Biotechnology Bulletin

Research Progress on Genetic Transformation of Allium Vegetables

Gao Xingying, Li Meilan, Wang Tingting, Hou Leiping

2013, 0(5): 1-6.

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Allium vegetables which are riched in nutritions and have high medicinal value play an important role in people’s life. This paper summarized the types of Allium and characters that have been genetically transformed successfully. At the same time, the transformation methods and the explant types used were stated in the paper. In addition, the paper completely presented the related factors to Agrobacterium-mediated transformation in details and prospected the genetic transformation of Allium vegetables.

Advance and Some Strategies of Monocotyledons Transformation Mediated by Agrobacterium tumefaciens

Zhang Jie, Zhou Yan

2013, 0(5): 7-14.

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In plant gene engineering, the Agrobacterium-mediated method is studied more deeply at present, and its technology is relatively perfect and effective. But the monocotyledons are not Agrobacterium natural host, so the research applications in monocot genetic transformation by Agrobacterium-mediated method are restricted. In recent years, with the rapid development of molecular biology, the transformation mechanism and influencing factors of Agrobacterium-mediated method are continuously explored and researched, making the Agrobacterium-mediated method is gradually applied in monocotyledons, especially gramineous crop, which bring breakthrough. In this paper, the Agrobacterium transformation mechanism, characteristic and factors influencing the monocotyledons conversion were introduced；The research progress in three crops genetic improvement such as wheat, maize and rice by Agrobacterium-mediated method were reviewed；The strategies improveing the efficiency of the monocotyledons transformated by Agrobacterium including ultrasonic treatment, addition of surface active agent, antioxidant and acetyl clove ketone to the culture media were summarized. It could be valuable for the further improvement and optimization of Agrobacterium-mediated monocotyledons transformation.

Type and Function of Root-specific Promoters

Wang Chunyan, Wang Xiaokun, Li Qiaoling, Xie Chengjian, Yang Xingyong

2013, 0(5): 15-21.

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Root is a significant organ in plant growth and development, which anchor the plant body and provide its only access to water and nutrients needed for the completion of a life cycle. The knowledge of root-specific genes, especially its promoters has important value on crop improvement, such as disease and insect resistance, salinity and alkaline stress tolerance, raising production and improving nutrition of root vegetable. This review describes a series of root-specific genes, emphasizes type and functions of root-specific promoters, and establishes the theoretical basis for further plant genetic engineering.

The Prospect and Development of the Human Acid Fibroblast Growth Factor

Tan Yaqing, Liu Dehu

2013, 0(5): 22-27.

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Fibroblast growth factor is a large protein family, human acidic fibroblast growth factor is an important member of it, which has extensive biological functions and potential clinical value. In this paper we review the progress in the study on the structure, function and genetic engineering of human acidic fibroblast growth factor. Some prospects and problems about its application are also discussed.

Research Progress of Anti-HIV Therapy Mediated by RNAi

Yang Wensi, Wang Yi, Wang Yang

2013, 0(5): 28-33.

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Human immunodeficiency virus(HIV)is the pathogen of Acquired Immune Deficiency Syndrome(AIDS), which is an incurable disease and has become a serious public health problem. RNA interference (RNAi) is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. RNAi technology has been widely used in biological and medical research. At present, many researchers have taken RNAi technology as a possible anti-HIV method and tested it in experiments. The main means in RNAi mediated anti-HIV experiments included siRNA targeted therapy and shRNA plasmid mediated therapy. This article will review the research progress of anti-HIV therapy mediated by RNAi.

Fibroblast Activation Protein—the Accomplice of Malignant Tumor

Huang Tianming, Mo Farong, Luo Guorong

2013, 0(5): 34-39.

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Fibroblast activation protein(FAP) is a kind of serine protease, mainly secreted by activated fibroblast, widely exist in epithelial tumors. The close relationship of FAP with the invasion, metastasis, angiogenesis and immune escape of tumors have been confirmed. This is a review about FAP of the structure and functions, the expression and mechanism of action in tumors, the application in diagnosis and treatment for tumors. At last, prospects for the usage of FAP in tumors.

Research Progress of Isobutanol Biosynthesis

Tian Yu, Wang Yiqiang, Wang Qiye

2013, 0(5): 40-44.

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As an important chemical raw material, isobutanol has always been used in many fields, which can be used as a new generation of biofuels in recent foundation. The unique advantages of high calorific value, easy mixing and high octane number have huge development potential and more attention. In this paper, the author gave presentations and discussion about the new bio-fuel production present situation of isobutanol and research progress in biosynthesis of isobutanol at home and abroad, having prospects of the trends for biosynthesis of isobutanol.

Research Progress on Biological Effect of Endosulfan

Xu Dan, Li Shuai, Zhang Min, Sun Yeqing

2013, 0(5): 45-51.

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Endosulfan is a class of persistent organic pollutants (POPs), implicated in a variety of human diseases with widespread concern. Endosulfan has been added to Annex A of the Stockholm Convention in 2011, but the impact of endosulfan on the environment and human health still should not be ignored by people. Endosulfan might affect environment and human health for a long time due to its characteristics of the environmental behavior including persistence, bioconcentration and high biological toxicity, as well as potential carcinogenicity, mutagenicity and endocrine disruptor effects. Here, we summarize the recent research overviews on environmental behavior, biological toxicity and harm to human. Especially, we focus on cytotoxicity of endosulfan to collect, sort out and perform further analysis, followed by valuable suggestion about future direction of research work. Hopefully, this review will be helpful to understand the reason that endosulfan is disabled nowadays and provide new idea of revealing the mechanism in biological effects of POPs including endosulfan.

Long Amplicon Real-time Quantitative PCR Technology and Its Application in DNA Damage Detection

Liang Jiao, Wang Shuhong, Zhang Ziping, Wang Yilei

2013, 0(5): 52-57.

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DNA damage as an endpoint for evaluating genotoxicity of environmental compounds and organism levels of environmental exposure as well as the risk of tumor is always the research focus. Several methods were developed for detecting DNA damage, PCR technology, due to its little DNA needs and high accuracy is widely used in DNA damage detection recently. Here, we review the application of PCR on DNA damage detection, more over, the technical key steps of a newly developed technology called the long amplicon real-time quantitative PCR (LA-real time QPCR) also were detailed .

Biological Effects of Simulated Microgravity on Arabidopsis Seedlings

Wu Chengfei, Guo Shuangsheng, Zhao Qi, Sun Jianfeng

2013, 0(5): 58-62.

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Under normal conditions and simulated microgravity on the growth of Arabidopsis seedlings, the experimental results show that simulated microgravity can develop the following results：(1) affects the growth and development of Arabidopsis seedlings；(2) simulated microgravity influences Arabidopsis photosynthetic characteristics；(3) simulated microgravity affect physiological and biochemical indices of Arabidopsis；(4) simulated microgravity affects the calcium distribution of Arabidopsis. In addition, we use gene chip technology to investigate the influence of simulated microgravity on the Arabidopsis gene expression.

Influence on Wheat Seed Embryo Proteins During the Germination Period by Changed NaCl Stress

Liu Xiangbiao, Niu Wenting, Duan Jiangyan

2013, 0(5): 63-68.

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For the comparison of different concentrations of wheat under salt stress seed germination stage embryo protein expression, “Jinmai -47” as the test material. Select the 3 concentrations of 0, 0.1 mol/L, 0.2 mol/L treated seeds, with the technology of 2-DE 3 concentrations under the embryo protein content changes have been studied. By PDQuest image analysis software analysis under salt stress protein changes, detected in normal seed embryo has 121 points, 0.1 mol/L under NaCl stress 32 protein spots abundance down, 21 protein spots increase in abundance；0.2 mol/L NaCl stress condition, is detected by a 46 point cut protein abundance, 16 protein spots abundance increase.Research results show that salt stress on seed germination of wheat embryos during part of the protein content and the expression have varying degrees of inhibition and activation.

Construction and Preliminary Analysis of cDNA Library of Puccinellia tenuiflora Roots Grown in Saline-alkaline Land

Fu Chang, Wang Shengnan, Guo Min

2013, 0(5): 69-72.

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Puccinellia tenuiflora roots gathered from saline-alkaline land were used to construct a cDNA library by using SMAT technology. The titer of the amplified cDNA library was 1.747×10⁹ CFU/mL, the inserted fragments were distributed from 0.5 kb to 2 kb, and the recombinant rate is 92%. Preliminary analysis of ESTs indicated that S-adenosylmethionine synthetase 2 gene and caltractin gene associated closely with plant salt-responsive reactions were screened in this cDNA library, which can be used to further screen salt-tolerant genes from Puccinellia tenuiflora. This cDNA library of Puccinellia tenuiflora roots laid important foundations for revealing salt tolerant mechanism in Puccinellia tenuiflora, mining salt tolerant genes, and cultivating engineered salt-tolerant plants.

Evolution of RNA Editing in Land Plant Chloroplasts

Wan Ping

2013, 0(5): 73-76.

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RNA editing has been observed in the chloroplasts of all land plants analyzed to date with the exception of liverworts. In this study, we analyzed the 1 514 chloroplast RNA editing sites from non-vascular plants (hornwort and moss) and vascular land plants (fern and eudicot) in features of the probability of amino acid transition, the probability of codon transition, the probability of position of editing site occurring in the codon, the probability of the occurrence of four bases at the minus 1 position near the RNA editing site, and the probability of the occurrence of four bases at the plus 1 position near the RNA editing site. We found that the codon transition of chloroplast RNA editing eudicot is significantly different from that of other plant groups.

Expression Analysis of a Banana Ubiquitin-conjugating Enzyme Gene MaUCE2 Under Abiotic Stress

Wang Anbang, Jin Zhiqiang, Liu Juhua, Jia Caihong, Zhang Jianbin, Miao Hongxia, Xu Biyu^{2, 3}

2013, 0(5): 77-80.

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In order to study the relationship between ubiquitin-conjugating enzyme E2 and stress resistance of plants, the banana seedings were treated with abiotic stress(drought, low temperature and salt stress). The expression levels of MaUCE2 in banana root under different stresses were analyzed by real-time qPCR. The results showed that MaUCE2 expression increased with the drought degree. In addition, the expression levels of MaUCE2 under severe drought is the highest. MaUCE2 expression is induced by low temperature stress, which increased with the decrease of the temperature. When the temperature down to 5℃, the MaUCE2 expression reached to the highest level. The expression levels of MaUCE2 under salt stress increased slightly compared with the control group. These results indicate that MaUCE2 was induced by abiotic stresses and might provide a foundation for the further research and application of biological function.

Cloning，Expression，Purification of Sec7 Domain of hEFA6A Protein

Xie Changlin, Rui Bin, Jiang Na, Zhao Chen, Tang Yajun, Wen Han

2013, 0(5): 81-85.

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As a GEF for the small GTPase Arf6, hEFA6A contains two domains which are pH domain and Sec7 domain. Sec7 domain is the key central domain that executes the function as a GEF. By analyzing the prediction results from Jpred and Uniprot, we chose the boundary contained 214 aa (506-719) from the full-length (1 024 aa) of hEFA6A. Sec7 gene fragment was cloned from homo brain cDNA library and ligated to p28a vector after Nde I and Xho I double digestion. Then the constructive vector p28a-Sec7 was transferred to BL21-Gold (DE3) competent cell for expression. The final concentration of IPTG was 0.3 mmol/L and E.coli cells were cultivated at 16℃ for 24 hours. The recombinant protein was then purified by Ni-NTA affinity chromatography and GE superdexS200 gel filtration column. Our results showed that Sec7 gene and the constructive vector p28a-Sec7 were acquired successfully. The sequence of Sec7 was completely matched with the sequence reported in NCBI. We had a high expression of 70 mg/L and harvested the 95% purity target protein successfully.

Cloning and Sequence Analysis of 5'-Flanking Region of Bovine FATP1 Gene

Gong Ting, Xu Houqiang, Chen Wei, Zhao Jiafu, Luo Heng, Chen Xiang, Zhang Yong

2013, 0(5): 86-92.

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The experiment was conducted to clone 5'-flanking region of bovine FATP1 gene to study regulatory mechanism of the specific transcriptional regulation region. The 5'-flanking region of bovine FATP1 gene was amplified with PCR, and then the 2 164 bp (-1969- +194 bp) 5'-flanking promoter sequence was obtained by product purification, ligation, transformation, and sequence comparison. The obtained cloning vector of 5'-flanking region of bovine FATP1 gene was analyzed by the promoter software in this study. The results indicated that there were 4 transcription start sites and 40 potential transcription factor binding sites. Comparing bovine with human, mouse, rat and dog, the homology of 5'-flanking promoter sequence were 74.1%, 71.1%, 72.0%, and 62.8%, respectively. The conservative region was mainly concentrated in -578- -93 bp of the upstream of transcription start sites, and the highly conserved region was in -265- -162 bp of the upstream of transcription start sites, which may be conclude that the -265- -162 bp of the upstream of transcription start sites was the core promoter region of the gene. The 2 164 bp 5'-flanking region of the bovine FATP1 gene was cloned successfully and the core promoter region was preliminary predicted in the gene.

Prokaryotic Expression and Antimicrobial Activity of a Novel Ribonuclease Rdrlec from Rana dybowskii

Yin Xuewei, Peng Yanli, Ran Shuai, Tao Fengyun

2013, 0(5): 93-98.

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Rdrlec is a novel RNase gene form Rana dybowskii (GenBank accession No.EU384704) and its biological function has not been identified. In order to explore the biological activity of its encoded protein, the Rdrlec gene was adjusted according to Escherichia coli codon bias without changing its amino acids. The synthetic gene was inserted to the pET-28a (+) expression vector through the EcoR I and Hind III site, and the resulting recombination expression plasmid was named pET28-Rdrlec and transferred into Escherichia coli BL21 (DE3) strains. After induced with 0.5 mmol/L IPTG at 34℃ for 6 h, the fusion protein was found expressed mainly in inclusion body form. After a series of steps including refolding, enterokinase cutting and Ni-NTA affinity chromatographic purification, the wild type Rdrlec recombinant protein was obtained, and it showed a single band on SDS-PAGE. It showed enzymatic activity to degrade RNA intonucleotides, which suggests that this molecule has formed the correct spatial structure. The recombinant Rdrlec shows significant antibacterial activity against Escherichia coli and Staphylococcus aureus.

Cloning and Sequence Analysis of Chalone Synthase Gene from Cassia tora

Zhong Dexin, Fang Yuanmengmeng, Guo Zhuanghao, An Hongqiang, Dong Yinsong, Ding Ruofan ,Wang Wanjun, Liao Hai, Zhou Jiayu

2013, 0(5): 99-104.

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The chalcone synthase (CHS) gene was cloned from the fresh cotyledons of Cassia tora, using genomic DNA as a template and a pair of specific primers for PCR amplification. The sequencing results showed that the Cassia tora CHS gene is 1 766 bp, containing two exons and one intron. The CHS gene of Cassia tora was submitted to GenBank with an accession number of (JX676773). The intron is located between 188-765 bp, in line with the GU-AG rule, and contained multiple enzyme digestion sites and cis-regulatory elements which may be involved in expression regulation. The intron of CHS gene had polymorphism, which may be led by the diversity of life history and living environment of different plants. The exon 2 of Cassia tora CHS was more conservative, encoding almost all functional sites of CHS. The NJ phylogenetic evolutionary tree of the amino acid sequence encoded by exon 2, accurately reflect the genetic relationship of the different plants, which can be used in different plant genetic differentiation and molecular evolution research.

Synthesis，Expression and Directed Evolution of the Formate Dehydrogenase Gene from Pseudomonas sp. 101 by Saturation Mutagenesis

Li Tianming, Zhu Linghuan, Liu Tianjia, Dai Baoxin, Feng Huiyong

2013, 0(5): 105-110.

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Formate dehydrogenase (FDH) is a key enzyme in NAD⁺-NADH coenzyme regeneration system, which has an important application value and industrial application prospect. In the paper, the gene (fdh) encoding format dehydrogenase (FDH) was artificially synthesized by overlapping PCR. Recombinant plasmid pET30a-fdh was constructed and transformed to E.coli BL21 (DE3). The recombinant FDH expressed in E.coli induced with IPTG at 16℃, mainly existed in a soluble form and reached a specific activity of 8.7 U/mgPro after purification. To improve the stability of FDH, the saturation mutagenesis of fdh was performed by using the overlapping PCR and the mutant enzyme Cys146AlaCys354Ala was screened. The activity of the two had no change, but the mutant gave rise to 2.5 fold enhancement of stability compared to that in its wild type.

Establishment and Optimization of the Pear SSR System of by Orthogonal Design

Bi Hongyuan, Wang Changbiao, Duan Yonghong, Guo Huangping⁴, Sun Yi

2013, 0(5): 111-115.

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In order to establish and optimize the SSR-PCR amplification system for pear total DNA, an orthogonal design experiment of 5 elements (Taq enzyme, Mg²⁺, template DNA, dNTP, primers)with 4 levels L₁₆(4⁵) was conducted. The PCR result was analyzed by the software DPS to select the best levels of responsive factors and established the optimal SSR reaction system. The optimal SSR-PCR system of 10 μL volumes consists of template DNA 30.0 ng, Taq DNA polymerase 1.0 U, each primer 0.2 μmol/L, Mg²⁺ 2.0 mmol/L, and dNTPs 0.4 mmol/L. PCR amplification procedures was：pre-denaturation for 4 min at 94℃, followed by 30 cycles of denaturation for 45 s at 94℃, anneal for 30 s at 48℃, extension for 30 s at 72℃, the amplification was completed after extension for 10 min at 72℃, then stored at 4℃. Total DNA of 24 pear varieties was tested to amplify SSR markers, which verified the robustness of the system.

Porcine Fetal Fibroblast Culture and Sex Identification of Banna Mini-pig Inbred Line

Xin Jige, Xiao Jing, Zha Xingqin, Cheng Wenmin, Qing Yubo, Pan Weirong, Zeng Yangzhi, Wei Hongjiang

2013, 0(5): 116-120.

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The present study was designed to culture fetal fibroblast of Banna mini-pig inbred line and built sex identification method. Experiments were conducted to isolate fetal fibroblast of Banna mini-pig inbred line from embryos by collagenase digestion and identify their sex using multiplex PCR through optimizing conditions, in which two pairs of primer were designed relatively according to specific fragments in SRY gene and β-actin gene (as internal control). The results demonstrated that it could successfully isolate fetal fibroblast of Banna mini-pig inbred line and the embryo of number 2, 3 were male that could amplify specific DNA sequences of SRY gene (309 bp) and reference gene (132 bp) by multiplex PCR, and the number 1, 4, 5, 6, 7 were female with only reference gene. The PCR products were sequenced and aligned with SRY gene sequence published in NCBI. It shared 99% homology and this made to further verify the validity of results. The study provided a reliable technical basis for the culture of special sex fetal fibroblast in transgene and gene knock-out research.

Genetic Characteristics of GOLA-DQA1 Gene at Exon2 in Hexi Cashmere Goat

Chai Wenqiong, Wang Jiqing, Hu Jiang, Liu Xiu, Li Shaobin, Luo Yuzhu

2013, 0(5): 121-125.

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Genetic Characteristics of GOLA-DQA1 gene at exon2 and the association of alleles with pregnancy in Hexi cashmere goat were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing technology. Nine alleles were identified at GOLA-DQA1 gene at exon2 in Hexi cashmere goat and 3 new DQA1 alleles were identified (GOLA-DQA1*B, GOLA-DQA1*E and GOLA-DQA1*H). Sequence analysis indicated that there were 42 single nucleotide polymorphism sites, which occupied 12.65% of exon 2 sequences which include twenty three transition sites, thirteen transversion sites, five coexist sites and one insertion site. Correlation analysis showed significant statistic difference between different alleles (P<0.05). Frequencies of GOLA-DQA1*A were significantly in abortion group higher than those in normal group (P<0.05). In contrast, GOLA-DQA1*H allelic frequencies in abortion group were significance lower than those in normal group (P<0.05). The result suggested that the allele GOLA-DQA1*A might associated with abortion and GOLA-DQA1*H might related to pregnancy protection in Hexi Cashmere Goat.

Single Nucleotide Polymorphism Analysis of Duck Adiponectin Gene

Zhang Yinhong, Li Huifang, Zhu Wenqi

2013, 0(5): 126-129.

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It was to study the Adiponectin gene polymorphism in duck population. The single nucleotide polymorphism(SNP) of Adiponectin gene T523C SNP on exon2 of Adiponectin was investigated in seven national duck breeds, including Beijing duck, Gaoyou duck, Putian black duck, Liancheng white duck, Shaoxing duck, Youxian Sheldrake and Jianchang duck by PCR-LDR method. And the gene frequency and genotype frequency distribution was analyzed by Hardy-Weinberg equilibrium. Results showed TT, CC and TC genotypes of Adiponectin gene T523C were all detected in seven populations. TC was dominant in six populations except Liancheng white duck, TT was dominant only in Liancheng white duck. T gene frequency was higher than C gene frequency in Beijing duck, Putian black duck, Liancheng white Duck and Youxian Sheldrake. At the other hand, C gene frequency was higherter than T gene frequency in Gaoyou duck, Shaoxing duck and Jianchang duck. The gene at T523C of Adiponectin gene is in genetic balance in 7 duck populations (P>0.05), which will provide an experimental basis on duck molecular genetics.

Analysis for Differentially Expressed Proteins of HSC-T6 Cell Affected by Nerve Growth Factor

Xu Jin, Zhang Xuerong, Hu Rentong, Luo Xiaoling, Chen Ying, Lin Xing, Liao Ming, Fu Rao, Fu Daoying

2013, 0(5): 130-136.

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It was to develop the 2-DE profiles of proteome from hepatic stellate cell and preliminarily analyze the affect of NGF on the protein expression of HSC-T6 cell. We established groups of control and NGF (4、8、16 mg/L), respectively, then acting on the HSC-T6. The differential expression of proteins were analyzed by imaging analysis and MALDI-TOF-MS after the total protein was extracted from the blank control group and NGF treated by 2-DE. The differential protein spots were classified with GO and analyzed with Signal transduction pathway. Results showed that HSC-T6 cell proliferation is inhibited evidently. Forty-seven differentially expressed protein were found in the proteome profile analysis of these two types of blank control group and NGF treated, among which 22 protein spots were up-regulated and 25 protein spots were down-regulated in the HSC-T6 treated by NGF, part of the different expression of protein is increasing with increasing of NGF concentration and part is decreasing with increasing of NGF concentration. Eighteen protein spots with the differential expressions more than 1.8 times were analyzed by peptide mass fingerprint (PMF) and thirteen differentially expressed proteins were found to be related with cell signal transduction, cell proliferation and oxidative stress. The cobra venom NGF can change the expression of the different protein expression of HSC-T6 cell in signaling pathway, providing a theoretical basis for the antifibrotic mechanism of NGF at protein level.

The Purification of GST-NDPK-A Fusion Protein and Detection of its Interacting Proteins in vitro

Lü Fen, Liu Zhong, Yin Xingfeng, Zhao Zhenling, Chen Miaojuan, Zhang Jiaxuan, Chen Wei, Qian Chuiwen, Xiong Sheng

2013, 0(5): 137-143.

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It was to understand the function of nucleoside diphosphate kinase A (NDPK-A) and explore its interacting proteins in vitro through GST-Pull down technique. The target genes were amplified from the templates pBV-NDPK-A and inserted into the prokaryotic expression vector pGEX-4T-2 with glutathione-S-transferase(GST)tag to construct the expression plasmid. After identified by restriction endonuclease digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 strain and exogenous protein expression was induced by IPTG. After purification using Glutathione Sepharose 4B affinity chromatography, the GST-NDPK-A fusion protein was identified by SDS-PAGE and Western blot then incubated with A549. GST-Pull down assay was performed to explore the interacting proteins in vitro followed by LC-MS/MS identification. Results showed that the prokaryotic expression vector of GST-NDPK-A fusion protein was successfully constructed and expressed effectively in E. coli BL21 strain. The fusion protein with bioactivity was purified using Glutathione Sepharose 4B beads. Furthermore, GST-Pull down assay indicated that NDPK-A could bind to Fussel-18 and Rrp12 in vitro.

Study on Sites of the Tolerate Peptide Insertion in the Fluorescent Protein of mCherry

Liang Junting, Li Luzhi, Chen Shaopeng, Jiao Zhen

2013, 0(5): 144-148.

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The fluorescent proteins(FPs)and their color variants have been widely used in cell biology, molecular biology and medicine. Various new technologies based on these proteins such as bimolecular fluorescence complementation (BiFC), fluorescence resonance energy transfer (FRET)-based techniques and optical highlighter are enlightening the whole biology technology. In this paper, we used transposon-based mutagenesis and flow cytometry to verify thirteen tolerate sites of mCherry with five amino acids inserted which fluoresced red. The inserted variants with fluorescence can be served for BiFC and provide more selective sites for designing of bio-sensors.

Transformation System of Cordyceps cardinalis Strain C033 Mediated by Agrobacterium tumefaciens

Guo Wei, Wang Lei, Wu Hongqing, Bai Ling, Zhang Weimin

2013, 0(5): 149-154.

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By using Agrobacterium tumefaciens-mediated transformation (ATMT), the transformation system of Cordyceps cardinalis strain C033 with A. tumefaciens EHA105 was successfully established. The results of geneticin-resistance-selection, gus activity detection and PCR analysis showed that nptⅡin T-DNA was integrated into the transformants and the transformants was genetically stable. Meanwhile, the factors affecting the transformation efficiency such as the concentration of strain C033 spores, the OD value of A. tumefaciens, the concentration of acetoyringone (AS) and the co-culture time were analyzed. The transformation efficiency was about 100 transformants per 10⁵ spores under the optimal condition. The application of ATMT in C. cardinalis will provide a powerful tool for the functional gene analysis of this fungus.

Breeding of High Epothilone B-producing Strains by Genome Shuffling

Wang Dahong, Liu Fei

2013, 0(5): 155-160.

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The mutation of Sorangium cellulosum AHB125 was induced using the classical UV-mutation method plus selection pressures of 2% sodium acetate. Two mutants, SC4-14 and SC4-56, were used as original strains to screen and breed high Epothilone B-producing strains by genome shuffling and explore the basic rule of genome shuffling in strain breeding. Five hereditarily stable strains with high Epothilone B production were obtained after four cycles of genome shuffling. Strain F4-28 yielded 67.4 mg/L of Epothilone B, which was 8-fold higher than that of the initial strain S. cellulosum AHB125 and 4 to 5-fold higher than the mutants used as the original strains for genome shuffling. In addition, the fermentation period of F4-28 was one day shorter than that of SC4-56. The technique of genome shuffling was an efficient method to screen and breed high Epothilone B-producing strains.

Rapid Deletion of nmpC Gene in Escherichia coli Chromosome

Zhang Danfeng, Chen Guoping, Hua Xiuting, Chen Yang

2013, 0(5): 161-164.

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It was to obtain the nmpC deleted strain of E. coli by using both Red and Xer recombination system. The sequence of nmpC from E. coli K-12 BW25113 was first amplified by PCR then inserted into pMD-18 vector to obtain pMD-nmpC. This vector was then digested by EcoR I and Pfu DNA polymerase was used to create blunt ends after digestion, following ligation with difGm fragment to form T-nmpC∷difGm recombinant. nmpC∷difGm was then amplified by PCR and transformed into E. coli by electroporation, following PCR analysis to confirm the designate ΔnmpC strain of E. coli without antibiotic resistance gene was obtained. Results showed the recombinant vector T-nmpC∷difGm was successfully constructed using a process mediated by Red and Xer recombination system. nmpC gene was deleted by homologous recombination and selectable marker was removed from experiment strain, which was confirmed by PCR. The ΔnmpC strain of E. coli without antibiotic resistance gene was obtained for the first time. This strain would be a promising tool for further investigating the function of NmpC outer membrane protein.

Identification of Polyphasic Taxonomy for a Chelatococcus sp. HB-4

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

2013, 0(5): 165-170.

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Abacterial strain HB-4 was isolated from stratum water in Huabei Oilfield, and identificated by polyphasic taxonomy basing on morphologic and physiologic methods, chemical composition analysis, genetic characteristicstest 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-4 were DPG, PE, PG and PC. Major fatty acids were C_18：1 ω7c (47.01%)and C_19：0 cyclo ω8c(21.83%), and ubiquinone Q-10 was the predominant quinone. HB-4 had enzyme activity of oxidase, catalase, esterase (C4), Esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphate hydrolase.

Isolation，Identification and Characteristics of Denitrifying Polyphosphate-Accumulating Organisms

Zhang Wugang, Hong Wulin, Zheng Chunli

2013, 0(5): 171-176.

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Being screened by a phosphate uptake experiment, where nitrates were denitrified to gas and through inspection of metachromatic and Polyphydroxybutyrate (PHB) granules, denitrifying phosphate-accumulating organisms (DNPAOs) P1-1 was isolated from a water sample in Lake Wuliangsuhai of Inner Mongolia. According to its morphological, physiological characteristics, BIOLOG identification and the analysis of its 16S rRNA sequence, it was identified as a strain of Pseudomonas fluorescens. The growth curve shows that strain P1-1 enters stationary phase in 12 hours’ training. Meanwhile, some important factors, temperature and pH value were studied to evaluate their effect on the growth and phosphorus removal ratios of strain P1-1. The results showed that the optimal conditions for the strain’s growth and phosphorus removal are 30℃ and pH8.0.

Isolation and Identification of Excellent Lactic Acid Bacteria from Silage and Its Biological Characteristics Research

He Yiqun, Lei Zhaomin, Wu Run, Wan Xuerui, Diao Xiaolong, Ai Wenna

2013, 0(5): 177-183.

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To screen excellent microbials for silage additive usage, 12 bacteria strains were isolated from the silage. According to the results of morphological observation, physiological and biochemical tests, and sequence analysis of the 16S rRNA gene of the strains isolated, 6 strains were screened and compared through acid yield tests and bacteriostatic tests. The results showed 5 strains were Pediococcus pentosaceus, 4 strains were Lactobacillus plantarum, 1 strain was Lactobacillus fermentum, 1 strain was Leuconostoc mesenteroides subsp. mesenteroides, 1 strain was Enterococcus faecium；Strain B1-7 and B5-2 which reached a stable period after 8 h were fastest-growing, others were 14 h；The B1-7 stain no longer grow at temperature higher than 45℃, medium pH>10, salinity>0.08 g/mL. Other stains could be used as bio-straw silage additives to do further research which showed strong resistance and silage potential.

Effects of Co-culture with Helper Bacteria on the Secondary Metabolites of Myxococcus fulvus

Huang Yan, Hu Jianwei, Zhu Honghui,

2013, 0(5): 184-189.

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The research of secondary metabolites of myxobacteria was restricted due to it's low content under the routine laboratory culture conditions. This study discussed the effect of two helper bacteria to Myxococcus fulvus producing secondary metabolites in liquid fermentation culture conditions. The results showed that the yield and diversity of secondary metabolites were improved by adding the two helper bacteria. After the co-culture of M. fulvus with 42 or 43 helper bacterium, the yield of the secondary metabolites increased from 0.05 g/L to 0.40 g/L and from 0 g/L to 0.45 g/L, respectively. The maximum yield was obtained for 42 (0.64 g/L) and 43 (0.69 g/L) helper bacterium, when the former was inoculated between 72 to 84 h or the later was cultured with M. fulvus simultaneously. However, the effect of the two helper bacteria on diversity of the secondary metabolites was different. The original secondary metabolites component of M. fulvus was changed by adding 43 helper bacteria but not 42. In conclusion, co-culture with helper bacteria can improve the secondary metabolism of M. fulvus, which would help analyze the secondary metabolites of myxobacteria.

Research on Biological Activity of Antibiotic Analogues in SC-04 Culture

Li Wenbin, Li Zengbo, Liu Xianjun

2013, 0(5): 190-193.

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The extraction and separation method of SC-04 antibiotics was optimized, based on the analysis of the bacteria fermenting liquid stability and its antibacterial material polarity. The bacteriostatic spectrum analysis showed that the bacteriostatic spectrum of the separation product is wide, and had satisfactory inhibition effect to the most pathogenic fungi such as pepper phytophthora cactorum and bacteriostatic activity was strong.

Rapid and Efficient Recycling DNA Fragments from Non-denaturing Polyacrylamide Gel

Zhou Yi Wang Yiping

2013, 0(5): 194-198.

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Obtaining specific DNA fragments is a prerequisite to carry out varieties of molecular biology experiments and polyacrylamide gel electrophoresis is the first choice to purify specific DNA owing to its high resolution. In this assay, by exploiting the curing effect of liquid nitrogen, an approach applying grinding to break the structure of polyacrylamide gel electrophoresis to extract and purify DNA is introduced. The DNA purified according to the method above and the counterpart based on the purification of agarose gel electrophoresis are subjected to further detection through polyacrylamide gel electrophoresis, with the result manifesting, the DNA purified via the method above has a higher purity and specificity as well as an equal efficiency comparing with the one purified by agarose gel electrophoresis. The method described here improves the specificity of DNA, and simultaneously being economical, efficient and less time-consuming, thus it worth using widely.

Study on the Technology of CS-PLGA Nanospheres Immobilized Alkaline Phosphatase

Gao Qiyu, Li Hongbin, Chen Hongli, Kong Yu, Zhou Chenyan

2013, 0(5): 199-204.

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Alkaline phosphatase expressing in recombinant E. coli was immobilized on Chitosan-poly (lactic-co-glycolic acid) by using glutaraldehyde as cross-linking reagent. The immobilization conditions and characterization of the immobilized enzyme were obtained through single factor and orthogonal experiment. The results showed that the best condition of immobilization was optimal when the CS-PLGA nanoparticles concentration was 50 mg, the volume fraction of glutaraldehyde was 4%, cross-linked time was 2.0 h and immobilized time was 9.0 h, the immobilized enzyme can keep activity about 76.2%. At the same time the optimal temperature, the optimal pH, the thermal stability and pH stability of alkaline phosphatase were comparatively improved after immobilization. So it confirmed that the technology would form basis of the further application of immobilized alkaline phosphatase.

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