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    16 July 2015, Volume 31 Issue 7
    Review
    Biotechnology and Kiwifruit Breeding in China
    Jing Zhaobin, Lei Yushan, Li Yongwu
    2015, 31(7):  1-10.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.036
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    Kiwifruit(Actinidia Lindl.)is an important fruit tree crop in China. With the development of modern biotechnology,traditional breeding of kiwifruit is mainly based on the selection from wilding and seedling,these methods presents coexist situation the challenge and opportunity compared with the application of molecular breeding such as the genomics and transgenesis. Compared with the other fruit trees,the researches for the genomics of kiwifruit are still at a relatively low level. This paper summarized the method,application,and the latest research progress of modern biotechnology in kiwifruit genetic breeding,and discussed the breeding strategy of kiwifruit in China,in order to provide the method and thought reference for kiwifruit conversional and transgenesis breeding in China.
    Research Progress of Genetic Engineering on Plant Salt Tolerance
    Guo Wenfang, Nong Wanting, Li Gangqiang, Liu Dehu
    2015, 31(7):  11-17.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.002
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    With the increase of global population and the decrease of arable area, growing serious food crisis leads to the exploration and utilization of salinized lands on Earth received attentions. People keep improving salinized lands through a variety of measures, and cultivate crops of high salt tolerance. In this paper we mainly introduce salt tolerance mechanisms in plants, as well as overview and future direction of researches on genetic engineering of plant salt tolerance.
    Research Progress of Plant Histone Acetyltransferases
    Xia Dean, Liu Chunjuan, Lü Shibo, Zhang Yanni, Liu Yijia, Ma Xujun
    2015, 31(7):  18-25.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.003
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    Histone acetylation is a dynamic and reversible process, including histone acetylation and de-acetylation. Histone acetyltransferases mediate histone acetylation and act in concert with histone deacetylases to regulate the status of histone acetylation, which modifies chromatin structure, affects gene transcription and thus plays a crucial regulatory role in plant growth, development and stress responses. In this review, we summarize the current research progress of histone acetyltransferases in respects of cellular distribution, classification, substrate specificity and functions.
    Direct Reprogramming of Somatic Cells into Neurons and Neural Stem Cells
    Zhou Zhenning
    2015, 31(7):  26-32.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.037
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    The direct reprogramming of somatic cells is a process that one differentiated cell type can be directly converted into another kind of differentiated cell type,which bypasses the phase of induced pluripotent stem cells(iPSCs). Compared with the iPSC technology,the direct reprogramming of somatic cells has higher efficiency of reprogramming,and avoids introducing oncogenes.Modern medicine has no effective treatment for nerve cell damage which due to neurodegenerative diseases,neurological genetic diseases and traumatism. Somatic cells directly reprogramming into neural cells may provide another way to treat these diseases. Recently,several studies have shown that the ectopic expression of specific neural and pluripotent stem cell transcription factors can directly convert differentiated cells into induced neurons(iNs)and induced neural stem cells(iNSCs). Here,we will review the most recent progress in the somatic cells directly reprogramming into iNs and iNSCs and also discuss the application of iNs and iNSCs in future.
    A Review on Correlation Between Long Non-coding RNAs and Cancer as well as Stem Cells
    Wan Bo, Liao Shiqi, Yuan Hongxia, Ma Meilan, Zhu Jian, Zeng Jiayu
    2015, 31(7):  33-39.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.004
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    The human genome contains more than 20 000 protein-coding genes, only approximately 2% of the total genes, while over 90% of the transcripts are long non-coding RNAs(lncRNAs). lncRNAs are transcripts commonly distributed in genomes of most mammals, with length of 200 to 100 000 nt, but not possessing function of encoding proteins. Studies have shown that lncRNAs were abnormally expressed in numerous types of cancers, playing a potential oncogenic or suppressor role in tumorigenesis. Meanwhile, lncRNAs have been found to be critical regulators of various biological processes, and they were inseparable with the occurrence and development of tumors. Besides, lncRNAs play a vital role in maintaining pluripotency and regulating the gene expression, self-regeneration and differentiation in stem cells, and they are becoming the new hot spot in cancer researches after microRNA. In this review, we summarize the functions and underlying mechanisms of lncRNAs in cancer and in stem cells biology, which aims to provide new ideas about diagnosis, treatment, and prognosis of the tumor.
    Research Progress on Diversity of Microbial Community During the Pickled Processing of Salted Fish Products
    Wu Yanyan, Qian Xixi, Li Laihao, Yang Xianqing, Ma Haixia
    2015, 31(7):  40-44.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.005
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    Salted fish is one of traditional aquatic products in China. All along it has been received people's favor due to its easy preservation, unique flavor and rich nutrition. However, microbial species in pickled processing are complex and have a significant effect on the quality of the products. Combining with the latest researches at home and abroad, here we expound the changes of microbial community in traditional salted fish and fast pickled fish by microbial fermentation, as well as the latest technology and application in microbial diversity researches. On this basis, we propose the research direction on diversity of microbial community during the salted fish processing, and provide the theoretical basis for the researches on microbial action mechanism in salted fish and its optimization control of production.
    Detection Methods of Genome-wide DNA Methylation Level Based on High-throughput Sequencing Technology
    Ma Langlang, Liang Zhenjuan, Xian Xin, Luo Shiwen, Li Qingchao, Liang Qianyun
    2015, 31(7):  45-50.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.007
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    DNA methylation has been being the research focus of epigenetics study in the recent years. There are over 10 kinds of methods for detecting DNA methylation level, and detection of methylation status on genome-wide level has been achieved with the development of the NGS(Next-Generation Sequencing). At present the major detection methods of DNA methylation level on genome-wide scope based on NGS include BSP-seq(Bisulfite Sequencing PCR), MeDIP-seq(Methylated DNA Immunoprecipitation Sequencing)and MBD-seq(Methylated DNA Binding Domain Sequencing). In this paper, the research progress of principles, procedures, advantages and disadvantages, optimization of the usage and some bioinformatics resources on these 3 methods were reviewed, which aims to provide some beneficial ideas to researchers who are studying DNA methylation models while employing methods of high-throughput sequencing.

    Research Progress of Cultivation Technology of Taxus and Its Distribution in China
    Liu Xinxing, Yu Xianghua, Liu Xueduan
    2015, 31(7):  51-57.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.008
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    The development of cultivation technology of taxus(Taxus chinensis)is of great significance on increasing taxus forest area and relieving the shortage of medicinal herb resources for paclitaxel. The basic biological characteristics of taxus and forest distribution in China are introduced. Then the research progress on cultivation technologies of taxus are summarized, including traditional seeding and cuttage technology, the latest research achievements on contemporary forest cultivation technologies and methods, especially the application of asexual reproduction in the cultivation of taxus forest. Finally, the aspects which should be strengthened urgently in taxus forestry breeding are pointed out. All of above works aim to provide the references for extending forest cultivation technology and enlarging cultivation area of taxus.

    Rapid Detection and Identification of Fusarium oxysporum f. sp. niveum
    Cao Yuexia, Ling Jian, Xie Bingyan, Yang Yuhong
    2015, 31(7):  58-63.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.009
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    Based on the genome comparison analysis between Fusarium oxysporum f. sp. niveum(Fon)and other closely-related pathogens, the specific sequences of Fon were obtained. Then we developed specific primers that could amplify Fon-specific DNA bands. Combined the Fon-specific primers with the universal primer of Fusarium oxysporum (W106R/W106S), we established a duplex PCR method that might rapidly and accurately detect Fon by 1 PCR reaction, which is beneficial to technically support the rapid identification of Fon by method of molecular detection..
    A Multiplex PCR for Rapid Detection of Genetically Modified Ingredient in Flue-cured Tobacco
    Yu Jing, Guo Yushuang, Lin Shifeng, Yu Shizhou, Zhao Jiehong
    2015, 31(7):  64-68.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.010
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    In order to improve the efficiency of detecting genetically modified ingredient(GMI), we established a multiplex PCR detecting 3 exogenous gene ingredients in flue-cured tobacco. By optimizing the concentrations of Mg2+, dNTP, rTaq, and primer as well as annealing temperature in the multiplex PCR system, the result showed that the optimal conditions for the experiment were:4 pairs of primers pre-mixed in ratio of 1∶1∶1∶1(V/V), Mg2+ 1. 5 mmol/L, dNTP 125 μmol/L, rTaq polymerase 1 U, mixed primers 1 μmol/L, DNA 100 ng annealing temperature 65℃, and 35 reaction cycles;under the above conditions, the system detected 1 tobacco endogenous gene NR, exogenous gene 35S promoter, NOS terminator and selectable marker NPT II gene by 1 PCR reaction. In this study, the optimized multiplex PCR could efficiently detect the GMI of 0.9%(V/V)in transgenic flue-cured tobacco.
    Establishment and Optimization of ISSR Reaction System for Valeriana officinalis L. var. latifolia Miq of Guizhou Province
    Qian Zhiyao, Zhou Daotang, Huang Xiuping, Zhou Mei, Chen Zuyun, Qin Ronggui
    2015, 31(7):  69-75.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.011
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    This study is to establish a stable and reliable ISSR system for Valeriana officinalis L.var. latifolia Miq. Based on the single factor experiment in 10 levels, L16(45)orthogonal design was applied to conduct combinatorial optimization in 4 levels of 4 major factors, and the results were analyzed by visual method, range analysis and variance method. The primer and the optimal annealing temperature were screened by optimized reaction system. The stability and reliability of this system were tested by 9 samples from different origins. The optimal ISSR conditions in the experiments were as following: in 25 μL reaction system containing 2.5 μL 10×Taq Buffer, 2.0 mmol/L Mg2+, 0.28 mmol/L dNTPs, 0.3 μmol/L primer, 1.75 U Taq DNA polymerase, 60 ng DNA template, and ddH2O completed. The order of the effect by the factors was in Mg2+ > primer > dNTP s > Taq DNA polymerase. The optimal annealing temperature for each primer was determined, and 17 primers with distinct and stable amplified bands were screened. Stability test showed that the optimized system was stable and reliable. By this study we may reach the conclusion that the optimal ISSR reaction system for V. officinalis var. latifolia was generated, and the desired primers also were screened, which may provide the technical foundation for the application of ISSR molecular marker technology in the study of genetic diversity of V. officinalis var. latifolia.
    Research report
    Arabidopsis AMP1 Negatively Modulates Plant Responses to Salt Stress
    Xia Jinchan, He Jinhuan
    2015, 31(7):  76-82.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.012
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    Salt stress severely affects the growth and development of plants and crop yield, therefore it is crucial to identify the related genes responding to salt stress in plants. Altered Meristem Program(AMP1)of Arabidopsis, encoding a putative glutamate carboxypeptidase, is involved in plant growth, photomorphogenesis and seed dormancy. In this study, we revealed new function of AMP1, i.e., its deficiency increased the capacity of salt resistance in deletion mutant amp1. The study convinced that phenotype of solid salt resistance resulted from 2 aspects: firstly the accumulating more betaine and proline in mutant than in wild one led to the decrease of cell potential in the mutants, and secondly the expression of downstream gene RD29A and AHA3 in the mutants was higher than that in wild ones and the latter promoted the exocytosis of Na+. The salt stress also triggered the oxidation stress in the plants; the study discovered that the deletion of AMP1 up-regulated the expression of antioxidant gene ZAT10/12, consequently lowered the accumulated level of peroxide in amp1 mutants, and thereby diminished the damages to cells and the prohibition of growth. All of them jointly enhanced the capacity of salt resistance in amp1 mutants. Our results proved that Arabidopsis AMP1 negatively regulated plant responses to salt stress.

    Cloning and Prokaryotic Expression of Sweet Sorghum Succinic Semialdehyde Dehydrogenase SbSSADH
    Wang Longhai, Yang Zewei, Zhu Li, Huang Dafang, Lang Zhihong
    2015, 31(7):  83-90.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.013
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    Gamma-aminobutyric acid(GABA)shunt is a metabolism bypass of tricarboxylic acid cycle(TCA)and widely existed in animals, plants and micro-organism. The activity of the GABA shunt is drastically enhanced in response to biotic and abiotic stress. Succinic semialdehyde dehydrogenase(SSADH), which can oxidize succinic semialdhyde into succinate, is one kind of key enzymes in the metabolic pathway of the γ-aminobutyric acid(GABA)shunt, and plays an important role in biological functioned of GABA shunt in organisms. Objective:This study aimed to construct the prokaryotic expression vector of Sweet Sorghum SSADH and express the soluble protein in E.coli. With the primers designed according to the homologous genes sequence provided by MaizeGDB and Gramene databases and mRNA from sweet sorghum as a template, SSADH gene cDNA was cloned with RT-PCR. Sequence analysis showed that SbSSADH gene have 94.77% similarity with Corn and Sugarcane SSADH gene, but the forward 200 bp signal peptide sequence of SSADH gene are different. The construct harboring the truncated SSADH gene fragment without the signal peptide sequence of SbSSADH were transformed into E. coli Rosetta cell, respectively. The expression and purification of proteins were detected with SDS-PAGE, and the enzyme activity of soluble protein SSADH was analyzed with enzymatic dynamics analysis. Results showed that we cloned the cDNA of SSADH from sweet sorghum, constructed the pET-28a-SSADH prokaryotic expression vector, and obtained the soluble protein:the result of enzymatic kinetics analysis indicated that the expressed protein has activity of Succinic semialdehyde dehydrogenase and activity have been inhibited by AMP and ATP. In this study the SSADH prokaryotic expression vector was constructed successfully and recombinant expression soluble protein of SSADH has Succinic semialdehyde dehydrogenase activity.

    Gene Cloning, Bioinformatic Analysis and Preliminary Functional Characterization of Gene Encoding Galactinol Synthase 3 in Jatropha curcas
    Wang Haibo, Zou Zhurong, Gong Ming
    2015, 31(7):  91-100.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.014
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    It was to study the roles of soluble sugars in the cold-tolerance of Jatropha curcas and their related applications in genetic engineering. The full length cDNA of J. curcas, a gene member of such enzyme family(JcGS3)was cloned herein, with a size of 1 053 bp covering the entire open reading frame(ORF)of 1 008 bp. This ORF encoded a polypeptide of 335 amino acids, with theoretical molecular weight of 38 kD, pI value of 5.44, and the typical DxD motif. Further promoter analysis of the genomic sequence of JcGS3 indicated the occurrence of TATA-box, CAAT-box, cold responsive elements of CRT/DRE, and ABA responsive elements. In addition, JcGS3 was characterized with a significant effect on improving the cold-tolerance of the recombinant yeast strain.
    Bioinformatics Analysis and Expression Study of DHDDS of Dunaliella viridis
    Pan Yifang, Gong Yifu, Yu Kai, Zhu Shuaiqi, Wang Heyu
    2015, 31(7):  101-108.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.015
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    Dunaliella viridis is a unicellular green alga producing valuable secondary metabolite carotenoids, and dehydrodolichyl diphosphate synthase(DHDDS)is an enzyme in the synthesis pathway of carotenoids. It was to investigate the correlation between expression of DHDDS gene in D. viridis treated with different concentrations of MeJA and the content of carotenoids. Total RNA was extracted from D. viridis and the full length of DHDDS was acquired by de-novo transcriptome sequencing, then the sequence was analyzed by bioinformatics website and software to predict the protein structure. Transcriptional levels of D. viridis treated with different concentrations of MeJA were examined by Real-time quantitative PCR(RT-qPCR). The total length of gene DHDDS was 2 211 bp, containing a 1 740 bp open reading frame(ORF)that encoded 579 amino acids. Theoretical isoelectric point of DHDDS protein was 7.63. Relative molecular mass was 62 472.7 Da. Bioinformatics prediction revealed that there was no signal peptide and transmembrane domain in DHDDS protein, and it was located in cytoplasmic matrix. Sequence alignment showed that DHDDS of D. viridis shared the highest homology of 59% with that of Chlorella variabilis. The RT-qPCR showed that the expression of DHDDS gene in D. viridis treated by 100 mol/L of MeJA was the highest with significant difference:at this concentration, the content of carotenoids was higher than other groups. There was certain correlation between the expression of DHDDS gene and the content of carotenoids. Conclusively, the DHDDS of D. viridis is an enzyme located in cytoplasmic matrix, relating to synthesis pathway of carotenoids. Within certain concentration, MeJA in lower concentration shows promoting effect on the expression, and inhibiting in higher concentration, there is a correlation between the expression and the content of carotenoids.
    Construction and Application of the RNAi Vector for BBS1 Gene in Chlamydomonas reinhardtii
    Chen Lihong, Li Lili, Gao Lifen, Zhou Junfei, Li Tiantian, Peng Hai
    2015, 31(7):  109-114.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.016
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    Bardet-Biedl syndrome is a disease caused by mutations of various genes and associated with afunction of primary cilia. There are 12 disease-causing genes of BBS1-12 respectively. This study aimed to understand the function of BBS1 gene in Chlamydomonas reinhardtii through RNAi interference technology. Firstly, the fast and simple RNAi backbone vector was constructed. Subsequently, a cDNA in BBS1 gene was cloned into media vector pEASY-T1 using RT-PCR. After sequencing, the target fragment was inserted into the RNAi backbone vector through the twice enzyme digestion. Verified by PCR testing, enzyme digestion and sequencing, the RNAi vector of BBS1 gene was successfully constructed. The phenotype of transgenic C. reinhardtii CC503 transformed into BBS1 RNAi plasmid by gene gun was significantly different from that of the control and their phototaxis changed compared to the control.

    Cloning and Expression Analysis of Full-length cDNA Sequence of a Low Temperature Resistant Candidate Gene △ 6FAD in Trachinotus ovatus
    Han Hongbo, Wang Zhongliang, Chen Gang, Tang Baogui, Zhang Jiandong , Huang Jiansheng, Zhou Hui, Shi Gang, Pan Chuanhao
    2015, 31(7):  115-123.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.017
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    The full-length cDNA of delta 6 fatty acid desaturase(△6FAD)in Trachinotus ovatus was cloned by RACE(Rapid Amplification of cDNA Ends), and the sequence structure of the gene and protein was determined through bioinformatics analysis. RT-PCR(Real-time PCR)was employed to analyze its expression differences in tissues under different temperatures and the expression patterns in low temperature. Ultimately, the results indicate that full length of cDNA sequence of △6FAD in T. ovatus is 1 908 bp, of which has 3' non-coding region 431 bp, 5' non-coding region 135 bp, and open reading frame 1 329 bp that encodes 442 amino acids. The protein is hydrophobic without signal peptide, and containing abundant protein secondary structure, typical histidine cluster motifs in this family, and cytochrome b5 domain. Phylogenetic results from the BLAST protein database reveal that the deduced amino acid sequence of △6FAD of T. ovatus is highly homologous with Lates calcarifer and Rachycentron canadum:lowly homologous with Scophthalmus maximu, Thunnusthynnus and Epinephelus coioides:but little homologous with Danio rerio, Mus musculus and Homo sapiens. RT-PCR results manifest that gene expression of △6FAD is significantly correlated with temperature and stress time in environment, the expression level of the gene at first slowly decreases, then rapidly rises as temperature lowering in the experiment of temperature gradient. However, there is different expression pattern in different low-temperature environment:for example, at 13℃ the gene expression of △6FAD increases to its original level after first decreasing with time:and while at 16℃ and 19℃ it is gradually decreasing with time, i.e., feedback adjustment way.
    Cloning and Prokaryotic Expression of Cathelicidin Gene from Japanese Eel I,Anguilla japonica
    Zhang Dongling, Yu Dahui
    2015, 31(7):  124-131.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.018
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    Cathelicidin, an utmost family of antibacterial peptide so far, possesses multiple biological functions. In order to explore and exploit the antibacterial mechanism and potential biological functions of Cathelicidin, full-length sequence of cDNA of Cathelicidin gene from Japanese eel Anguilla japonica I(AjCathI)was obtained by RACE-PCR. The full cDNA of AjCathI was 842 bp and the ORF contained 570 bp encoding 189 amino acids. Analysis of amino acid sequence demonstrated that AjCathI contained 4 conservative cysteine residues at C terminal, and the mature antibacterial peptide had the highest similarity with Plecoglossus altivelis by homology of 65.57%. Phylogenetic analysis revealed that AjCathI shared a common evolutionary origin with other teleost fishes. AjCathI was cloned into pET-28a and expressed in Escherichia coli BL21(DE3). The result manifested that AjCathI protein was expressed in inclusion bodies. The protein was isolated by Ni column, identified by Western blot and re-natured by dialysis, the protein of high purity was gained. This study laid the foundation for further verifying mature peptide sequence of AjCathI and studying its antibacterial activities as well as other biological functions.
    Cloning and Expression Analysis of a Transferrin Receptor 1 Gene from Swamp Eel, Monopterus albus
    Jiang Ao, Li Wei
    2015, 31(7):  132-137.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.019
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    For investigating the function and tissue expression specificity of transferrin receptor 1 gene(TfR1)from swamp eel, TfR1 gene was isolated from the liver of swamp eel using homologous cloning and RACE techniques, the bioinformatics method was used to analyze the putative amino acid sequence, and semi-quantitative RT-PCR was used to study the expression of TfR1 gene in different tissues. The results showed that the full length of cDNA(GenBank accession number:KF819396)was 2 839 bp including a 134 bp 5' un-translated region(UTR)and a 380 bp 3' UTR, and it encoded a polypeptide of 774 amino acids. Sequence alignment by amino acid indicated that the inferred amino acid sequence of TfR1 from swamp eel shared high identity with those of other teleosts in 55.68%-68.41%. Semi-quantitative RT-PCR analyses demonstrated that the TfR1 transcripts were significantly varied in different tissues, the highest in blood cells, moderate in kidney, spleen and intestine, and little in liver, stomach, skin, brain, heart and muscle.
    Optimization of Prokaryotic Expression of Antibacterial Peptide hyastatin Gene in Scylla paramamosain
    Peng Yinhui, Cai Xiaohui, Xiong Xiangying, Liu Xujia, Huang Guoqiang
    2015, 31(7):  138-142.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.020
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    This study aims to optimize the prokaryotic expression of hyastatin gene in Scylla paramamosain. The fragment of cloned mature peptide in S. paramamosain hyastatin was ligated with the expression vector pET-28a. This recombinant was transformed into Escherichia coli BL21(DE3), and the gene was expressed by inducing under the optimal conditions. The expression of this protein was the highest under the 37℃ and in 0.2 mmol/L of IPTG for 4 hours. The molecular weight of the expressed product was identical to that of expected protein by SDS-PAGE analysis. The fusion protein was efficiently expressed as inclusion bodies, and further purified by HisTrap HP column. The result of Western blot showed that the fusion protein could be bound specifically with mouse anti-His-tag Mab. In conclusion, the prokaryotic expression product of hyastatin gene in S. paramamosain may be obtained by optimizing expression conditions.
    Immunogenicity and Immunoprotection of Translocation Protein B(TolB)in Vibrio alginolyticus
    Pang Huanying, Zhou Zejun, Zhang Yanfei, Li Jing, Qiu Mingsheng, Ding Yu, Jian Jichang, Wu Zaohe
    2015, 31(7):  143-149.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.021
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    To investigate the possibility of translocation protein B(TolB)from Vibrio alginolyticus HY9901 as a candidate antigen for vaccine production, the full length tolB gene was amplified by PCR and designed specific primers according to the whole genome sequence of V. alginolyticus published in GenBank. Sequence analysis revealed that tolB gene was 1 353 bp and encoded a putative protein of 450 amino acids(GenBank accession number:JQ846501). BLAST analysis showed that the tolB of V. alginolyticus had high genetic relationship with other known Vibrio, its sequence was conservative, and might be a common antigen candidate protein. The TolB protein of prokaryotic expression was injected into SPF mice to prepare polyclonal antibody, and its titer of ELISA reached 1∶40 000. Western blot indicated that TolB serum reacted specifically with the induced recombinant protein. Further the TolB protein was used as an antigen to immunize grouper(Epinephelus awoara)twice, and ELISA detection found that the antibody titer of the immunized grouper increased to the highest(up to 1∶2 048)in the fourth week. Challenge assay test showed that the immunoprotection rate of TolB to E. awoara was 76%. Therefore the TolB of V. alginolyticus has solid immunogenicity and immunoprotection, and it can be used as a candidate antigen for vaccine of Vibrio subunits.

    Molecular Cloning,Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)
    Ma Yuanyuan, Cheng Feiyue, Xing Wanjin
    2015, 31(7):  155-160.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.023
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    The purpose of this study is to clone sheep sperm equatorial segment protein 1 cDNA(SPESP1), express SPESP1 in prokaryotic cells. Firstly, SPESP1 cDNA was amplified with total cDNA of sheep testicular as template and the designed primers according to the sequence of sheep genome in GenBank. Then the amplified SPESP1 cDNA was cloned into the expression vector pGEX-4T1 to construct pGEX-SPESP1, which was transferred into Escherichia coli BL21 for expression and the conditions of expression were optimized. The purified fusion protein by SDS-PAGE gel-slicing was identified by Western blotting. By sequence alignment, two base pairs of cDNA sequence were different between the cloned SPESP1 and the predicted one in GenBank, which led to 1 amino acid varied. The expression of the recombinant fusion protein in E. coli BL21 succeeded under the optimal condition of 37℃ and 0.005% of IPTG for 4 h. SDS-PAGE and Western blotting analysis revealed that a protein at band of approximate 64 kD could be recognized by the antibody of anti-GST and anti-sheep-SPESP1, which was in accord with the prediction, and this implied that expression of fusion protein was achieved. In conclusion, successful gain of purified fusion protein GST-SPESP1 lays a foundation for the functional studies of SPESP1 in the membrane fusion of sperm and egg.

    Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella
    Zhai Qi, Huang Bing, Dong Hui, Zhao Qiping, Zhu Shunhai, Liang Siting, Li Sha, Yang Sihan, Han Hongyu
    2015, 31(7):  161-168.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.024
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    The objective of this study is to construct eukaryotic recombinant expression plasmids of a conserved hypothetical protein gene(EtCHP559)of Eimeria tenella, and study the expression of EtCHP559 gene in transfected DF-1 cells. The full-length cDNA of EtCHP559 was cloned using 5'RACE approaches. Bioinformatics analysis revealed that the gene contained 1 746 bp, of which the ORF was 1 224 bp(position 104 - 1 327 bp)encoding 407 amino acids with molecular weight of 46 kD. The deduced amino acid sequence of the protein had a transmembrane region and signal peptide. The cDNA fragment consisting of complete ORF was amplified by RT-PCR, and ligated to the eukaryotic expression vector pcDNA3. 1-flag. The recombinant plasmid pcDNA3. 1-flag-EtCHP559 was identified successfully by PCR and restriction enzyme digestion, and the target band of approximate 1 224 bp was observed. Subsequently, the recombinant plasmid was transfected into DF-1 cells, Western blot recognized target protein of around 47 kD, and immunofluorescence also showed that green fluorescence in transfected DF-1 cells was observed. These results indicated that the full-length sequence of EtCHP559 was obtained, and the recombinant plasmid of EtCHP559 was successfully constructed and expressed in eukaryotic cells, which laid a foundation for the future research on functions of EtCHP559.
    Isolation,Identification and Phosphate-solubilizing Capacity of Phosphate-solubilizing Bacteria from the Rhizosphere of Camellia
    Liu Xiaoyu, Fu Dengqiang, Chen Liangqiu, Yang Weibo, Li Dongxia, Fu Haiquan
    2015, 31(7):  169-173.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.025
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    Phosphate-solubilizing bacteria were isolated from the rhizosphere of camellia, and 17 strains were screened using traditional method of isolation and culture of microorganisms. By utilizing the method of transparent zone, phosphate-solubilizing bacteria were preliminarily screened from the rhizosphere soil. Subsequently, the bacteria were re-screened by using method of molybdenum-antimony anti-spectrophotometric to measure content of soluble phosphorus in fermentation liquor, and strain 6-Y-09 with the highest capacity of solubilizing phosphate was gained. According to morphology of the colony, physiological and biochemical properties, 16S rDNA sequence and phylogenetic analysis, strain 6-Y-09 was identified as Burkholderia capacia. The strain has great potential in further development of microbiological fertilizer because of its stronger phosphate-solubilizing capability, which will provide an outstanding strain resource for improving phosphorus supply of camellia and promoting its growth.
    Screening and Identification of a Sponge-associated Fungus HMP-F66 Inducing Oxidative Burst in Tobacco Cell Suspensions
    Pang Zhiwei, Lu Xu, Hu Jiangchun, Cheng Xiaoqi, Wang Nan, Song Yanling
    2015, 31(7):  174-179.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.026
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    The plant innate immune system provides an alternative way for crop protection, and the plant immunity triggers have been an important source of biorational pesticide leads. In order to find new plant resistance inducers, fifty sponge-associated fungal isolates were screened for their ability to induce oxidative burst in the tobacco suspensions using the H2DCF-DA fluorescence assay. Strain HMP-F66 showed significant activity in inducing H2O2 production in tobacco suspensions. HMP-F66 was assumed to be Aspergillus oryzae by its morphological, physiological, cultural characters, and rDNA ITS sequence analysis.
    Isolation and Identification of Serratia marcescens Yj1,and Separation and Purification of Organophosphate Degrading Enzymes
    Yu Ting, Wang Chunhong, Zhang Tingting, Ji Tian, Wu Zhihai, Yang Meiying
    2015, 31(7):  180-187.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.027
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    The strain Yj1 isolated and purified from the soybean soil could degrade soy lecithin and dimethoate. The 16S rDNA identification of the strain, optimization of growth conditions and examination of enzyme activities were performed and organophosphate degradation enzyme from Yj1 was separated and purified. The results revealed that the similarity of 16S rDNA in Yj1 and Serratia marcescens WW4(CP003959. 1)was 99%. The orthogonal experiment for the culture medium demonstrated that the optimal growth condition of Yj1 was the combination of mannose, peptone and pH8. The growth rate of Yj1 was poor in either of 2 phosphorus sources;however, the growth rate in soy lecithin was better than that in the dimethoate within 72 h. Moreover, the activities of acid phosphatase, alkaline phosphatase and organophosphate degrading enzyme(ODE)were higher when the soybean lecithin as the sole phosphorus source than those of dimethoate, and the activity of alkaline phosphatase was obviously greater than that of acid phosphatase and ODE within 72 h. ODE was successfully purified from Yj1 by combining ammonium sulfate precipitation and SP Sepharose Fast Flow. SDS-PAGE results showed that the purified protein was in a single band. The purified multiple by SP Sepharose Fast Flow was 5. 303 times as many by ammonium sulfate precipitation, and by the latter 1. 416 times as many by crude enzyme.
    Screening of a Strain Producing Acid-stable α-Amylase and Optimization of Its Fermentation Condition
    Qu Jianhang, Yin Yi, Jiao Guobao, Ding Changhe, Zhang Qian, Qu Lingbo, Liu Zhongmin
    2015, 31(7):  188-192.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.028
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    A strain SH3 producing acid-stable α-amylase was isolated from the soil nearing a starch factory. It was preliminarily identified as yeast. The pH range of crude enzyme reaction was 3. 8-8. 0 with the optimum of 5. 0, and the activity of the enzyme was still available in the temperature of 80℃ with the optimum of 50℃. The optimization of single-factor fermentation revealed that the optimal condition for the enzyme production was 37℃, pH 5. 0, soluble starch as carbon source, peptone as nitrogen source and 200 mL liquid volume. Orthogonal test showed that the optimal fermentation condition was as soluble starch of 15 g/L, peptone of 30 g/L, 37℃ and pH4. 5, under which the enzyme activity was 96.8 U/mg.
    Cloning,Expression and Characterization of Glucoamylase from Gliocladium roseum
    Zhang Liang, Hua Erbing, Wang Huaming
    2015, 31(7):  193-200.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.029
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    Glucoamylase from Gliocladium roseum was secretively expressed in Aspergillus niger G1 strain. A putative glucoamylase gene(about 1. 8 kb)was amplified by PCR using genomic DNA of G. roseum as template. The amplified products were ligated into the clone vector pGm to construct recombinant plasmid pGm-3440. The recombinant plasmid transformed into A. niger G1 strain, and amdS screening and PCR validation confirmed that an engineering Aspergillus strain of expressing glucoamylase was obtained. Fermentation of recombinant strain indicated that secretive expression of the glucoamylase gene was available in A. niger, and its enzyme activity was measured by method defined in national standard(QB/T 1803-1993), and it reached 292 U/mL. Further enzymatic analysis demonstrated that the optimum pH and temperature for the recombinant enzyme were 5. 0 and 50℃, respectively. Meanwhile, it had poor thermal resistance, but promising pH stability.
    Optimizing the Method of Acetone Powder to Extract Polyphenoloxidase from Honeysuckle Using Response Surface Methodology
    Qu Zheng, Luo Lei, Yang Bin, Kang Xinyan
    2015, 31(7):  201-206.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.031
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    In this study, the method of acetone powder was optimized to extract the polyphenoloxidase(PPO)from honeysuckle flowers(Lonicera japonica)by response surface methodology. The results showed that three operating parameters had great effects on the PPO specific activity, and their effects were ranked as follows:extraction time > buffer pH > material-to-liquid ratio. The established regression model was highly fitting(R2 = 99. 31%), and the interaction effect between buffer pH and material-to-liquid ratio was extremely significant. The optimal extraction condition was pH 7. 49, material-to-liquid ratio 1∶120 and extraction time 14. 5 min, under which the PPO specific activity was 137. 389 U/mg.
    Expression, Purification and Immunoprotection Potential of Pseudomonas aeruginosa Outer Membrane Lipoprotein I
    Chen Chunlin, Liu Xiang, Wang Chengxiang, Zhang Xiaojuan, Ju Xiong, Zhang Tao, Wu Sanqiao
    2015, 31(7):  207-213.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.032
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    Outer membrane lipoprotein I(OprI)in Pseudomonas aeruginosa has solid immunogenicity and holds prospects in the development of vaccine. The OprI-expressing strain was obtained by molecular cloning. Western blotting verified that antibody specifically combined with OprI. The purified OprI was gained by the SDS-PAGE gel slices. Immunizing mice with OprI, specific immunity activated by OprI protected 57. 14% mice from P. aeruginosa infection, which reached significant protection compared with the control group. The orthogonal experiment was employed to obtain the optimal inducing expressing condition of OprI strain:strain OD600 value,IPTG final concentration,inducing time and temperature,were 0. 8,0. 3 mmol/L,8 h,and 28℃,respectively;the optimal culture condition was that the rotation rate,glucose concentration,and medium volume were 230 r/min,0% and 50 mL,respectively. This work laid the groundwork for the researches on industrial fermentation,protein function and vaccine development of OprI.
    Construction of LSPA1 Early Gene gp22 Bacterial Two-hybrid Bait Vector and Screening Library
    Feng Mengdie, Mao Pujia, Hong Yu, Xu Zeyang, Zhao Jihua, Yang Hongwen, Song Wuzhan, Huang Fen, Jing Shenrong, Zeng Weikun
    2015, 31(7):  214-219.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.033
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    This study aims to construct a bait vector pKT25-gp22 and the gene library of Salmonella paratyphi A in bacterial two-hybrid system for further screening study. The gp22 gene was obtained by PCR amplification and then cloned into pKT25 to constitute the bait plasmid pKT25-gp22. The genomic DNA of S. paratyphi A was extracted and partially digested with Sau3AⅠ. The fragments between 250 to1 500 bp were re-natured and ligated to BamHⅠsite of pUT18C plasmid, and the genomic DNA library was obtained. The bait plasmid pKT25-gp22 and pUT18C were co-transformed to BTH101, and the self-activation of bait plasmid pKT25-gp22 and the toxicity of bait protein to the host bacteria were detected. The library plasmids were transformed to JM109 competence and the library diversity was identified by PCR. The results revealed that the bait vector was successfully constructed without self-activation of the transcription of reporter gene and non-toxic to the growth of bacteria. The genomic library covering 8 times of S. paratyphi A genome met the requirements of screening. In conclusion, the bait plasmid pKT25-gp22 and the genomic DNA library of S. paratyphi A were successfully constructed, which can be utilized in bacterial two-hybrid study.

    Cloning,Expression and Purification of Urease B Subunit of Helicobacter pylori
    Yan Jinjin, Yan Dongming, Zou Xue, Liu Dan, Peng Chao, Su Yanan
    2015, 31(7):  220-225.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.034
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    In order to obtain high-purity and bioactive urease B subunit protein(UreB), the fragments of ureB amplified by PCR was inserted into expression vector pET28a, and the recombinant plasmid pET28a-ureB was constructed successfully. The identified plasmid was transformed into Escherichia coli, which was induced to express by IPTG. The expressed protein UreB was purified by Q Sepharose High Performance anion exchange chromatography and desalted by G-25 gel filtration chromatography, then analyzed by SDS-PAGE and double immunodiffusion. The results indicated that the molecular weight of the protein was about 64 kD and its purity was 98. 5% after desalting, which was in accord with the prediction. Double immunodiffusion showed that protein UreB possessed a favorable bioactivity and specificity. The finalized purification process achieved the goal of 1-step purification may obtain high-purity and bioactive protein, and it was simpler than the existing purification process as well as effective.
    Expression, Purification, Crystallization of SPD1587 Protein from Streptococcus pneumoniae
    Huang Jian, Huang Meirong, Luo Shilu, Zhu Jiehua, Min Xun
    2015, 31(7):  226-230.  doi:10.13560/j.cnki.biotech.bull.1985.2015.07.035
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    SPD1587 protein is a transcription factor of streptococcus pneumoniae D39 strains, which plays an important role in nasopharyngeal colonization and lung infection. It has a conserved domain that was similar to transcription factors Mga of group A streptococcus, but the protein three-dimensional structure is unclear. In this study, the protein expression vector of PET28a-spd1587 was successfully constructed and a soluble form of SPD1587 protein was acquired in Escherichia coli BL21(DE3)strain. High purity SPD1587 protein was obtained by Ni-NTA affinity chromatography and DEAE negative ion exchange chromatography. The crystal of SPD1587 protein was obtained by the vapor diffusion method.
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