Table of Content

15 August 2014, Volume 30 Issue 8

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Research Advances on Phototropin Receptor and Phototropin Signaling Mechanism in Plant

Qiao Xinrong, Duan Hongbin, Ye Zhaowei

2014, 30(8):  1-7. 

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Phototropin（PHOT）is blue-light receptors found following the phytochrome and cryptochrome in plant. PHOT mediate phototropism，chloroplast movement，stomatal opening，leaf expansion and leaf positioning induced by blue-light in higher plants. Research on molecular mechanism of physiological response mediated by PHOT were highly focused in recent years. This paper reviewed research advances of structure characteristics of light sensitivity and signaling mechanism of Arabidopsis phototropin.

Research Progresses of Stress-induced Epigenetic Regulation Mechanism in Plant

Ran Liping, Kong Yueqin, Fang Tingting, Wang Youping

2014, 30(8):  8-15. 

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Plant as sedentary organisms, needs to adapt their gene activity to the adverse or stressful environmental challenges. Epigenetic regulation accompanies stressful environments, such as extreme temperature, drought, salinity, heavy metal, pathogen and hormones etc., which lead to the impressive development and phenotype variation of different plant species with adaptability to unfavorable conditions. In this paper, the current research status of epigenetic changes induced by stresses, including DNA methylation, histone post-translational modification, chromatin modification, non-coding RNA, as well as the interaction between these epigenetic incidences were reviewed.

Protein Phosphatases Ⅱ C in Plants are Involved in Abiotic Stress Tolerance of Several Signaling Pathways

Du Chi, Zhang Fuchun

2014, 30(8):  16-22. 

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Abiotic stress could cause a series of changes to plants in morphological, physiological, biochemical and molecular level. Especially the abiotic stresses would lead protein phosphorylase PP2C related gene expression change. At the same time, the proteins related biosynthesis induced by the abiotic stresses would improve plants resistance. However, the different PP2C through the different signaling pathways involved in abiotic stress. This article introduced the regulative mechanisms of PP2C mediated the abiotic stress signal pathways.

Tag Selection of Tandem Affinity Purification and Its Application in Plant Protein-Protein Interactions

Zhang Zhifei, Zhao Zhili, Wen Zhaozhu

2014, 30(8):  23-27. 

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Tandem affinity purification（TAP）is a technology for the purification of protein complex under the normal physiological conditions, which has been extensively utilized to identify protein-protein interactions（PPIs）and reveal interaction network of protein complexes. In recent years, TAP technology has been improving in the PPIs study, with the development of new labels, TAP method improvements and jointed use with other technologies associated. TAP technology is also being increasingly used in the studies of plant PPIs. This paper reviews the selection of TAP affinity tag and advance of research that TAP has been successful used in the study of plant PPIs.

The Current Situation and Prospects of Banana Chilling Stress

Wang Anbang, Jin Zhiqiang, Liu Juhua, Jia Caihong, Zhang Jianbin, Miao Hongxia, Xu Biyu

2014, 30(8):  28-33. 

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Most banana plantations in China belonging to the non-suitable growing areas often suffers from chilling stress caused significant economic losses. The banana chilling stress is one of the most principal natural disasters. The present paper reviewed the advances on such field as symptoms and types of chilling stress, physiological and biochemical research, chilling-resistant measures, cold resistance related genes in banana. The problems in banana chilling stress research and the prospect of the future research on banana chilling stress were simply discussed in order to provide references for chilling stress research and production.

Research Progress of Genes Related to Melanin Synthesis in Sheep

Meng Haohao, Xu Ruixia, Dai Rong, Li Hui, Li Liangyuan, Wan Pengcheng, Shi Guoqing

2014, 30(8):  34-39. 

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As a genetic marker， coat color plays an important roles in determining cross combination，breed purity and phylogenesis. Coat color of mammals is determined by the distribution and proportion of eumelanin and pheomelanin， which produced by melanocytes. This article summarized the biological functions and the genetic variation of Tyrosinase gene（Tyr）， Melanocortin 1 receptor gene（MC1R）， Agouti signaling protein gene（Agouti）and Tyrosinase-related protein gene（TYRP1）.

Advances of the Studies on Structure and Function of Promoter

Wang Jing, Li Bing, Liu Cuicui, Zhu Zhen, Zhang Jiyu

2014, 30(8):  40-45. 

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Gene expression and its regulation are dependent, on to a great extent, on transciption. An elementary transciption reaction is initiated by the recognition of regulatory regions and especially promoters. Promoters is the cis-regulatory element of gene expression and its regulation, exsit in eukaryotes and prokaryotes. This paper expounded on the general characteristics and the function of promoter segment and cis-element functions.

Research of 3C Protease Picornaviruses and Its Cleavage Substrates

Liu Yan, Li Bingqing, Meng Hong

2014, 30(8):  46-51. 

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Picornavirus is a big family of animal viruses, viral protein synthesis needs its own proteases’ cleavage to form sructural and functional proteins. 3C protease is one of picornaviruses’ own proteases. 3C can also cleave some host proteins to benifit viral replication. 3C protease’s structural characteristics, active center, cleavage sites and cleavage substrates’ functions are key of further understanding 3C protease mechanism. Here follows the review on these issues.

A Review of Development in WSSV Immediate-early Gene Research

Ran Xiaozhuo, Li Fang

2014, 30(8):  52-58. 

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White spot syndrome virus is one of the main pathogens which causes great damage to the shrimp aquaculture. During the infection, WSSV immediate-early genes（IE genes）utilize the host cell transcription regulators to increase their own transcription. Regulatory proteins encoded by IE genes are critical for the virus life cycle. The IE proteins may either regulate the expression of viral genes, or interact with regulators in the host cells to create a proper environment for viral replication. Here we reviewed the recent progress in WSSV IE gene research and especially focused on five most-studied WSSV immediate-early genes：ie1, wsv051, wsv083, wsv249 and wsv403. The function of IE proteins and the transcription regulation of their genes were discussed.

A PCR-RFLP Method for Channa argus and Channa maculate Discrimination

Dong Chuanju, Zhang Songhao, Chen Kunci, Song Yingnan, Xu Peng, Sun Xiaowen

2014, 30(8):  59-64. 

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In order to identificate Channa argus and C. maculate efficiently. We developed a PCR-RFLP method to conduct research in molecular biology. Briefly, two genomes of C. argus and C. maculate were aligned and compared, a SNP was identified which can discriminate two Channa species. A pair of primers was designed to amplify the mitochondrial DNA of both species, then digested by restriction enzyme EcoR I and test the digestion result by agarose gel of 1.5%. The result showed that the PCR product of C. maculate was digested by EcoR I that generate two fragments of 315 bp and 875 bp, while the PCR product of C. argus can not be digested by EcoR I. Thus, two Channa species were clearly discriminated.

Optimization of ISSR-PCR Reaction System of Anise（Illicium verum Hook. f.）Based on Orthogonal Design

Feng Dan, Ning Delu, Chen Shaoyu, Li Yongjie, Zhang Yanli, Wu Tao, Chen Haiyun

2014, 30(8):  65-69. 

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Based on orthogonal design experiments, five main factors in ISSR-PCR amplification system, including Mg²⁺ concentration, Taq DNA polymerase, DNA template, primer（UBC 868）and dNTPs, were studied to optimize the system for Illicium verum Hook. f.. The results showed that the optimized system was a total volume of 25 μL containing 3 mmol/L Mg²⁺, 0.5 U Taq DNA polymerase, 0.016 ng DNA template, 0.8 μmol/L primer and 0.2 mmol/L dNTPs. The optimal amplified procedure was started with predegeneration at 94℃ for 5 minutes, followed by 40 cycles of 30 seconds for denaturalization at 94℃, 45 seconds of annealing at 46℃, 60 seconds of extension at 72℃；7 minutes of extension at 72℃ and indefinitely remain at 4℃.

Comparison of Four Methods for Whole Genomic DNA Extraction from Bemisia tabaci

Dai Tianmei, Lü Zhichuang, Wan Fanghao

2014, 30(8):  70-75. 

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Obtaining high-quality DNA is a prerequisite to study the molecular mechanisms of many organisms. For tiny insects, low DNA concentration（from one single insect）is unable to meet the need of some experiments. To find a convenient and efficient method for whiteflies genome extraction, KAc, salting-out, chloroform-isoamylol and phenol methods were used to extract bull whiteflies genome DNA, respectively. The efficiency of different DNA extraction methods were evaluated by comparing the concentration, purity, PCR products of DNA, extraction time and the toxicity of the reagents, respectively. The results showed that the average concentration of DNA extraction of salting-out method was 521 ng/μL, and the purity could meet the experiment requirements, and the PCR product brand of MSAP primer amplification was bright. Compared to other methods, salting-out method was more simple, rapid and non-toxic. Therefore, salting-out method was the best ideally method to extract the whole genomic DNA from whiteflies.

Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis

Li Ji, Lu Xu, Huang Tiandai, Hua Yuwei, Huang Huasun

2014, 30(8):  76-81. 

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Southern blot technology based on the DIG-chemiluminescent detection has been widely used in numerous species. Using Hevea brasiliensis as materials, the DIG-labeled Southern blot analysis was improved through optimizing several key steps, including extraction of genome DNA, usage of DNA samples amount, digestion system, the comparison between PCR and random-primed labeled method and a serial of procedure in the process of the hybrid. The results showed that desirable digestion result could be achieved when at least 40 μg DNA samples with high quality were enzymed in 300 μL reaction system for 12 hours. The efficiency of probe labeled with the method of PCR was higher than that of probe labeled with random primer obviously, while the Southern blot result of PCR labeled was clear, and the background was lightly, which was conducive to read the copy number accurately. Finally, Southern blot with DIG-labeled was optimized, and good result with high sensitivity and low background was achieved．

Cloning and Sequence Analysis of Yak PAFR Gene and Expression in Uterus Before and After Pregnancy

Wei Bo, Pan Yangyang, Yu Yao, Tian Feng, Yu Sijiu

2014, 30(8):  82-88. 

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Platelet-activating factor receptor（PAFR）gene were cloned from uterus of domestic yak by reverse transcription polymerase chain reaction（RT-PCR）. The results shows that domestic yak PAFR gene was 1 029 bp in length, encoded predicted mature protein of 342 amino acids, which encoded a hydrophilic protein. Compared with the reference sequence of PCR primer designing homology of the domestic yak PAFR coding sequence was 99.7% at the amino acid level, and also showed high homology of with other species. The PAFR has 5 transmembrane domains, features G protein-coupled receptor domains, no signal peptide. The result of the quantitative Real-time Polymerase chain reaction（QRT-PCR）shows that PAFR was highest expression in gestation and lowest in follicular phase in uterus of domestic yak. That suggested PAFR play an important role in implantation and gestation.

Relative Quantification of mRNA Transcription of TLR3 and TLR5 in Different Tissues of Yak

Chen Yabing, Lan Daoliang, Lin Baoshan, Huang Cai, Huang Yong, Li Jian

2014, 30(8):  89-93. 

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According to the sequences of yak TLR3 and TLR5, the real-time PCR primers were designed to establish a method for the detection of mRNA transcription of TLRs by Real-time quantitative PCR, and the transcription level of TLRs in different tissues of yak was analyzed. The results showed that two genes have different expression pattern and expression level in tissues. The TLR3 mRNA was detected in examined tissues except mammary gland, and it was expressed at a higher level in heart, large intestine, stomach and muscle. The TLR5 mRNA level was higher in heart, lung, large intestine, small intestine, stomach, muscle and ovary. The results indicated that there exists difference in the transcription level of TLRs in tissues, which may be associated with the recognition of pathogens. For the same gene, the mechanism of gene may bring difference in expression level in different tissues.

Cloning and Bioinformatics Analysis of MAPK13 Gene in Lanzhou Fat-tailed Sheep

Jin Fangyuan, Xu Hongwei, Da Xiaoqiang, Zang Rongxin, Bai Jialin, Gao Xin, Cai Yong, Feng Yulan, Yang Jutian

2014, 30(8):  94-101. 

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The objective of this study is to reveal the biological function of mitogen-activated protein kinases（MAPK）gene in sheep and provide theoretical data for application by cloning MAPK and analyzing its sequence as well as genetic features. The specific primers on the CDS template of MAPK13 gene were designed and then the MAPK13 gene sequence of Lanzhou fat-tail sheep was cloned using RACE and RT-PCR technology. After that, its genetic characteristics were analyzed by bioinformatics methods. The 1 397 bp full-length cDNA of MAPK13 of Lanzhou fat-tailed sheep was obtained, in which the length of CDS is 1 102 bp. It encodes 367 amino acids in total. It was predicted that the molecular weight of MAPK13 protein of Lanzhou fat-tail sheep is 42.29 kD and its theoretical isoelectric point is 8.82. It is a non-transmembrane hydrophobin with no signal peptide, which locates mostly in cytoplasm by the result of subcellular level prediction and does not belong to the secretory protein. Its amino acid sequence contains 19 phosphorylation sites, 3 glycosylation sites, 3 phosphorylation domains, 4 other domains and 1 LCR domain. Also, its secondary structure is mainly randomly curly. Nucleotide sequence analysis revealed that the gene nucleotide sequence of Lanzhou fat-tail sheep has some different from the known sheep MAPK13 mRNA（Genbank No.：NM_001139455.1）. Base transition occur in the 852th site is（C vs. A）, respectively, the corresponding amino acids in the 265th is（S vs. T）. Base transition occur in the 951th site is（T vs. G）, but the corresponding amino acids do not change.The phylogenetic tree indicates that Lanzhou fat-tail sheep is close to bos. The fact that the structure of MAPK13 of Lanzhou fat-tail sheep highly similar with other species indicates that the gene is highly conservative. The S_TKc domain in the sequence can transfer the gamma phosphoryl of ATP to the serine / threonine residues of protein, leading to function imbalance and expression change in a series of obesity related gene.

Transfection of Bovine Fetal Fibroblast with the Vector for Expressing Human GDNF Specifically in Mammary Gland

Yu Fei, Li Bin, Zhang Jingjing, Ding Haimai, Zhang Xueming

2014, 30(8):  102-107. 

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In order to prepare bovine mammary gland bioreactor for production of human recombinant GDNF, the female bovine fetal fibroblast cells were successfully isolated using tissue bulk attachment. The fibroblast cells were cultured consecutively for 75 days for further morphologic observation and chromosome analyzing. The plasmid vector pNR-GDNF, which contained the Neo^r gene and the DsRed2 gene as positive selection marker genes and human GDNF cDNA gene regulated by bovine beta-casein promoter for specific expression in mammary gland, was transfected into the bovine fetal fibroblast cells by electroporation. After selection with G418 for 7 days, resistant cells expressing red fluorescence protein were isolated, cultured, expanded and cryopreserved by standard procedures. The transgenic cells were indentified by PCR. The results showed that bovine fetal fibroblast cells possessed normal morphology, multiplication characteristics and chromosome number and the foreign gene was integrated into the genome.

Cloning，Sequence Analysis and Prokaryotic Expression of Musca domestica Allergen Tropomyosin Gene

Wei Chuanchuan, Xiu Jiangfan, Wang Yu, Guo Guo, Peng Chuanlin, Wu Qinyi, Wu Jianwei

2014, 30(8):  108-112. 

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It was to probe into the homologous cloning of the Musca domestica allergen Tropomyosin gene, and then construct the prokaryotic expression vector which was expressed in Escherichia coli. Screening and isolation of Musca domestica Tropomyosin gene from Musca domestica cDNA library was carried out. The gene and encoded protein sequence of Tropomyosin was analyzed by bioinformatics methods , including general physical and chemical properties, signal peptide, secondary structure, tertiary structure, epitope and subcellular localization. Then plasmid pEASY-E1-Tropomysin were transformed into Escherichia coli BL21(DE3) competent cells for expression. The open reading frames of the Tropomyosin gene was 828 bp that encded a putative protein with 275 amino acids. The protein, with predicted molecular weight 31.6 kD and pI of 4.65, has the conserved Tropomyosin domain so belongs to Tropomyosin family. The result showed that the recombinant prokaryotic expression vector pEASY-E1-Tropomysin was successfully constructed and fusion protein was expressed in E. coli.

RNA Interference of Antifreeze Protein Gene in Tenebrio molitor Mediated by Bacterially Expressed dsRNA

Shi Meng, Liu Xiaoning, Ma Ji

2014, 30(8):  113-119. 

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In order to further study the non-freezing function of antifreeze protein（AFP）genes in Tenebrio molitor using RNAi technology, the RNAi fragment of Tmafp433 from T. molitor was inserted into L4440 dsRNA expression vector, and transformed into E. coli HT115（DE3）. The dsRNA corresponding to Tmafps, designated as Tmafp-dsRNA, was expressed by IPTG induction, and injected into the larvae after purification. The mRNA level of Tmafps was detected using real-time quantitative PCR. The results showed that the E. coli HT115（DE3）containing L4440-Tmafp recombinant plasmid can express Tmafp-dsRNA. After 24 h of injection, the expression of Tmafps in T. molitor was significantly decreased to 60.8% of the control, suggesting that the expression of Tmafps was inhibited by injecting the bacterially expressed Tmafp-dsRNA. The present results laid foundation for further research in afp gene function.

cDNA Cloning and Construction of Eukaryotic Recombinant Expression Vector for Hepcidin Mature Peptide from Tilapia（Oreochromis niloticus）

Liu Jingjing, Tao Yan, Wen Ya

2014, 30(8):  120-125. 

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Hepcidin is a small cysteine-rich cationic antimicrobial peptide with the regulative function for iron metabolism. It is expressed predominantly in the liver of animals. Hepcidin plays an important role in the host’s immune response against microbial invasion. Thus, it is considered to be good substitutes for traditional antibiotics. TH1-5, one of the three different hepcidin cDNAs from tilapia（Oreochromis mossambicus）. The cDNA encoding hepcidin mature peptide（mTH）containing 22 residues was cloned from hepcidin full-length cDNA of tilapia（Oreochromis niloticus）liver by PCR. The forward and reverse primers were designed with reference to the nucleotide sequence of #br#O. mossambicus hepcidin TH1-5. This cDNA fragment carrying Xba I and Xho I sites was inserted into pGAPZ-A plasmid with the same restriction sites to construct a recombinant expression plasmid “pGAPZ-A-mTH”. The colony PCR, Xba I and Xho I restriction endonuclease digestion and DNA sequencing demonstrated that the “pGAPZ-A-mTH” recombinant expression plasmid was constructed successfully. In addition, the recombinant expression plasmid was transformed into Pichia pastoris GS115 by an electroporation system. PCR using yeast DNA as template demonstrated that the recombinant expression plasmid was inserted into the yeast chromosome.

Expression，Purification，Crystallization of Acetylcholine Binding Proteins from Lymnaea stagnalis in Bac-to-Bac System

Lin Bo, Meng Hailing, Wu Yong, Bing Hui, Zhangsun Dongting, Luo Sulan

2014, 30(8):  126-131. 

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It was to construct a recombinant bacmid that expresses acetylcholine binding protein from Lymnaea stagnalis（Ls-AChBP）and express the Ls-AChBP functional genes in Bac-to-Bac expression system to obtain the crystal. Synthesized Ls-AChBP cDNA was inserted into the correct position of the baculovirus expression vector pFastBac1. After the positive recombinant bacmid was screened, the recombinant bacmid was transfected into the insect cells to generate restructured baculovirus for high expression of the recombinant protein Ls-AChBP（rLs-AChBP）, which was captured by nickel-charged resin, and further purified by gel filtration chromatography. The rLs-AChBP pentamer was obtained and performed crystallization by robot screen. Recombinant Ls-AChBP expression in Bac-to-Bac system was successfully and we got Ls-AChBP crystal. Ls-AChBP is one of the AchBPs. The crystal structure of rLs-AchBP is an available template of description of nAChRs structure and function.

Molecular Identification of 23 Marine Fungal Strains and Their Activities Against Plant Pathogenic Fungi and Cytotoxic Activities

Yang Xiaolan, Chen Yuchan, Li Haohua, Zhang Weimin

2014, 30(8):  132-137. 

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Twenty three marine fungal strains were isolated from the sediments of the South China Sea and identified by intergenic transcribed spacer（ITS）sequencing. The fermentation broth extracts of these marine fungal strains were tested for their inhibitory activities against growth of 4 plant pathogenic fungi, namely Colletotrichum gloeosporioides, Alternaria alternate, Curvularia lunata, Cylindrocladium scoparium, and investigated for their cytotoxic activities against SF-268, MCF-7, NCI-H460 and HepG-2 tumor cell lines by the SRB method. The results showed the extracts of 11 strains presented higher than 50% inhibitory rates against at least one of those plant pathogenic fungi at a concentration of 50 mg/mL and the extracts of 9 strains presented higher than 80% inhibitory rates against at least one of those tumor cell lines at a concentration of 100 μg/mL. Among all strains studied, Eupenicillium sp. FS100, Penicillium sp. FS105, Dichotomomyces cejpii FS110 and Acaromyces ingoldii FS121 exhibited obvious inhibitory activities against plant pathogenic fungi and/or tumor cell lines.

Endophytes Isolation and Broad-spectrum Antagonistic Bacterias Screening from Banana

Wang Mengying, Zhou Dengbo, Jing Tao, Hu Yifeng, Gao Zhufen, Xie Qingyi, Zhang Xiyan, Qi Chunlin

2014, 30(8):  138-145. 

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In order to determine the main distribution of endophytes and their broad-spectrum antimicrobial activity, endophytes were obtained from healthy and diseased tissues of two disease-resistant and one disease susceptible banana cultivars. Endophytes were separated from roots, corms, pseudostems, leaves and store in the ultra-low on Luria-Bertani（LB）, Yeast Extract with supplements（YE）, and Potato Dextrose Agar（PDA）strain store medium. Then screened broad-spectrum antagonistic bacteria which against Fusarium oxysporum f. sp. Cubense, Curvularia lunata, Curvularia fallax, Corynespora cassiicola（Berk & Curt）Wei, Alternaria musae, Deightoniella troulosa, Colletotrichum musae, Pestalogiopsis sp., Btoryosphaeria dothidea. Taxonomy identification of 041, 04-1, 19-1, 03A-1 was conducted by evaluating morphologic characteristics and 16S rDNA gene sequences for phylogenetic analysis. After purification, total of 438 endophytes were obtained. The total of isolates showed that we obtained 240 strains bacteria, followed by 142 strains actinomycetes, and 56 strains fungi. The richest number of endophytes that isolated from diseased NanTian banana cultivars（128）. Ten actinomyces and two bacterias were determined to possess antibiotic activity against Ten banana pathogens. Isolates 041 was the most effective and had 28.13±1.89 mm width of inhibition zone. Isolated 041, 04-1, 19-1, 034-1 were identified as Streptomyces misionensis.

Isolation of Diesel Degrading Strain Acinetobacter sp. AK5 and Its Degrading Performance

Xu Xiaoyu, Chen Jinghua

2014, 30(8):  146-151. 

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A diesel degradable bacterial strain was isolated from activated sludge and identified as Acinetobacter sp. AK5 through physiological, biochemical identification and 16S rDNA sequence analysis. Experiments of the different pH values, NaCl concentrations, culture time and diesel concentrations were detected to evaluate the diesel degradability by Acinetobacter sp. AK5. The results show that the optimal initial pH scope for the bacterial growth is from 5 to 9, the optimum NaCl concentrations is between 3% and 4%. When the diesel concentration is 5 g/L, the 7 d diesel degradation rate can reach 99%, while when the concentration of diesel is 20 g/L, 7 d diesel degradation rate can be 67%. The Acinetobacter sp. AK5 can grow well in artificial seawater medium and inorganic salt culture medium, therefore it has promising application prospect in seawater and freshwater oil pollution treatment.

Heterologous Expression and Characterization of Phenylalanine Hydroxylase from Chromobacterium violaceum

Cao Shuhui, Zhou Li, Cui Wenjing, Liu Zhongmei, Zhou Zhemin

2014, 30(8):  152-158. 

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Phenylalanine hydroxylase（PAH）derived from Chromobacterium violaceum has potential pharmaceutical value due to its simple structure and the closer property to human PAH. We cloned the pah gene from C. violaceum and constructed recombinant expression vcectors pET24a-pah, finally, the pah gene was efficiently expressed in Escherichia coli BL21（DE3）. The specific activity of recombination protein was 503.2 U/mg after purification. The studies of enzymatic properties showed that the enzyme displayed maximum activity at 40℃, and its half-life was 15 min at 50℃. The optimal pH was pH7.5, and the enzyme activity was stable at the range of pH6-8. Under the conditions of 37℃ and pH7.5, its K_m was 1.5 mmol/L, V_max was 0.5 mmol/min, k_cat was 5.05/s, and the catalytic efficiency（k_cat /K_m）was 3.37 L/mmol·s.

Breeding and Fermentation Characterization of Mutants of Bacillus licheniformis for 2，3-Butanediol Production

Guo Wenyi, Sun Qinghui, Song Lina, Gao Songsong, Yang Hongjiang

2014, 30(8):  159-163. 

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Currently, most isolated 2,3-butanediol producing strains are pathogens and they cause certain risks to public health and environments. In this work, Bacillus sp. 127-7 was isolated for 2,3-butanediol（BD）production from raw milk samples. It was identified as Bacillus licheniformis by analyzing its 16S rRNA gene sequence. By UV-mutagenesis, mutants that could endure high glucose concentrations and produce relatively high amounts of acetoin were isolated for further analysis. In shake-flask fermentations, 2,3-BD production was increased by 41.1% in mutant strain BL41 compared with the parent strain 127-7. Additionally, BL41 yielded much less amounts of lactate acid in the cultures without acidity control and 2,3-BD reached 81.4 g/L in the fed-batch fermentation. Moreover, the minimal residual glucose concentration was incre ased to 30 g/L in the culture, the fermentation time was significantly reduced to 46 h with 83.4 g/L 2,3-BD. The highest productivity is up to 1.9 g/L·h. The results showed that B. licheniformis BL41 may be used as a candidate strain for industrial production of 2,3-butanediol.

Optimization of Fermentation Conditions of Antagonistic Bacterium AM53 Against Colletotrichum gloeosporioides

Yang Jing, Zhou Guoying, Tan Yimin, Lu Zongyan

2014, 30(8):  164-168. 

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It was to optimize the fermentation conditions of Bacillus licheniformis. The Plackett-Burman design was used to optimize the fermentation conditions, and the results showed that culture temperature, the initial pH and the rotating speed had significant influence on the count of the living AM53. After the steepest ascent test and Box-Behnken design, the optimal fermentation conditions of AM53 that we got were：culture temperature was 30.5℃, the initial pH was 8, rotating speed was 185 r/min and inoculation volume was 2%, cultured 24 hours. Under this condition, the average of OD₆₀₀ was 1.861, which means the experimental values agreed with the predicted values, the predicted model was reliable and available for the optimization of AM53 fermentation conditions

Optimization of Fermentation Medium by Bacillus amyloliquefaciens Strain C101 Using Response Surface Methodology

Liang Changcong, Guo Lijia, Liu Lei, Zhang Jianhua, Yang Laying, Wang Guofen, Huang Junsheng

2014, 30(8):  169-174. 

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The fermentation medium components for spore content by Bacillus amyloliquefaciens strain C101 was optimized by response surface methodology（RSM）. Firstly, three of the most significant influence factors were screened by the method of Plackett-Burman design as MnSO₄, KH₂PO₄ and（NH₄）₂SO₄. The path of steepest ascent was applied to approach the optimal region of the three significant factors. Lastly, the optimal concentration and correlations among three factors were identified by RSM. The optimal fermentation conditions were determined as followed：sucrose 20 g/L, urea 4.0 g/L, soybean meal 4.0 g/L, KNO₃ 2.0 g/L, Na₂HPO₄ 2.4 g/L, KH₂PO₄ 0.52 g/L, （NH₄）₂SO₄ 0.55 g/L, NaCl 1.0 g/L, MgSO₄·7H₂O 0.50 g/L, FeSO₄ 0.005 0 g/L, MnSO₄ 0.005 4 g/L. Under these conditions, the maximum theoretic spore number was 14.67×10⁸ /mL. After three parallel verifications, the maximum theoretic value was consistent with mean value of verification test and the spore number was increased by 188% compare to that before optimization.

Optimization of β-cyclodextrin Production by Recombinant β-cyclodextrin Glycosyltransferase

Yang Yulu, Wang Lei, Chen Sheng, Wu Jing

2014, 30(8):  175-181. 

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The βcgt gene encoding β-CGTase from Bacillus circulans strain 251 was cloned into the expression vector pET-20b（+）.The vector was then transformed into Escherichia coli BL21（DE3）for extracellular production of β-CGTase. The activity in the supernatant of recombinant E. coli BL21（DE3）was 20 U/mL. Furthermore, the condition for β-cyclodextrin（β-CD）preparation by this recombinant β-CGTase was optimized. At 15% potato starch, pH5.5, 30℃, 2.5%-5%（V/V）cyclohexane, 10 U β-CGTase per gram substrate incubated for 24 hours, 75.3% of the starch was transformed into β-CD. This is the highest level of β-CD conversion by enzyme method at home and abroad.

Purification of GST-TRAF6 Fusion Proteins from Inculsion Body Expressed in E. coli

Zhang Xixuan, Li Ye, Zhang Zhenqi, Dong Shirui, Zhao Pei, Ruan Haihua

2014, 30(8):  182-188. 

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There was only a minor fraction of GST-TRAF6 fusion proteins were observed soluble when expressed in E.coli. Most of the GST-TRAF6 fusion proteins were present in the inclusion body, in which the expressed proteins aggregate and exist in inactive. To obtain GST-TRAF6 fusion protein in a high yield and high purity, a method to purify the GST-TRAF6 fusion proteins from inclusion body without any loss of its bioactivity was constructed. Firstly, the inclusion body was denatured with 8 mol/L urea. Secondly, the GST-TRAF6 fusion protein in inclusion body were gradually solubilized by gradient dilution renaturation. Once the GST-TRAF6 fusion protein was fully dissolved in renaturation solution, it was conventional to purify the GST-TRAF6 fusion protein with glutathione sepharose 4B. The specificity and the bioactivity of the purified GST-TRAF6 fusion protein were testified by western blot and in vitro ubiquitination assay, respectively. The results indicated that GST-TRAF6 fusion protein were successfully solubilized by gradient dilution renaturation following the denaturation of 8 mol/L urea, the concentration of the purified GST-TRAF6 fusion protein was achieved a concentration of 396 ng/μL with a purity over 90%. The specificity of this GST-TRAF6 fusion proteins were identified with anti-GST western blot. In vitro ubiquitination assay indicated that the GST-TRAF6 fusion protein in a concentration of 17 ng/μL could rapidly catalyze the formation of free ubiquitin chains within 5 minutes. In conclusion, it was found that the GST-TRAF6 fusion protein could be successfully renatured with the method of gradient dilution renaturation with the recovery of its capability of ubiquitin ligase. This results would supply a useful method for the large-scale purification of proteins from inclusion body when expressed in E. coli.

The Expression and Purification of Recombinant Myoglobin and Preparation of Its Quality Control Samples

Ren Yanna, Cai Lei, Wu Jianwei, Qian Wei, Zhang Ronghua, Wang Jihua, Tang Shixing

2014, 30(8):  189-195. 

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We intended to provide a cheap, stable, highly specific and sensitive lyophilized protein as quality control for diagnositic kits. The myoglobin gene code was optimized and two-step methods were used in this study for the synthesis of myoglobin gene. The DNA fragment of myoglobin was inserted into pET-28a vector forming a recombination plasmid, then, transformed into BL21（DE3）bacteria. Protein expression was induced by 0.5 mmol/L IPTG and purified by Ni Sepharose 6 Fast Flow（FF）. After confirming its specificity, we studied the stability of the lyophilized protein at different storage temperatures（37℃, 25℃, 4℃）. Results showed that the target protein is approximately 20 kD with high specificity, good morphology and stability after freeze-drying under the optimized conditions and protein diluting solution. The lyophilized myoglobin protein with low cost, good stability, high specificity and sensitivity, canbe used as quality control for both qualitative and quantitative myoglobin testing kits.

Optimization Screening of Peptides Inhibition to H₃₇Ra

Wu Congmei, Li Lingling, Guan Xiaoxia, Liu Xintao, Chen Ji, Yin Yuhe

2014, 30(8):  196-201. 

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By inhibition experiments to determine the inhibitory effect of peptide compounds under different culture conditions, and measure theirs minimal inhibitory concentration（MIC）. Adding Vc to compare inhibitory effect and future optimize screening H₃₇Ra inhibitors. According to the results of ELISA test screen the phage peptide library, and then uses the Molecular docking software to simulate peptide docking with ICL protein crystal（1F8I）. The successful docking peptide uses the solid-phase synthesis method of Fmoc for synthesis, and tests its biological activity. By preparative reversed phase HPLC purifies the synthesized peptides and detects mass spectrometry. Four peptides were screened, all of which have bacteriostatic effect, and correlated with the dose. The results showed that when the concentration between 800 and 1 500 μg/mL all group were bacteriostat. When the concentration is 500 μg/mL, only one peptide has bacteriostatic effect in the normal medium, while all cannot inhibit in the limit carbon medium. The inhibitory effect of No.2 peptide is best, and its MIC is 200 μg/mL in normal culture, while it is 500 μg/mL in carbon limitations. Positive control group, two media’s inhibitory effect were consistent, RIF’s and INH’s MIC were 0.8 μg/mL and 0.5 μg/mL, respectively. According to MIC results, the inhibitory effects of peptide, RIF and INH were improved when adding Vc to the normal culture, and to be a dose-dependent increase.

Study on Antimicrobial Resistance and Plasmid-mediated Quinolone Resistance of Foodborne Salmonella Isolates

Liu Guishen, Yu Tao

2014, 30(8):  202-207. 

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A total of 638 food samples were collected randomly to determine the prevalence of Salmonella. The overall percentage of Salmonella prevalence was 9.7%（n=62）. Among 16 different serotypes identified, S. Anatum was most common. The isolates were frequently resistant to sulfamethoxazole, trimethoprim/sulfamethoxazole, streptomycin, and ciprofloxacin. Five different types of amino acid substitutions were identified in quinolone resistance-determining region（QRDR）of GyrA and ParC of 16 ciprofloxacin-resistant isolates. Double mutations of Ser83Phe and Asp87Gly in GyrA accompanied by an additional mutation of Ser80Arg in ParC were found most frequently. The qnr genes were present in seven（11.3%）of 62 isolates, and among them, two isolates carried qnrA and five carried qnrS. qnrB, qnrC, and qnrD were not detected in the isolates in this study. Eight isolates were positive for aac（6'）-Ib, of which three isolates carried the-cr variant. The results suggested the diversity of serotype distribution, serious situation of antimicrobial resistance and the presence of plasmid-mediated quinolone resistance（PMQR）genes in foodborne Salmonella from Xinxiang.

Rapid Quantitation and Dynamic Accumulation Analysis of Neutral Lipids in Yeast

Du Xiuxiu, Fang Zhijia, Chen Zhongxiang, Kuang Xin, Huang Zhiwei

2014, 30(8):  208-214. 

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A colorimetric method for rapidly detecting neutral lipid content in yeast was developed, which was based on the staining of yeast cells by lipophilic dyes Oil red O or Sudan Black B and extraction with ethanol after cell rupturing using the method of combining hydrochloric acid or sodium hydroxide with heating. The extraction of Oil red O is better and the colorimetric results were more accurate, while Sudan Black B also can be used as an alternative dye for estimating neutral lipid content. Furthermore, the changes of neutral lipids in yeast mutated phospholipid fatty acid phosphatase（PAH1）and triglyceride lipase（TGL）, which are known as two key genes to regulate triglyceride metabolism, were studied using this method. Disruption of PAH1 resulted in a remarkable decrease in triglyceride content, although total neutral lipid levels did not change；while triglyceride and total neutral lipids contents were significantly accumulated in TGL knockout mutant. Finally, the dynamic changes of yeast neutral lipids content were successfully monitored with the change of growth phase by this method, suggesting that the neutral lipids were obviously accumulated after exponential phase.