes in GSH-Px family. These results would be thetheoretical foundation for the further study on the genetic control of PHGPx gene expression.

Transcriptional Regulation Analysis and Prokaryotic Expression ofahpC Gene Putatively-encoding Alkyl Hydroperoxide Reductase inXanthomonas oryzae pv. oryzae

Wang Yanli, Wu Maosen, Tian Fang, Chen Huamin, He Chenyang

2012, 0(12): 114-119.

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The ahpC gene involved in degradation of hydrogen peroxide was cloned from the wild-type strain PXO99^A of Xanthomonasoryzae pv. oryzae in this study. The gene putatively encodes the alkyl hydroperoxide reductase AhpC. The sequence of ahpC protein is highlyconserved in different kinds of pathogenic bacteria tested. To find out the structural characteristics of transcription of the gene cluster oxyR-ahpFahpC,reverse transcription PCR（RT-PCR）analysis revealed that ahpC and ahpF gene, the enzyme electron donor gene are co-transcribed inthe same operon and same transcriptional unit. The activity of ahpCp-lacZ was detected, suggesting the ahpC promoter activity was positivelyregulated by transcriptional regulator factor OxyR. In addition, construction of expression vector of ahpC using pET-28a（+）and prokaryoticexpression was performed. The soluble recombinant proteins were expressed, which could be purified and used for further biological functionanalysis.

Study of Arabidopsis BAND7 Gene Sequence Analysis and MutantPhysiological Function

Sun Hongju, Xue Qiong, Zhang Yantao, Wang Yan, Kang Yan, Qi Zhi

2012, 0(12): 120-124.

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BAND7 is one kind of proteins with SPFH structural motif and wildly distributed in various animal cells, which coupledwith other membrane proteins and regulated ion transportation and signaling transduction. By sequencing comparison, four putative BAND7genes were identified in Arabidopsis, At1g69840, At3g01290, At5g6274 and At5g51570. Semi-quantitative reverse transcript PCR analysisindicated that they expressed in both the leaf and root with comparable higher expression in the leaf. Through PCR-based detection method,T-DNA knockout homozygous mutants SALK_088328 and SALK_104547 were identified in the At1g69840 and At5g51570 genes, respectively.Comparing growth phenotype of the wild type and these mutants revealed a role of At5g51570 in the calcium homeostasis.

Cloning and Sequence Analysis of the South Xinjiang Strain ORF3Gene of Porcine Reproductive and Respiratory Syndrome Virus

Zhao Li, Liu Yonghong, Chen Yanzhou, Wen Yaqin, Liu Bo, Li Jiangtao, Cui Haoran, Cheng Bo, Zhang Chunguang

2012, 0(12): 125-130.

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Grasp the characteristics of pandemic strain of the South Xinjiang PRRSV, provide a theoretical basis for the effectiveprevention and control of the South Xinjiang PRRS. In this study, five complete sequences of ORF3 genes of the South Xinjiang PRRSV wereobtained by cloning and sequencing and sequence analysis. The results showed that the gene length of five complete sequences of ORF3 genes ofthe South Xinjiang PRRSV were both 765 bp, nucleotide homology between them was 90.3% to 99.5% ；The nucleotide homology of ORF3 geneswith LV of Europe type represents strains, ATCC VR-2332 of American type represents strains, CH-1a of China type represents strains, JXA1 ofthe HP-PRRS China type represents strains, attenuated vaccine strains used wildly in Chinese pig farms was 61.2% to 62.2%, 88.6% to 89.2%,92.5% to 95.8%, 90.5% to 99.2%, 87.8% to 99.6%, respectively ；The amino acids homology of ORF3 genes with the American type strainandand Europe type strains was 80.3% to 99.2% and 53.5% to 55.1% ；The five ORF3 genes of the South Xinjiang PRRSV were both belongs to theAmerican type subgroup II strains. The molecular epidemiology investigation result of the South Xinjiang PRRSV for the choice of vaccine forSouth Xinjiang PRRS made a further adjustment or guidance.

Cloning and Construction of Prokaryotic Expression Vectors forChannel Catfish BPI Antibacterial Peptide

Gao Bei, Tao Yan

2012, 0(12): 131-138.

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Bactericidal/permeability increasing protein（BPI）, an antimicrobial protein expressed in organism cells, shows particularantimicrobial activity against gram-negative bacteria. BPI contains two domains, N-terminal domain and C-terminal domain. The antibiotic andendotoxin-neutralizing functions of BPI are attributed to the N-terminal domain. The present study was performed with reference to the reportfor the recombinant DNA expression of human BPI. A cDNA fragment “BPI_Ntd” encoding the N-terminal active domain of BPI was clonedfrom the gill of channel catfish（Ictalurus punctatus）by RT-PCR and nested PCR. This fragment encodes a peptide consisting of 175 aminoacid residues, in which 63 residues can form a three-dimensional apolar lipid-binding pocket which is used to bind lipopolysaccharide（LPS）located in outer membrane of gram-negative bacteria. The comparison of the channel catfish BPI with the BPIs from other organisms showed that40 conserved amino acid residues appear at the N-terminal domain, and among them 9 highly conserved residues located in the LPS-bindingregion. The pET-32_a（+）and pET-28_a（+）with different characteristics were chosen as the prokaryotic expression vectors to constructthe prokaryotic expression system. “BPI_Ntd” fragment was inserted into these two plasmids respectively. The colony PCR, EcoR I and Hind IIIrestriction endonuclease digestion and DNA sequencing demonstrated that the “pET-32_a-BPI_Ntd” and “pET-28_a-BPI_Ntd” recombinant expressionplasmids were constructed successfully.

Expression of Plant des-pGlu1-Brazzein Gene in Alcohol OxidasedefectiveStrain of Pichia pastoris and Analysis of Its Biological Activity

Xu Tingting, Chen Ruibo, Bao Yuanyuan, Zhao Weidong, Zheng Zhenyu, Li Chunli

2012, 0(12): 139-143.

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Digested by Bgl Ⅱ, the linear recombinant expression vector pPIC9K-Bra was transformed into Pichia pastoris GS115 byelectric shock to produce alcohol oxidase -defective expression strain GS115-pPIC9K-Bra. The strain was identified by PCR and induced by 0.5%methanol. The results indicated that the secreted target protein accounted for 95% of total protein in supernatant fluid and showed biologicalactivity. The alcohol oxidase -defective expression strain GS115-pPIC9K-Bra was successfully constructed which was foundation for furtherstudy.

Screening and Identification of a Strain Producing L-glutamateOxidase and Its Enzymatic Properties

Lu Chan, Zheng Pu, Sun Zhihao

2012, 0(12): 144-149.

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Chromogenic 4-Aminoantipyrine/phenol reaction was used in the procedure of strain screening. A strain named Str-6 wasobtained. Taxonomy of the strain, based on its morphological, physiological and biochemical characteristics, as well as the sequence of 16SrDNA, was classified to Streptomyces collinus. The preliminary fermentation test showed that strain Str-6 could produced extracellular L-glutamateoxidase, and the maximal yield could reached 0.023 1 U/mg protein cultured at 28℃ for 36 h. It has strong substrate specificity for L-glutamate.The optimal pH was 6.0 and the temperature of the L-glutamate oxidase was 35℃ . The enzyme exhibited high thermal stability at 35-40℃ andstable at pH6.0--7.0.

Screening and Identification of a Rare Actinomycetes ExhibitingIMPDH Inhibitive Activity

Yuan Lijie, Liu Aihua, Zhang Yuqin , Yu Liyan, Wang Qian, Zhang Yueqin

2012, 0(12): 150-155.

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One actinomycete strain I06-03147 was isolated from rhizosphere soil of medicinal plant Aconitum carmichaeli Debx. collectedfrom Chongqing. The fermentation broth of strain I06-03147 exhibited inhibitive activity of IMPDH. In view of morphology, physiological andbiochemical characteristics, chemotaxonomic data, phylegentic analysis based on 16S rRNA gene sequence, the studied strain belonged to genusKineosporia.

Isolation，Identification and Characterization of a Crude-oildegradatingThermophiles in Petroleum Reservoirs

He Hua, Qiao Fadong, An Mingli, He Weihong, Du Xun, Jia Bin, Wang Yanan1

2012, 0(12): 156-162.

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A high temperature-resistant bacteria strain P98-18A1 capable of growing in petroleum hydrocarbons at 55℃ was isolated fromhot produced fluid from Zhongyuan oilfield. This strain was identified as Bacillus sp. based on phenotypic, physiological, biochemical and 16SrDNA sequence analysis. With crude oil and several alkanes degradation experiments, results showed that this strain can effectively utilize C6-C16 n-alkanes, and selectively utilize of the chain longer than C16 alkanes to some extent under the optimum conditions.

Modified Method for the Efficient and Fast Extraction of Total RNAfrom Saccharomyces cerevisiae with Hot-Phenol

Li Weiwei, Dun Baoqing, Wang Zhi, Qu Juanjuan

2012, 0(12): 163-166.

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To research an efficient method for total RNA extraction from Saccharomyces cerevisiae, haploid CEN.PK1and diploid CEN.PK2 as the object of study was used to study the new modified hot-acid-phenol method. The total RNA concentration of CEN.PK1 and CEN.PK2was 7.63 mg/mL and 3.77 mg/mL, OD260/280 was 2.10 and 2.04 and OD260/230 was 2.32 and 2.24, using the modified method by spectrophotometerdetermination．It is proved that the RNA was fit for the requirements of follow-up experiments in molecular biology by PCR and fluorescencequantitative PCR detection without DNA contamination compared with the Trizol method. The extraction time 1.5 hours is shorter than that ofconventional method 2.5 hours used and the modified method has the advantages of simple operation, high efficiency and high quality. Repeatedexperiments proved that this method can also be used to extract the total RNA amount of Saccharomyces cerevisiae and can be used for practicalapplications．

The Preparation and Identification of P2X7 Short Peptide ReceptorMonoclonal Antibody

Tan Chao, Han Li, Shen Xiejing

2012, 0(12): 167-172.

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The aim was to prepare and identify the monoclonal antibodies of the P2X7 receptor. Our method was to use the extracellularfragment of human beings’ P2X7 receptor to produce the short peptide as an antigen to immunize mouse for three times. Then B lymphocyteswere isolated from the mouse spleen, and then mixed with myeloma cells and cultured. The positive hybridoma clones were selected and widelycultured to prepare and identify the biological effects of P2X7. The supernatant was collected and purified by HiTrap protein G affinity column.The results were as follows. Firstly, a stable hybridoma cell line secreting mAb against human P2X7 receptor was obtained, and the heavy andlight chains of the secreted mAb were IgG1 and κ respectively. The titers of the ascites was 1∶6.4×104, which was remained stable aftersubculture of hybridoma cells for 30 passages or storage at liquid nitrogen for 6 months. Secondly, the western blotting test showed that the mAbcould specifically bind to P2X7 receptor. The conclusion was that the prepared mAb against P2X7 receptor showed high specificity and stability,which laid a foundation for the development of the antibody drug aimed at P2X7 receptor as a target antigen.

Molecular Identification of Nervilia fordii HanceSchltr. and Its Adulterants Based on lTS2 DNA Barcodes

Huang Qionglin, Ma Xinye, He Rui, Zhan Ruoting, Chen Weiwen

2012, 0(12): 173-179.

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It was to establish a novel identification method for Nervilia fordii and its adulterants through ITS2 sequence analysis. TotalDNA was extracted from fresh leaves, and ITS2 region was amplified and sequenced with a pair of universal primers. The sequences wereassembled and aligned by DNAMAN and CLUSTALX, and NJ tree was constracted using MEGA 4.0. Secondary structures of ITS2 sequenceswere predicated in the database built by Schultz et al. Significant differences were observed in the ITS2 sequences between Nervilia fordii andits adulterants. The interspecific genetic distance was far more than the intraspecific disatance. Both NJ tree and secondary structure based onITS2 sequences can distinguish Nervilia fordii and its adulterants intuitively. ITS2 is an effective biomarker to identify Nervilia fordii and itsadulterants.

Construction of the Mutant of the Salmonella typhimurium BacteriaspvB Genetic Defect

Qiu Qiaocheng, Ye Ying, Chu Yuanyuan, Liao Li, Wu Shuyan, Huang Rui

2012, 0(12): 180-183.

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Highly conserved spvB gene is an important virulence factor of infected cells apoptosis, which encoded product with the ADPribosyltransferaseenzymatic activity. To further study the pathogenic mechanism of the spvB gene, this study began to build a mutant of the spvB gene in Salmonella typhimurium. According to the spvB gene sequence in GeneBank, two pair’s primers were designed with Primer Premier 5.0, upper- and down-stream of the spvB gene to amplify two homologous DNA fragments. The homologous recombinant DNA fragment of the defective target gene was cloned into the suicide plasmid PGMB151, which was then transduced into the target cell of S. typhimurium SR-11 for homologous recombination. The recombination was identified by PCR. A deletion mutant of the spvB gene in SR-11 was confirmed by PCR and sequencing analysis.

AMB-1 Expression of Superoxide Dismutae Fe-SOD of Magnetospirillum magneticum AMB-1 in Escherichia coli and Its Enzymatic Characterization

Wang Qilei, Li Xiangqian , Xu Linyu, Xue Yemin

2012, 0(12): 184-191.

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In this study, superoxide dismutaes genes fesod from Magnetospirillum AMB-1 were cloned and expressed in E.coil BL21 （DE3）. The SOD gene was obtained from the genome of AMB-1 through PCR amplification, and the resulting amplified gene was cloned into an IPTG-inducible expression vector pET-20b, respectively. The expression vectors pET-20b-fesod-histag was further transformed into E.coil BL21 （DE3）. In contrast to the control BL21（DE3） / （pET-20b）, the growth curves of the recominent strains showed that the growth rates of BL21 （DE3） / （pET-20b-fesod-histag）was obviously higher than that of control. Superoxide dismutaes activity result showed that the SOD activity level of BL21（DE3）/（pET-20b-fesod-histag）per OD₆₀₀ was higher than that of control. SDS-PAGE analysis showed that fesod gene was expressed effectively and the expression product was about 22 kD. Moreover, the expression level of an hypothetical SOD protein derived from the host bacteria was gradually increasing during the IPTG inducible expression in BL21（DE3） / （pET-20b）. Molecular cloning of superoxide dismutaes genes from AMB-1 and their expression in E.coil.

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