Table of Content

24 March 2016, Volume 32 Issue 3

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Review

Research Progress of the Physiological and Molecular Regulation Mechanism of Hevea brasiliensis in Response to Ethephon Stimulation

WEI Ming-ming, LI Wei-guo, GAO Xin-sheng, HUANG Xiao

2016, 32(3):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.002

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Application of the ethephon stimulation, as an important means to increase the yield of rubber tree, has been widely used in the production of natural rubber. However, the regulatory mechanism of rubber tree in response to ethephon stimulation is still unclear, which has become one of problems that need to be solved urgently in developing the new rubber tree stimulation agent and breeding new rubber tree varieties with ethephon stimulation resistance. This paper summarized such researches in recent years, the genes related to the ethylene signal transduction pathway of rubber tree, the biological regulation mechanism of rubber tree in response to ethephon stimulation, and the side effects of ethephon stimulation to rubber tree, aiming to provide references for deeply studying the molecular mechanism of the rubber tree in response to ethephon stimulation.

Research Progress on Regulatory Function of Bacterial Small RNA Resistance to Nutritional Stress

FU Zhu-qing, YU Yun-mei, WEN Xiao-jun, YUAN Wei-xi, ZHAO Zu-guo

2016, 32(3):  12-17.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.003

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The survival environment of bacteria is complex and volatile, and it is usually in a state of over-nutrition or denutrition. Therefore, bacteria only are able to survive by using sophisticated regulation system to timely and accurately adjust its gene expressions to adapt to the changing nutritional environment. Bacterial small RNA(small RNA, sRNA)playing an important role in regulating expression of bacterial gene in response to nutrient stress, is a class of RNA regulators discovered in bacteria in recent years. This article reviews the characters and the functions of bacterial small RNA resistance to nutrient stress.

Research Progress on Expression Regulation Mechanism of Genes Encoding Granule-bound Starch Synthase in Plants

MIAO Hong-xia, SUN Pei-guang, ZHANG Kai-xing, JIN Zhi-qiang, XU Bi-yu

2016, 32(3):  18-23.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.004

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Granule-bound starch synthase(GBSS)is a key enzyme to determine the amylose synthesis of plants. In monocots, GBSS includes two isoenzymes, designated as GBSSI and GBSSII. Dicot plants contain only one of GBSSII isoenzyme. The expression of gene GBSSI mainly controls the amylose synthesis in the storage organs such as seeds, embryos, and endosperms etc, while the expression of gene GBSSII mainly controls the amylose synthesis in the vegetative organs such as roots, stems, and leaves etc. In this paper, the latest research progress on expression regulation mechanism of GBSS genes in model plants and crops was reviewed. These results are expected to provide a reference for the study of GBSS genes from other plants.

Research Progress of Efficient Expression and Optimization of Production of Antibacterial Peptide

YANG Ping, YUAN Yi-hao, YANG Xiao-li, ZHONG Yue, LIAO Yu-xin, GAO Rong

2016, 32(3):  24-30.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.005

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Antimicrobial peptides(AMPs), a class of polypeptides induced by external conditions, are encoded by specific genes. In the era of facing the issue of “drug resistance” caused by antibiotics, AMPs own huge potential of benefits to mankind and replacing the antibiotics due to their characteristics of broad-spectrum antibacterial activity, scarce generation of drug-resistance, and antitumor virus, etc. However, the antibacterial activity and instability of natural AMPs should be enhanced, their toxicity should be reduced, and the large-scale production may be achieved by improving the industrial process. Therefore, the recent researches on the molecular modification and design, production and application of AMPS are summarized in detailed here.

Off-target of CRISPR/Cas9 System

YIN Shen, HE Gui-fang, LAI Fang-nong, XIE Feng-yun, MA Jun-yu

2016, 32(3):  31-37.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.006

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As an adaptive immune system discovered in bacteria, clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology has been used as the most effective genome-editing tool. By now, CRISPR/Cas9 has shown a great promise in healing human genetic diseases, however, off-target is a critical issue while applying it. The off-target of CRISPR/Cas9 leads to the false phenotype and wrong interpretation. Optimization of on-target activity and minimizing off-target activity will be challenges while CRISPR/Cas9 is applied in the future. In this review, we focused on the off-target related issues, including factors involved in the target specificity, strategies and tools of minimizing the off-target of CRISPR/Cas9.

Technique

Application of Electron Microscopy Technology in the Research of Plant Diseases

MA Dan-dan, DENG Yu-qing, ZHOU Yan, ZHOU Chang-yong, LI Zhong-an

2016, 32(3):  38-43.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.007

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Electron microscopy(EM)is the most important invention in the 20th century. The EM plays a great role in detection of the plant disease and observation of ultrastructure and morphology because of its high resolution. The principles of electron microscopes commonly used at present and their applications in the plant disease research were introduced in this article, including transmission electron microscope(TEM), scanning electron microscope(SEM), environmental scanning electron microscope(ESEM), scanning tunneling microscope(STM)and atomic force microscope(AFM). This article is aimed to provide reference for the better utilization of electron microscopy.

Research Progress on Enzymatic Synthesis of RNA

WANG Jing, PAN Xiao-ming, LIU Yu, LIANG Xing-guo

2016, 32(3):  44-51.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.008

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With the rapid development of RNA related studies, RNA synthesis technology is further challenged from two aspects of quality and quantity. At present there are mainly two methods for RNA synthesis, chemical synthesis and enzymatic synthesis. By the commercialized chemical way, the synthesis of RNA with < 90 bases is achieved；however, the cost of the synthesis is relatively high, which leads the initiation of many researches in difficulty. Compared to the chemical way, the enzymatic synthesis presents the characteristics of the high efficiency and mild reaction conditions, and by it a long sequence of RNA can be synthesized, thus, it is an efficient and low-cost measure for RNA synthesis. The enzymatic production of RNA is in two steps, transcription and processing of initial transcripts. The transcription may be further classified into the linear transcription and the rolling circle transcription model, with which the characteristics of initial transcripts vary, and the corresponding processing methods should adapted to the varied initial transcripts, thereby, the accurate target RNA products may be obtained. Recently, as the exploration of enzymatic synthesis continues, many novel transcription and processing strategies have been developed. In this review, the mechanisms, characteristics and problems of the transcription models and processing methods in enzymatic synthesis were summarized, aiming at providing the references for further study and selective utilization of enzymatic synthesis of RNA.

Improving the Percentage of Exerted Stigma in CMS Lines of Japonica Hybrid Rice by Molecular Marker-assisted Selection

CAI Zhi-jun, LI Jin-jun, ZHOU De-yin, FU Hao-wei

2016, 32(3):  52-57.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.009

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It is the most effective way to improve the yield of seed production of Japonica hybrid rice by enhancing the percentage of exerted stigma in its CMS lines. In this research, QTLs(qPES3, qPES9, qPES12), regulating the percentage of exerted stigma in indica rice, were transmitted into CMS lines of Japonica hybrid rice through crossing and backcrossing, aiming at selecting the CMS lines of Japonica hybrid rice with high percentage of exerted stigma by pedigree breeding and molecular detection technique, i.e., exploring the feasibility of improving the percentage of exerted stigma in CMS lines of Japonica hybrid rice by molecular marker technique. The research results showed that Jialong17A and Jialong43A, two CMS lines developed in this study, carried both qPES3and qPES12. The phenotypic values of percentage of exerted stigma and outcrossing rate indicated that QTLs expressed in Japonica rice, but displaying difference in various genetic background. Conclusively, molecular marker-assisted selection(MAS)is an efficient method to improve the percentage of exerted stigma in CMS lines of Japonica hybrid rice.

A Simple Method for Preparing PCR Template of Sugarcane Leaf Tissue

CUI Xue-qiang, ZHANG Shu-zhen, FENG Cui-lian

2016, 32(3):  58-62.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.012

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Using transgenic sugarcane leaves as materials, a small amount of young leaves were treated by alkali and transient heat, then neutralized, and cracking mixture was formed. The mixture was directly used as the template of PCR, and transgenic sugarcane exogenous gene of bar, KP4, CryIAc-2A-gna and endogenous gene Shactin as target genes, the amplified results were stable, accurate and reproducible, which reached the same effect of DNA amplification by conventional CTAB method. The templates by this method were still stable at room temperature for 2 weeks and at 4℃, -20℃ for 1 month. A series of exogenous gene PCR reactions were tested, which verified that this method was widely available in the detection of transgenic sugarcane. The method is fast for preparation of PCR templates without DNA extraction process, owing the advantages of requiring less material sample, low cost, simple, rapid, and efficient.

On the Rapid Propagation Technique of Stem Segments of Dendrobium officinale

OUYANG Fan, DONG Wen-bin, FU Yu, FAN Cheng, LI Yu-hong

2016, 32(3):  63-67.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.010

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A rapid propagation system was established by tissue culture in order to provide raw materials for better use and development of Dendrobium officinale. Using stem segments of D. officinale as explants, effects of the differential kinds and concentrations of plant growth regulators and natural additives on the induction of adventitious shoots growth and proliferation of cluster shoots were studied；and the optimal mediums at different culture stages were selected. Results showed that the optimal hormone ratio for induction of adventitious shoots was NAA 1.0 mg/L and BA 2.0 mg/L, a combination of NAA 0.5 mg/L, BA 2.0 mg/L, banana juice 100 g/L and activated carbon 100 g/L was the optimal medium for the proliferation of cluster shoots. It is feasible to obtain the efficient and stable plantlets by establishing high efficiency and rapid propagation system from stem segments of D. officinale.

On the Effect of Treating Petroleum-contaminated Soil by Different Bioremediation Technologies

SUN Xian-feng, YANG Bo-bo, ZHU Xin-jie

2016, 32(3):  68-72.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.011

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In order to explore the effect of bio-remedying petroleum-contaminated soil, the biological stimulation and biological enhancement technologies were employed to study the changes of petroleum hydrocarbon component, surface tension, the quantity of microorganism, and enzyme activity index in the remediation process. The results showed that biological stimulation was better than biological enhancement in the degradation of saturated hydrocarbon, the reduction of the surface tension, increasing the quantity of microorganism, and the improving enzyme activity；while biological enhancement degraded refractory aromatic hydrocarbon better than biological stimulation, each of them had its own advantages.

Research report

On Application of Herbicide Resistant Gene bar and EPSPS in Transgenic Sugarcane

WANG Wen-zhi, YANG Ben-peng, CAI Wen-wei, XIONG Guo-ru, FENG Cui-lian, WANG Jun-gang, WU Yuan-li, SHEN Lin-bo, ZHANG Shu-zhen

2016, 32(3):  73-78.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.012

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In this research gene bar and EPSPS were transferred into sugarcane cultivar via agrobacterium-mediated method, and the transgenic sugarcane lines resistant to glufosinate-ammonium and glyphosphate were obtained. Directly spraying two herbicides at application concentration for field onto the field planting the transgenic sugarcane lines demonstrated the genetic stability of exogenous gene in generation T0, T1, ratoon 1, and 2 of them, and the lines had the stable herbicide-resistant ability. Therefore, the transgenic sugarcane lines resistant to herbicide can be widely used in the field production as the other transgenic herbicide-resistant crops such as corn, soybean, rape and cotton.

Subcellular Localization and Expression Analysis of Hexose Transporter Gene ShHXT6 in Sugarcane

MA Xiao-wen, ZHAO Ting-ting, WANG Jun-gang, ZHANG Shu-zhen, YANG Ben-peng

2016, 32(3):  79-86.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.013

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The gene ShHXT6 of hexose transporter(HXT)of sugarcane was cloned with the specific primers designed according to the homologous species in NCBI. The cDNA of ShHXT6 was 1 929 bp, encoding 460 amino acids. The predicted molecular mass was 53.87 kD, and pI was 9.44. The deduced amino acid of ShHXT6 with the HXT from Zea mays, Setaria italica and Oryza sativa was in consistence by 80.85%, 76.89% and 72.86%, respectively. Phylogenetic tree analysis showed that the consistency of ShHXT6 amino acid and maize HXT6 protein was very similar. The expression of ShHXT6 was high in the immature tissues, especially in the immature leaves, but nearly no expression in the roots. The transient expression vector of this gene was constructed, and the subcellular localization of protein encoded by ShHXT6 was determined by the method of Agrobacterium injection into onion epidermis, and the results showed that the protein encoded by this gene was located in the cell membrane.

Isolation and Induced Expression of Ethylene Transcription Factor Gene CaERF18 from Capscium annuumm

ZHENG Jing-yuan, LIU Feng, ZHU Chun-hui

2016, 32(3):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.014

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To clarify the candidate genes and expression of one ERF transcription factor in pepper(Capsicum annuum L. HDA149)during the incompatible interaction between HDA149 and Meloidogyne incognita, the core sequence of an ethylene transcription factor gene CaERF18 was isolated from pepper induced by M. incognita with RT-PCR. The cDNA core sequence was 694 bp, encoding 231 amino residues containing a conserved ERF motif of 59 amino acids that shared 44.3%, 42.6% and 39.3% identities with the motif of NtERF118, SlERF4 and SlERF19, respectively. The expression analysis revealed that the expression of CaERF18 was significantly induced by M. incognita, the relative expression level of CaERF18 began to increase at 12 h with 3.8 times, and 7.2 times(peak)of the control at 72 h after inoculation with M. incognita. The expression of CaERF18 was strongly up-regulated by the nematode inoculation, which implied that the gene might play an important regulatory role in the resistant interaction of C. annuum to nematode.

The Mechanism of Lentinan Enhancing the Resistance of Cucumber Seedlings to Colletotrichum orbiculare

ZHANG Wen-hua

2016, 32(3):  93-97.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.015

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This study is to investigate the inducing effect of lentinan on the defense responses of cucumber(Cucumis sativus L. cv. Jinfeng)seedling leaves. The seedlings were treated with 0.75 g/L of lentinan. The concentration of H₂O₂ and salicylic acid in cells of leaves increased rapidly and reached the peak levels at 10 min and 4 h, respectively. Afterwards, the levels of both H₂O₂ and salicylic acid gradually declined to the ones before treatment. Moreover, the activities of defense related proteins, including β-1, 3-glucanase, chitinase, and phenylalanine ammonia lyase, significantly increased after lentinan induction, and still kept stable after 96 hours. In addition, the resistance of cucumber treated with lentinan to Colletotrichum orbiculare was notably enhanced after lentinan induction. Both the number and area of lesions on cucumber leaves significantly decreased to 53.1% and 51.3% in comparison with the control, respectively, which indicated that lentinan played a role in inducing the defense responses of cucumber cells.

cDNA Cloning, Sequence Analysis and Tissue Expression of Gene MSH4 and MSH5 in Goats

ZHENG Jie, LIU Shuang, LUO Bin, HU Liang, YANG Ke-wei, ZI Xiang-dong

2016, 32(3):  98-104.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.016

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The total RNA of ovary, endometrium, oviduct and pituitary extracted from 5 Tibetan goats and 5 Jintang black goats in the period of estrus were used to clone and analyze cDNA sequences of gene MSH4 and MSH5 by RT-PCR, and their expression levels in different tissues were determined by real-time PCR. The result showed that the coding region of gene MSH4 from Tibetan goat and Jintang black goat were 2 499 bp, encoding 832 amino acids；there were 5 base variations between two breeds, leading to 3 differences of amino acid. The coding region of gene MSH5 from Tibetan goat and Jintang black goat were 2 496 bp, encoding 831 amino acids；there were 9 base variations between two breeds, leading to 5 differences in amino acid. The nucleotide sequences of coding region of gene MSH4 from Tibetan goat were 99.8%, 99.8%, 99.4%, 98.1%, 94.4%, 85.1%, 84.7% and 93.5% homology with that of Jintang black goat, Capra hircus, Ovis aries, Bos taurus, Equus caballus, Mus musculus, Rattus norvegicus, and Homo sapiesn, respectively. The nucleotide sequences of coding region of gene MSH5 from Tibetan goat were 99.6%, 99.6%, 97.3%, 88.0%, 85.8%, 85.3% and 90.2% homology with that of Jintang black goat, C. hircus, B. taurus, Canis lupus familiaris, M. musculus, R. norvegicus, and H. sapiens, respectively. The mRNA of gene MSH4 and MSH5 were all expressed in ovary, endometrium, oviduct and pituitary of two breeds of goat, but there was no significant difference between two breeds(P>0.05). The results reveal that both gene MSH4 and MSH5 are conservative in the course of animal evolution, and the role of their effect on prolificacy of goats needs to be further studied.

Expression of microRNA-483 and microRNA-486 in the Cloned and fat-1-transgenic Bovine

Lü Yang, WANG Yu, SUN Jia-jia, GONG Chun-ling, LI Guang-peng

2016, 32(3):  105-108.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.017

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The expression levels of microRNA-483 and microRNA-486 in heart, liver, spleen, lung, kidney, placenta, cotyledons, endometrium from normal, cloned, and transgenic bovine were measured by real time quantitative PCR. The results showed that microRNA-483 and microRNA-486 were expressed in all tissues of normal, cloned and fat-1-transgenic bovine；significantly higher in the heart than other tissues. However, the expression levels of microRNA-483 and microRNA-486 in transgenic bovine were lower than normal one. Further, the expressions of microRNA-483 and microRNA-486 varied in different tissues and high in the heart, indicating that the expressions of microRNA-483 and microRNA-486 may be correlated with pathophysiological process of myocardial hypertrophy and myocardial infarction.

TGF-β1-induced Differentiation of Wapiti Antler Mesenchymal Stem Cells to Cartilage and Expression Profile of Gene c-myc

HAN Chun-mei, WANG Shan-shan, GAO Qing-hua, ZHENG Yong-fu, MA Meng-ting, ZHANG Qin

2016, 32(3):  109-114.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.018

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Antler mesenchymal stem cells(MSCs)play a pivotal role on the antler regeneration and ossification. To investigate the chondrogenic differentiation of antler MSCs and the regulation role of the proto-oncogene c-myc in this process, the second passage cells(MSCs, P2)of 60 d antler from adult Tarim wapiti were induced to chondrogenesis by the stimulation of transforming growth factor TGF-β1(10 ng/mL)in vitro. The inducing effects were identified by Alcian blue staining and immunohistochemics, and the expressions of gene c-myc during this process were detected by qPCR. The results demonstrated that, on the day 9 after induction, some MSCs begun to change from spindle-shaped to rounded or polygon, and the chrysanthemums-shaped pattern of the original MSCs gradually changed to paving stone like；the cartilage capsules were observed on the day 14, and the cartilage extracellular matrix was obvious and cell apoptosis appeared on the day 21. While the cells in the control group were observed to have apoptosis on the day 28, and the cavitations were discovered in the cells. On the day 35, massive cell apoptosis of both groups were observed, and the cell refraction became weak and the gaps between cells became larger. The identification by Alcian blue staining revealed that heavy positive staining emerged in the cellular matrix from the day 14 after stimulating. The detection by immunohistochemistry demonstrated that positive brown Col II reactant was in the cellular matrix from the day 21 after stimulating, and the color became darker along with the culture time, mainly distributed in the cells and their surround matrix. In the cartilage differentiation process from the day 7 to 28, the expressions of gene c-myc in the cells of the induced group were significantly lower than that of control group(P<0.05), while there was no significant difference after the day 35(P>0.05). In conclusion, Tarim wapiti antler MSCs differentiated into cartilage under the stimulation of TGF-β1. The down-regulated expression of proto-oncogene c-myc induced the apoptosis of antler MSCs and then differentiation to chondrogenic cells.

Cloning, Bioinformatic Analysis and Expression Patterns of Chaperonin Gene CCTδ from Musca domestica

TAO Ru-yu, ZHAO Xue-jun, YANG Yu-jin, WU Jian-wei, GUO Guo

2016, 32(3):  115-121.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.019

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This work aims to clone, have bioinformatic analysis and explore the expression patterns of chaperonin gene CCTδ from Musca domestica. We screened and isolated the gene CCTδ from the built cDNA plasmid library of M. domestica larva by EST sequencing, and having the plasmid as template, the amplification was conducted by PCR. Further, with bioinformatics we analyzed the structures and functions of gene CCTδ and the encoded protein, and constructed phylogenetic tree. We collected the bodies at different developmental stages of M. domestica life history(eggs, varied-instar larvae, pupae, female and male adults), and chose 6 tissues of 3-instar larvae(body wall, trachea, salivary glands, fat body, markov tube, and midgut), thus the expression patterns of gene CCTδ at different developmental stages and tissues of M. domestica were detected by real-time quantitative PCR. An about 1 600 bp specific fragment of housefly’s gene CCTδ was acquired by PCR amplification. The open reading frame of gene CCTδ was 1 602 bp that encoded a putative protein with 533 amino acids. The predicted molecular weight of the protein was 57.16 kD and pI was 7.50. The secondary structures were mainly composed of α-helix and random coil. Comparative analysis of the phylogenetic tree showed that it was conservative, and the genetic distance was closer with Anopheles darlingi. The spatial and temporal expression patterns of gene CCTδ revealed that gene CCTδ expressed in each developmental stage of M. domestica, while the highest at pupa stage；and it expressed the highest in trachea among various tissues of 3-instar larvae. Conclusively, in this study, the gene CCTδ of M. domestica was successfully cloned, and the spatial and temporal expression patterns of gene CCTδ were preliminarily explored.

Expression of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus(DpCPV1)Non-structural Protein p44 in Bac-to-Bac System and Localization in Infected Cells

PENG Han, WANG Hong-xiu, WANG Jin-chang, GUAN Li-mei, JIN Liang, WAN Cui-xiang

2016, 32(3):  122-128.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.020

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In order to study the function of the Dendrolimus puntatus cytoplasmic polyhedrosis virus(DpCPV1)protein p44, PCR primers were designed according to the sequence of genome segment S8, the prokaryotic expression vector for genome segment 8 of DpCPV1 was constructed, and the polyclonal antibodies were prepared by immunizing rabbits with the purified expressed protein. Three recombinant plasmids(Bacmid-p44, Bacmid-p44-eGFP and Bacmid-eGFP)were constructed using Bac-to-Bac Baculovirus expression system., and they were transfected into insect cell Sf9 for the expression. The detection and subcellular localization of the protein were determined by Western blot. The results from Western blot showed that the actual expressed protein of Bacmid-S8 in Sf9 was 35 kD, smaller than the one(44 kD)expressed in the prokaryotic expression vector. The subcellular localization of fusion protein of p44-eGFP was determined by laser scanning confocal microscope, and the fused green fluorescent protein(eGFP)of p44 concentrated in the cytoplasm of the cells, while infused eGFP distributed throughout the whole cell, indicating that p44 protein of DpCPV1 was localized mainly in the cytoplasm of the cell. This work was the first study of having the eukaryotic expression of DpCPV1 S8 and subcellular location of it in insect cells,

Cloning and Biological Characterization of Aquaporin 3 from Lampetra japonica

ZHAO Chun-hui, SHI Jin-jie, WANG Hao, LIU Xin, LI Qing-wei

2016, 32(3):  129-136.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.021

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Aquaporin 3(AQP3)belongs to the family of aquaglyceroporin, and it involves in the transport of water, urea and glycerol and promotes cell proliferation and migration. In this study, the complete cDNA sequence of Aqp3(designated as LjAqp3)was cloned from Arctic lamprey Lampetra japonica(GenBank accession number KR054618). The open reading frame of LjAqp3 was 900 bp, encoding 299 amino acids, with a predicted 6 transmembrane helix domains and a putative signal peptide. There were 2 highly conserved asparagine-proline-alanine(NPA)motifs and the aromatic/arginine(ar/R)structure in LjAQP3. Additionally, LjAQP3 shared high sequence homology with AQP3 of other species. Real-time quantitative PCR revealed the constitutive expression of LjAqp3 in multiple tissues and organs.

Preparation and Application of cRNA Probe Labeled with Digoxingenin for Gene F64 of Rice Field Eel

JIANG Jiao-yun, TIAN Wen-fei, FENG Long, QU Xian-cheng

2016, 32(3):  137-141.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.022

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The specific gene primers were designed based on the sequences of gene F64 of rice field eel, then the templates for synthesizing the cRNA probe were amplified, then recombinant F64/pGM-T plasmids were constructed and linearized by restriction enzymes, further the sense and anti-sense cRNA probes were synthesized by in vitro transcription with RNA polymerase, and finally labeled with Digoxigenin. The expression changes of gene F64 during the gonadal development of rice field eel were detected by in situ hybridization. According to our results, the expression was only detected by the anti-sense F64 cRNA probe, and the earliest signal was observed in stage V of ovarian, but no more in early stage of ovarian formation. Our study indicated that the cRNA probes can be synthesized using in vitro transcription, and the cRNA probes prepared in this study can be used for accurately detecting the spatial and temporal expression of gene F64.

Detection of Quorum Sensing Signal Molecules in Vibrio anguillarum Isolated From Litopenaeus vannamei

PAN Yu-rong, ZHANG Cai-li, ZHU Su-qin, SUN Xiu-jiao, ZENG Ming-yong

2016, 32(3):  142-147.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.023

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The aim of the present study was to detect and analyze quorum sensing of Vibrio anguillarum(VA)isolated from spoiled Litopenaeus vannamei. AHLs activities of VA were detected by reporter strains(Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136)combined with TLC method. Report strains V. harveyi JMH597 and V. harveyi JAF375 were used to detect the activity of AI-2 and CAI-1. The result indicated that AHLs(3-OH-C₆-HSL, C₈-HSL and 3-oxo-C₈-HSL)、AI-2 and CAI-1 were secreted by VA andthe activity of the signal molecules were density-dependent.

Development of a Lentivirus Vector-based Vaccine Carrying Follicle-stimulating Hormone Receptor and Assay of Its Immunological Effect

MA Xiao-ling, LIU Hong-chun, LI Jiang-wei

2016, 32(3):  148-154.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.024

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This work aims to prepare lentivirus vaccine with a follicle-stimulating hormone receptor(FSHR)and investigate the immunological effect of it on mice. The gene(fshr366)of extracellular region of FSHR was cloned into lentivirus vector. The recombinant plasmids were transfected into the 239T cells by the method of lipidosome transfection, and the virus particles(Lenti-FSHR366)with target gene were produced after encapsulation. The expressions of fshr366 mRNA and FSHR366 protein in 293T cells infected by Lenti-FSHR366 were detected by RT-PCR and Western blot. For evaluating the immunological effects, BALB/c mice were intraperitoneally inoculated with single immunization of fshr366-carring virus, collecting the blood samples from the orbits of mice at day 0, 14, 21 and 28 after immunization, then ELISA method was used to detect the specificity of the sera from immunized mice and determine the titer of the antibody. The RE digestion and DNA sequencing results showed the gene fragment of fshr366 was successfully cloned into lentivirus vector. After transfection to 293T cells with the encapsulated lentivirus particles carrying fshr366, the detection results by RT-PCR and Western blot indicated the expression of gene fshr in the cells at transcription and protein level. The results of ELISA revealed that the humoral immune response in mice rose on day 14 after single immunization through intraperitoneally injection of lentivirus particles carrying fshr366, the titer of antibody was 1:1600.

Prediction of Optimal Growth Temperature of Bacterium Based on the Homologous Proteins

CONG Hua-jian, WU Shuan-hu, TIAN Jian, CHU Xiao-yu, WU Ning-feng

2016, 32(3):  155-165.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.025

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The optimal temperature for each bacterium differs, which is related to its gene sequence. In order to explore the correlation between them, the known genome sequences of 92 bacteria with own different optimal temperatures were selected as the study material, then the common homologous protein from 92 bacteria were searched, and frequencies of the amino acids in homologous protein were calculated. A significant correlation between the frequency of the amino acid in homologous protein and the optimal growth temperature was realized. The analysis of the sites in homologous genes showed that the helix regions in the protein sequence were the most correlated with its optimal growth temperature. This study presents important significance on understanding the mechanism of the bacterial adaption to the temperature as well as designing the mutation to improve the protein stability.

The Expression and Immunoreactive Analysis of Recombinant Human C-reactive Protein in Different Prokaryotic Expression Vectors and Strains

LI Jiang-feng, JIAO Li-yuan, ZHAO Xiao-ni, WANG Ji-hua, CAI Lei

2016, 32(3):  166-170.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.026

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The prokaryotic expression vector of human C-reactive protein(CRP)was constructed and transferred into Escherichia coli BL21(DE3)and Rosetta gami2(DE3)pLysS, respectively, and then these recombinant strains were induced for the expression of CRP. Mainly gene engineering, affinity chromatography and dialysis refolding methods were employed. The expression vectors and recombinant strains of human CRP were constructed successfully, and the varied CRPs with different protein tags were acquired by IPTG. In conclusion, expressions of the same vector in different strains differed；it expressed in the E. coli BL21(DE3)and Rosetta gami2(DE3)pLysS respectively, and the inclusion bodies of recombinant CRP were obtained. The results of detecting the refolded recombinant CRP by Western blotting and ELISA revealed that the immunoreactivity of recombinant CRP in the prokaryotic expression vector was low, and CRP may have the immunoreactivity while it is in the pentamer form.

The Optimization of Enzyme Complex Formulation for Enzymatic Hydrolysis of Corn Stalk

WANG Qi, WANG Lin-feng, YAN De-ran, ZHANG Fei-yang, LIU Tian-tian, WU Jing-bo

2016, 32(3):  171-177.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.027

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Reducing the cost of enzymatic hydrolysis is the key step in bio-ethanol production from cellulose. In order to enhance the conversion of cellulose into ethanol, the enzymatic hydrolysis condition of corn stalk pretreated by steam explosion was optimized by enzyme complex formulation. The affecting rules of cellulase, xylanase and β-glycosidase on enzymatic hydrolysis efficiency were studied by single-factor experiment and orthogonal test. The results showed that the cellulose content of steam-exploded corn stalk reached 42.21%, and the hemicellulose content was only 3.65%. Cellulase was crucial to the enzymatic hydrolysis process, the enzymatic hydrolyzation yield was 75.45% when added by 40 FPU/g. Xylanase promoted more cellulose exposed, enzymatic hydrolyzation yield reached 78.03% when added by 1 500 IU/g. β-glucosidase was conducive to eliminate the feedback inhibition caused by cellobiose, when dosage was in 40 IU/g, the concentration of cellobiose was 0.330 4 g/100 mL, enzymatic hydrolyzation yield reached 76.45%. The optimal conditions for the process by orthogonal experiment were as follows：cellulase 30 FPU/g, xylanase 800 IU/g, and β-glucosidase 40 IU/g, under this and when the substrate concentration was 25%, glucose was up to 9.3 g/100 mL, i.e., increased by 57.63% compared to using a single cellulase while the glucose was only 5.9 g/100 mL. The affecting significance of three enzymes was ordered as follows：cellulase > xylanase > β-glucosidase.

Promotion of Soluble Expression of Laccase in Escherichia coli by Fusion of His-tag or S-tag on the N-terminal of It

YUE Qing-xia, ZHANG Li-jie, TIAN Jian, WU Ning-feng, YAO Dong-sheng

2016, 32(3):  178-183.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.028

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Laccase from Bacillus licheniformis has the advantages of high catalytic efficiency, wide range of substrates, etc., thus it owns the broad application prospect in industrial and agricultural fields. However, the expression level of the enzyme in the foreign gene expression system is low, which greatly limits its application in the agricultural and industrial fields. His-tag or S-tag is the small peptide with low molecular weight and usually can not affect the characteristics of heterologous fusion protein, and they are beneficial to be applied in the purification and detection of heterologous proteins. In this study, fusion of His-tag and/or S-tag on the N-terminal of the laccase CotA from B. licheniformis was constructed into pET-22b vector, and the vector was transferred into Escherichia coli BL(DE3), then we found that the soluble expression level of laccase significantly increased in E. coli BL(DE3). Compared with the construction without no fusion of any tags on N-terminal, the expression level with His-tag was about 37 folds, 20 folds with S-tag, and 28 folds with both His-tag and S-tag, respectively.

Prokaryotic Expression, Purification and Activity Analysis of Recombinant Carboxypeptidase G2

LI Shu-gang, WANG Yong, ZHANG Wei, XIN Yu, DAN Guo-ping, CHAI Xin-juan, GONG Hui-ying, YU Ting-he

2016, 32(3):  184-189.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.029

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This work is to express carboxypeptidase G2(CPG2)in prokaryotic expression system, purify it and detect its activity in vitro. The gene fragment encoding CPG2 was amplified by PCR, and the prokaryotic expressed plasmid pET-30a-CPG2 was constructed by gene recombinant technology, then it was transformed into Escherichia coli BL21(DE3)for the expression with induction. The target protein from grounded cells was purified by metals chelating affinity chromatogram and anion-exchange chromatography. Majority of the fusion protein was expressed in soluble form, and its specificity was confirmed via SDS-PAGE and Western blotting. The preliminary experimental result showed that the recombinant protein degraded MTX, and the specific activity reached 400 U/mg.

Optimization of Expression Conditions of Recombinant Bovine Pancreatic Carboxypeptidase A in Pichia pastoris by Response Surface Method

WANG Xiao-yun, XU Shi-han, LIANG Zhi-hong, HUANG Kun-lun

2016, 32(3):  190-197.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.030

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Central composite design(CCD)and response surface methodology(RSM)were used to optimize the expression conditions of bovine pancreatic carboxypeptidase A. The results of single-factor test showed that the optimal conditions as following：the inducible expression time was 96 hours, the methanol content was 0.5% every 24 h, the initial induction pH4, and OD₆₀₀ before induction expression was 7. Based on the above result, CCD was used to design the tests, and RSM to have regression analysis. The optimal parameters for the expression were obtained as：methanol content was 0.5% every 24 hours, the induction pH 5.90, OD₆₀₀ before inducible expression was 6.54, the expression level after 96 h reached 0.325 mg/mL. Compared with the theoretical value(0.326 mg/mL), the small relative error was 0.38%. Also, the expression of the target protein was confirmed by Western blot. Conclusively, using RSM for optimizing the expression condition of bovine pancreatic carboxypeptidase A was accurate and reliable, and conducive to seek the optimal condition, thus this lays a foundation for guiding the high-density fermentation production of bovine pancreatic carboxypeptidase A in fermenter.

Isolation and Identification of Microorganisms from Spoilage Vinegar

ZHAI Lei, SU Jiao-jiao, LIU Yang, CAO Yan-hua, YAO Su, CHENG Chi

2016, 32(3):  198-202.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.031

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The structure and diversity of microbial community in normal and spoilage vinegar samples were studied by PCR-DGGE technology combined with pure culture technology. By comparative analysis and tie-back test, we found that Lactobacillus acetotolerans was the polluting strain causing vinegar spoilage. This is the first domestic reports regarding that L. acetotolerans was the cause of the vinegar spoilage and provided theoretical basis for the enterprises of vinegar production to control the microbial pollution.

Function Analysis of Up-regulated Gene(URG11)Based on Its Bioinformatics

YAO Yang, SU Jie, LIU Kai-ge, Xu Rui

2016, 32(3):  203-208.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.032

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This work was to obtain differentially-expressed genes with hepatoma cell HepG­(HepG2-X)Y of stable transfected HBx and non-transfected hepatoma cell HepG­₂ using gene chip technology. The preliminary bioinformatic analysis demonstrated that the gene of the protein encoded 673 amino acids with a predicted molecular weight of 17.06 kD and pI 4.83. URG11 were mainly localized in the nuclear and played the role in transcription regulation, growth factor and signal transducer, and it showed 76%-97% identity with the others reported 12 species, as well as was consistent with the evolutionary relationship between species.

Expression of Protein Us11 and Preparation of Polyclonal Antibodies

TONG Hai-yan, LI Guo-yi, TANG Ying

2016, 32(3):  209-214.  doi:10.13560/j.cnki.biotech.bull.1985.2016.03.033

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The previous studies have discovered that protein Us11 of HSV-1(herpes simplex virus 1)played an important role in antagonistic host innate immune responses after HSV-1 infection. By synthesizing the gene Us11 of HSV-1, the polyclonal antibodies for Us11 were cloned and prepared, which provided the solid experimental basis for studying the functions of Us11 and the interactions between Us11 and host protein. The artificially-synthesized gene Us11 was as template and amplified, and then the recycled gene fragments of Us11 were cloned into the prokaryotic and eukaryotic expression vectors. Further, the induced expressed Us11 protein was purified, and polyclonal antibodies for rabbit were prepared. The recombinant plasmid was transfected into 293T cells；the expressed protein was detected by indirect immunofluorescence(IFA)and Western blot(WB). Plasmid pCold-Us11 and pFLAG-Us11 were successfully constructed, soluble Us11 protein expression was achieved, and the polyclonal antibodies targeting for Us11 was acquired by immunizing animal with purified Us11 protein. The results from IFA and WB revealed that prepared antiserum may specially detect the Us11 protein expressed in transfected 283T cells by pFLAG-Us11.

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Cover

2016, 32(3):  300. 

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Content

2016, 32(3):  400. 

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Copyright

2016, 32(3):  500. 

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