Table of Content

18 May 2015, Volume 31 Issue 5

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Studies on Biocontrol of Cotton Verticillium Wilt

Liang Hong, Huang Jing, Zhao Jia, Chen Zhe, Wang Changbiao

2015, 31(5):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.001

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With the enhanced awareness of environmental protection, biocontrol of Cotton Verticillium wilt is proved to be an important field. This paper reviews the research progress have been achieved, including the type of biocontrol bacterium；the research progress on antibacterial substances and antagonistic genes；the application on bio-organic fertilizer. Points out the trends of biocontrol Verticillium wilt and provides a reference for future research.

Studies on WUSCHEL-related Homeobox（WOX）Protein Family

Gao Li, Sun Yimin, Shao Tiemei, Kong Weina, Cui Runli, Lu Nan, Wu Tao

2015, 31(5):  7-12.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.002

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WUSCHEL-related homeobox（WOX）transcription factor, family members are essential in the many stages of plant development（for examples, stem-cell maintenance in shoot and root apical meristem, lateral organ development, floral organ formation and embryonic patterning）by promoting cell proliferation or preventing cell differentiation. We outline the phylogenetic analysis, the molecular characteristics, biological functions and the mechanism of action of WOX gene family members. We also provide prospective vision in this field.

Research Progress on the Harmless Treatment and Resource Reuse of Antibiotic Bacteria Residues

Chen Liwen, Fang Senhai, Wang Mingzi

2015, 31(5):  13-19.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.003

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The dozens of hazard bacteria residues are generated during the antibiotics fermentation process. An incorrect disposal of them would cause critical environmental and health problems. Here we summarize the recent progresses on the harmless treatment and resource utilization with this kind of waste, such as reusing it as nitrogensource in fermentation medium, producing energy, forage, biodegradable film and gypsum retarder, for enhancing the efficiency of harmless treatment and resource utilization of antibiotic bacteria residues.

Advances in Research of Straw Degradation with Cellulase and Its Genetic Engineering

Zhang Senxiang, Yin Xiaoyan, Gong Zhiwei, Yang Zhonghua, Hou Yali, Zhou Wei

2015, 31(5):  20-26.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.004

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Energy shortage and environmental pollution have become the public focusing issue. Straw biomass with its rich resources, non-polluting and renewable feature, has great application prospect in solving the energy crisis. Converting straw biomass to fermentable sugars by hydrolysis with cellulase and combing with fermentation may produce ethanol, hydrogen and other materials of energy, which has been a mature technology route. The crucial steps of utilizing straw biomass are the pretreatment of straw biomass and efficient obtaining of glycoside hydrolases. We summarize and analyze the current research from 3 aspects: the structural characteristics of straw and its biological pretreatment; the mechanism of cellulase in hydrolysis of straw biomass; and the gene engineering for cellulase. It has a guiding significance in the promotion of applying straw biomass for energy.

The Role of MicroRNA in the Cell Apoptosis Mediated by NF-κB

Qi Renli, Huang Jinxiu, Long Dingbiao, Huang Ping

2015, 31(5):  27-31.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.005

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As a key regulatory factor in cell, activated NF-κB induces the transcription of target genes involved in many cell activities, especially the inflammation, immunity and apoptosis. Previous studies proved that NF-κB plays various roles in different type of apoptosis. Moreover, expression and function of NF-κB could be systematically or specifically regulated by microRNA of non-encoding RNAS. The mediating effects of NF-κB on apoptosis were summarized, and the role of microRNA in the mediating process were further discussed .

Research Progress of Enzyme and Relevant Gene Screened from Rumen by Metagenomic Approach

Zhang Jun, Zhao Shengguo, Wang Jiaqi, Jin Di, Bu Dengpan

2015, 31(5):  32-40.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.006

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Rumen microbial community is a complex biological system with extremely rich biodiversity and large amount of genetic and ecological resources, and it is also an important treasury for the development of enzyme. Using metagenomic approach could avoid the restriction of microbial culturing, with directly extracting DNA, gene screening and expressing the uncultured microbes to enlarge the database of genes, which provides possibility for the development of novel biocatalyst. The study explained metagenomic approach, and summarized the application of metagenomic approach in screening functional enzymes and studying relevant genes from rumen.

Research Progress on Methods of Removing Marker Genes in Transgenic Plants

Lang Yaoling, Guan Xiaoyan, Bai Guohui, Liu Jianguo

2015, 31(5):  41-47.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.007

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With more and more varieties of transgenic plants emerging, environmental and food safety from using transgenic plants have been attracting extensive public attentions. One of the current problems is that the selectable marker genes, usually having the antibiotic or herbicide resistance, remain in transgenic plants. In order to improve the safety of using transgenic plants, removing marker genes from transgenic plants is not only conducive to the multiple using the same transgenic plants, but also more likely to allow the transgenic plants accepted by people. The current methods of removing marker genes in transgenic plants were reviewed, and the advantages and disadvantages of each method were compared from all aspects, which have a guiding significance for selecting a proper method in this field.

Biological Wastewater Treatment at Low Temperatures：Advances and Future Trends

Wang Shuo, Shi Wenxin, Wang Yan, Yu Shuili, Li Ji

2015, 31(5):  48-53.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.008

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The efficiency of wastewater treatment decreases due to the low temperatures in the winter season in most parts of China, which results of the increase of the operation cost, and hence an adverse impact on the environment and damages to human health. The recent advances on the biological wastewater treatment technologies at low temperatures were sammanzed. In terms of the morphological types of sludge（e.g.2 floc sludge, anaerobic granular sludge and aerobic granular sludge）, their advantages and disadvantages were compared and the trends on biological wastewater treatment at low temperatures were prospected.

Screening and Breeding Strains Producing High-yield Avilamycin by Genome Shuffling

Mao Lingqi, Li Cunzhi, Tao Xingwu, Yan Dazhong

2015, 31(5):  54-60.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.009

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In order to screen strain producing high-yield avilamycin by mutagenesis, a wild-type strain Streptomyces viridochromogenes Tü57 was treated by a series of traditionally physical and chemical mutagenesis, resulting in the acquisition of numbers of forward mutants while judged by the great improvement in avilamycin A production. Genome shuffling was then carried out to fuse different sources of forward mutants, following with various fusants harvestry. The screening strategy adopted the combination method of thin layer chromatography (TLC) and cylinder-plate (two dosages technique) as the primary test, and high performance liquid chromatography (HPLC) as the secondary test. A mutant producing high-yield avilamycin, designated as R708, was obtained. The yield of avilamycin A from this mutant reached to 0.208 g/L in the shake flask experiment, which was a 20-fold increment compared to that of the wild-type strain. Our conclusion is that genome shuffling could increase the genetic diversity of the offspring strains; therefore this method is more quickly and efficiently than traditional mutagenesis in breeding strains producing high-yield avilamycin.

Optimized Sample Preparation for Metabolome Studies on Streptomyces avermitilis by GC-MS

Guo Gang, Tang Dan, Tian Pingping, Shi Xiufeng, Cao Peng, Gao Qiang

2015, 31(5):  61-67.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.010

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We established a method for preparing metabolome sample of Streptomyces avermitilis, including cell separation, washing, quenching, disruption and extraction conditions based on GC-MS analysis system, and applied it in the analysis of the metabolome of industrial strain 9-39 and wild type strain. The optimal conditions for sample preparation were as follows: separating cells by centrifugation, washing 3 times, quenching 5 minutes with liquid nitrogen, cell disruption with bead mill（3 cycles and 48 seconds per each cycle）, and extraction of metabolites（chloroform:ethanol:water = 2:2:1）. By using this optimized protocol, 59 differential compounds were identified and quantified, including amino acids, organic acids, fatty acids and saccharides. Our established method can be used to effectively analyze the Streptomyces avermitilis metabolites.

A Method of Recovering Short DNA Fragments from Non-denaturing Polyacrylamide Gel

Xiang Xiaohua, Jiao Yushun, Wu Xinru, Cheng Yazeng, Hou Shuo, Liu Guanshan, Wang Yuanying

2015, 31(5):  68-72.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.011

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The polyacrylamide gel electrophoresis with high resolution is a top choice for isolating and purifying specific short DNA fragments, and also serves as a prerequisite for a variety of subsequent molecular biology experiments. In this study, three methods, i.e., modified boiling method（grinding with liquid nitrogen + boiling + concentrating at low temperature）, traditional boiling method（in which DNA is precipitated with absolute ethanol and 3 mol/L sodium acetate together）, and direct recovery method were used to extract short DNA fragments. The short DNA fragments were used as the template for a second PCR reaction. The results showed that recovered DNA fragments by the modified boiling method had higher purity and finer specificity, and the recovery rate was nearly the same as the traditional boiling method. Replacing the method of precipitation with ethanol and sodium acetate by method of concentrating at low temperature is feasible and novel.

Procedure to Prepare Samples for Two-dimensional Electrophoresis of Secreted Proteins from Saccharomyces cerevisiae

Du Wei, Piao Yongzhe, Huang Wei, Gu Yue, Zhao Changxin

2015, 31(5):  73-77.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.012

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Saccraomyces cerevsiae FFC2144 was cultured in nitrogen base medium without protein. The secretory proteins of yeasts were extracted by ammonium sulfate precipitation, ultrafiltration and lyophilization-phenol extraction respectively. Extraction rates by 3 methods were calculated and the proteins were separated by two-dimensional electrophoresis. The proteins isolated were confirmed by MALDI-TOF-MS. The extraction rate by lyophilization-phenol method was 73.67% and 114 protein spots were obtained with the most protein spots and clearest electrophoretogram. Lyophilization-phenol method could be an ideal separation method for studying secretory proteomics.

The Establishment of FAST-LAMP Method for Detecting Influenza A（H1N1）Virus

Lü Qinfeng, Luo Peng, He Lei, Yang Yongyao, Zheng Wei, Li Li, Wu Zhonghua

2015, 31(5):  78-83.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.013

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It was to establish a new loop-mediated isothermal amplification（FAST-LAMP）for rapidly detecting influenza A（H1N1）virus. A set of primers were designed according to HA gene, including the outer primers, internal primers, loop primers and nest internal primers. The nest internal primers increased the contact sites of primers and nucleic acid template, so that the amplification efficiency could be improved and detection time reduced. The FAST-LAMP method could be 45 less than LAMP method, and 20 minutes less than LAMP method with loop primers, i.e. detection time reduced significantly detecting sensitivity reached 1×10² copies cDNA template/tube. It proved that the established FAST-LAMP method is an efficient and reliable method for detecting influenza A（H1N1）virus.

Transcriptome Analysis of Sarcopyramis nepalensis via RNA-seq Technology

Jin Hong, Jiao Genlin, Chen Gang,

2015, 31(5):  84-92.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.014

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Transcriptome analysis of Sarcopyramis nepalensis leaves was performed via a newly developed high-throughput sequencing technology（Illumina RNA-seq）. A total of 51 305 unigenes were generated with 921 nt of average length and 1 490 nt of unigene N50 after filtering and assembly of original reads. These unigenes from the de novo assembly were further annotated using BLAST and BLAST2GO softwares. A total of 40 532 unigenes annotated with databases of non-redundant protein sequence（Nr）, non-redundant nucleotide（Nt）, Swiss-Prot, Gene Ontology database（GO）, Clusters of Orthologous Groups（COG）and Kyoto Encyclopedia of Genes and Genomes（KEGG）databases available at NCBI as references. The proportion of unigenes annotated in Nr, Nt, Swiss-Prot, KEGG, COG and GO databases were 77.53%, 56.18%, 53.14%, 46.58%, 29.69% and 60.72%, respectively. Total 39 302 CDSs were obtained using blast in protein databases, and 2 065 CDSs were predicted using ESTscan software. KEGG pathway parsing revealed that 2 323（9.72%）unigenes were involved in biosynthesis of secondary metabolites（KO01110）, and 78 unigenes encoding the cytochrome P450 family proteins were identified. These annotated information provided theoretical foundationfordetermining the vital genes involved in biosynthesis of secondary metabolites of medicinal plants.

Cloning，Prokaryotic Expression of Sorghum bicolor SbGABA-Ts

Yang Zewei, Wang Longhai, Zhu Li, Wang Hai, Huang Dafang, Lang Zhihong

2015, 31(5):  93-99.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.015

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GABA is a ubiquitous four-carbon，non-protein amino acid，and has been associated with growth and development，signaling transduction，and stress response in plants. GABA transaminase（GABA-T），the enzyme responsible for the catabolism of GABA，has not been fully explored，especially in several important crops. Two putative GABA transaminase genes（GABA-T）from Sorghum bicolor were obtained based on homology analysis with the identified GABA-Ts of other plants and RT-PCR. Subsequently，the two SbGABA-T genes and corresponding N-terminal truncated SbGABA-Ts were inserted into pET28a（+）vector and individually imported into E. coli strain BL21（pLysS，DE3）. Percentage improvement of soluble recombinant proteins were showed when removing the targeting peptide sequence of SbGABA-Ts. The fusion proteins were expressed partially in soluble form after incubating for 18 h at 16℃ by adding 1 mmol/L IPTG，and purified through nickel-affinity chromatography column.

Ectopic-overexpression of AmHsa32 from Ammopiptanthus mongolicus Promoted Heat Tolerance in Nicotiana benthamiana

He Qian, Wang Yanping, Chen Yuzhen, Lu Cunfu

2015, 31(5):  100-105.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.016

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Hsa32 is a novel heat-shock protein（Hsp）and mainly found in land plants and essential for acquired thermo-tolerance. AmHsa32 as one orthologue of the Hsa32 gene, was isolated from a desert plant, Ammopiptanthus mongolicus（Leguminosae）. In order to verify the function of AmHsa32, we constructed the plant expression vector Pcambia2300-35S-AmHsa32-OCS, and transferred it into Nicotiana benthamiana through the medium of Agrobacterium strain. PCR and Western blotting demonstrated that AmHsa32 had been integrated into the genome of Nicotiana benthamiana. Seed germination and seedling growth analysis indicated that overexpressing AmHsa32 resulted in enhanced tolerance to heat stresse in transgenic Nicotiana benthamiana. Our work suggests that AmHsa32 might be a valuable gene for plant resistant breeding with transgene technology.

Cloning and Expression Analysis of Proline Transporter Gene in Ammopiptanthus mongolicus

Yue Guangzhen, Jin Man, Li Junlin, Yang Shunying, Guo Manyuan, Su Yanhua

2015, 31(5):  106-112.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.017

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The cDNA of proline transporter gene in Ammopiptanthus mongolicus（GenBank accession No. KJ873133）was amplified by RACE technology. Bioinformatics analysis showed that the length of AmProT ORF was 1 329 bp, the gene encoded a putative polypeptide of 442 amino acids with a calculated molecular mass of 48.0733 kD and a theoretical pI of 9.32. It had 11 transmembrance domains, which is the typical characteristics of proline translocator. Phylogenic analysis indicated that AmProT was 86% similar to proline transporter gene of Glycine max. Real-time PCR analysis revealed that AmProT expressed in all tissues examined with more higher level in the above-ground part than the underground part; expression profiles under different stress treatments such as drought, salt and ABA were compared, and the results revealed that transcriptional level of AmProT was up-regulated. It is speculated that AmProT plays an important role in response to abiotic stresses such as drought and salt.

Cloning and Expression Analysis of Cytosolic 6-phosphogluconate Dehydrogenase Gene in Tobacco(Nicotiana tabacum)

Lin Shifeng, Fu Qiang, Yu Jing, Zhao Jiehong, Ren Xueliang, Wang Rengang

2015, 31(5):  113-119.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.018

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A cDNA sequence of cytosolic 6-phosphogluconate dehydrogenase（6PGDH）gene was cloned from Nicotiana tabacum by in silico cloning combined with RT-PCR and SMART RACE technologies, and designated as Nt6PGDH（Accession number KM211534）. The full length of its cDNA sequence is 1 932 bp, with a 1 455 bp open reading frame, and the gene encoded a protein of 484 amino acids with the sequence of highest identity, 95% as 6PGDH from Solanum lycopersicum and Solanum tuberosum. Bioinformatics analysis indicated that Nt6PGDH had no signal peptide, no transit peptide and notrans-membrane domain, and it was located in cytoplasm. Gene expression analysis showed that the expression levels of Nt6PGDH in roots, stems and leaves were higher at fast-growirg stage than at seedling stage. Moreover, at the same developmental stage, the highest level of Nt6PGDH expression were in the roots, then in the stems, the lowest in the leaves.

Cloning and Expression Analysis of Actin Gene in Cinnamomum camphora

Li Yongpeng, Zhang Liwei, Yao Yao, Huang Rui, Du Li

2015, 31(5):  120-127.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.019

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As a house-keeping gene, Actin has been always being used as an internal standard to normalize mRNA levels between different samples by quantitative real-time PCR, thus plays an important role in gene expression analysis. In this research, through a method of homology cloning, a pair of degenerate primers were designed based on the conserved sequences of Actin genes from other plants submitted to GenBank, and then 4 cDNA fragments from Cinnamomum camphora were obtained using RT-PCR. Molecular biological analysis showed that, each of the 4 cDNA fragments was 998 bp and encoded a putative protein of 332 amino acids. Moreover, according to the homology analysis, the 4 cDNA fragments belonged to Actin subfamily, and they were named CcACTa, CcACTb, CcACTc and CcACTd, and deposited in GenBank（Accession number：KM086736 KM086737 KM086738 and KM086739）. Quantitative real-time PCR results revealed that the expression level of CcACTc in different organs such as root, stem, leaf and leaves under low temperature treaments was relatively stable. It could serve as a candidate reference gene.

The Effect of the Fermented Liquid and Mycelium Extract of FX-139 on the Growth and Defense Enzymes of Ginseng callus

Guo Shuangshuang, Shi Ce, Han Mei, Yang Limin

2015, 31(5):  128-133.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.020

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Using the fermented liquid and mycelium extract of actinomycetes FX-139 isolated from ginseng rhizosphere as the donor, the effects of it on the growth and ability of inducing defense enzyme activity of ginseng callus were studied. The results showed that: （1）The treatment of FX-139 fermented liquid and mycelium extract at the concentration of 20 mg/L had the strongest promoting effect on Ginseng callus growth, increasing 68.96% and 51.60% higher than that in the control, i.e., there was significant difference. （2）There was an active peak of PAL, POD and PPO in 21 d, 14 d and 28 d respectively under the treatment with water saturation n-butyl alcohol extract of fermented liquid, it raised 1.29 times, 2.5 times, and 2.12 times against the control group respectively. There was an active peak of PAL, POD and PPO in 21 d, 14 d and 14 d respectively under the treatment with acetone extract of mycelium, it raised 1.9 times, 2.6 times, and 1.4 times against the control group respectively. （3）The change of defense enzyme activities in ginseng callus was detected. PAL, POD and PPO enzyme activity were obviously changed with the treatment of fermented liquid and mycelium extract. Overall, Ginseng callus defense responses could be induced by fermented liquid and mycelium extract of FX-139. Fermented liquid extract could effectively induced POD and PPO, and mycelium extract could effectively induced PAL, POD and PPO.

Preparation and Regeneration of Protoplasts Isolated from Embellisia Fungal Endophyte of Oxytropis glabra

Hu Jiya, Lu Ping, Niu Yanfang

2015, 31(5):  134-139.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.021

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The aim of this research is to obtain more active and larger amount of protoplasts of Embellisia fungal endophyte from Oxytropis glabra, investigate the optimal conditions of protoplast preparation and regeneration, and lay foundation for setting up later exogenous gene transformation system. The protoplasts are made by enzymes, and the influences on protoplast yield regarding different combined enzymes and their concentration, pH, temperatures, osmotic solutions, enzymolysis time and hyphal culture time were discussed. The protoplasts regenerated on TB3 medium were analyzed for understanding the impacts of different enzymolysis time on the regeneration. The protoplast concentration reached to 4.42×10⁵ /mL when medium mass was hydrolyzed by enzymes consisting of 2.5%（W/V）lysing enzymes, 2%（W/V）cellulose and 3%（W/V）helicase, the culture time was 10 days, the enzymolysis temperature was set to 30℃, the pH was 5.8, the 1.2 mol/L MgSO₄ solution was selected as osmotic stabilizer and the cultures were incubated in a flat shaker（80 r/min）for 8 h. The protoplast regeneration rate reached to 46.7% when enzymolysis time was 6 h.

Optimization of Agrobacterium-mediated Transformation in Castor(Ricinus communis L.)

Li Wei, Zhai Yujia, Li Pengcheng, Wang Changlu

2015, 31(5):  140-145.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.022

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The main factors affecting the efficiency of Agrobacterium infection were optimized, and the optimal infection conditions were 5-day-old pre-cultured explants cultured in bacteria solution with OD₆₀₀ 0. 8. Infected explants after 5 d of co-culture were transferred to medium with 250 mg/L Kan for bud induction and many strains with Kan resistance ability were obtained. By adding 20 mg/L AS, 124mg/L Na₂S₂O₃ and 152. 4 mg/L DTT in the common culture medium, browning phenomenon was effectively inhibited and survival rate of resistant shoots was enhanced. Regenerated plants with resistance to Kan were verified by PCR and Southern blot test. Total 18 positive transformants were obtained.

Screening，Identification and Optimization of Cellulase-producing Strains

Li Zhengming, Zhang, Juan, Deng Zhongyang, Lu Fan, Qin Wensheng

2015, 31(5):  146-152.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.023

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To isolate efficient cellulase-producing bacteria, 180 bacterial isolated from rotten wood, humus, and other soil samples, and 44 were screened from them and confirmed as cellulase-producing bacteria by Gram’s iodine solution staining. In the subsequent secondary screening tests, those 44 isolates were cultured in fermentation media and their total cellulase activities（FPase）were measured and compared. The strain J1-3-1, with the highest FPase activity, was identified as Sphingobacterium sp. by 16S rRNA sequence analysis. The optimal fermentation conditions for enzymes production of J1-3-1 were determined. Under the optimal conditions, its activities of FPase, CMCase, and β-glucosidase reached 8.76, 28.04和7.02 U/mL, respectively. The results demonstrated that J1-3-1 was a promising candidate for potential industrial production of cellulase.

Isolation，Identification and Fermentation Characterization of a Bacitracin Producing Bacteria

Zhang Yuming, Ni Zhihua, Li Baoku

2015, 31(5):  153-157.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.024

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Bacitracin has been widely utilized in livestock breeding and cultivation, and it is necessary to screen strains for improving bacitracin production. In this study, low concentration of bacitracin was used to screen bacitracin producer, and one strain was isolated and identified as Bacillus licheninformis strain Y822. The optimal culture condition for the strain was pH7.0, and the improvement of oxygen supply was favorable to the production of bacitracin. B. licheninformis Y822 was sub-cultured for 5 generations. Each generation of the strain was used to produce bacitracin using the optimized fermentation condition. The average value of the produced bacitracin from 5 generations of B. licheninformis Y822 was （783.51±7.34）U/mL, and the percentage of bacitracin A was about（75.04±0.83）%. The production of bacitracin by B. licheniformis Y822 was stable and efficient. Therefore, B. licheniformis Y822 was a potential candidate for producing bacitracin in industry.

Clone and Heterologous Expression of the Poly-γ-glutamic Acid Synthesis Gene pgsBCAE from Bacillus amyloliquefaciens C1

Wang Shifeng, He Jian, Chen Yilu, Zheng Tao, Shen Qirong, Yong Xiaoyu

2015, 31(5):  158-166.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.025

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Poly-γ-glutamic acid（γ-PGA）, a new type of biodegradable material with high molecular weight, is synthesized by many microbes; the construction of genetic engineering strain will be highly valuable to the research of synthesis mechanism and production of γ-PGA. The gene cluster, pgsBCAE, involving in the biosynthesis of γ-PGA in Bacilus amyloliqueficiens C1 were amplified by SEFA-PCR, which were then ligated in expression vector pET29a（+）and transformed into E. coli BL21（DE3）host cells. The recombinant clone, E. coli BL21（pET29a-pgsBCAE）induced by IPTG obtained the ability to synthesize γ-PGA by fermentation in shake flask, and the yield of γ-PGA was 0.14 g/L. Both Mn²⁺ and Zn²⁺ increased γ-PGA production of E. coli BL21（pET29a-pgsBCAE）. With the increase of the concentration of Mn²⁺ and Zn²⁺, this promotion became much more significantly. On the other hand, Mg²⁺ in low concentration（1 mmol/L）also promoted γ-PGA production, the promotion decreased with the increase of Mg²⁺ concentration, while high concentration of Mg²⁺（4 mmol/L）inhibited γ-PGA production of the recombinant clone significantly. The above results suggested that Bacilus amyloliqueficiens C1 contained the intact gene cluster pgsBCAE for the synthesis of γ-PGA, which can be cloned and expressed in E. coli BL21. The production of γ-PGA in the genetic engineering strain was affected by several metal ions.

Isolation and Identification of Novel Denitrifier Strain Rhizobium radiobacter and Its Nitrifying Characterisation

Luo Xiaoxi, Gao Jianzhong, Chen Zaizhong, Yu Yongliang

2015, 31(5):  167-172.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.026

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In order to screen and identify new strain with nitrification, a denitrifier was isolated from sewage treatment plant. The strain named N312 isolated by enrichment and plate culture was identified based on its morphology characteristics, 16S rDNA gene sequence analysis and Biolog. The conditions of medium composition were optimized by orthogonal experiment. The autotrophic denitrifier strain N312 was isolated and identified as Rhizobium radiobacter, which was rod, Gram-negative, colony round and white. The results indicated that the nitrite removal efficiency by the strain could reach 100% after 6 days. The best medium was NaNO₂ 1.5 g, Na₂CO₃ 1.5 g, Mg₂SO₄·7H₂O 0.25 g, FeSO₄·7H₂O 0.2 g，KH₂PO₄ 0.5 g. Strain N312 is a new autotrophic denitrifier with a highly research value.

Gene Cloning and Expression Analysis of Fesod Gene from Chlorella pyrenoidosa

Shen Jia, Jiang Lingzhi, Sun Xue

2015, 31(5):  173-178.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.027

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The Fesod gene of Chlorella pyrenoidosa was cloned by RACE method. Total 1 215 bp Fesod sequence was obtained, containing 52 bp 5' untranslated region, 452 bp 3' untranslated region, and an open reading frame of 711 bp. The bioinformatics analysis showed that the molecular weight of FeSOD was 26.42 kD and isoelectric point was 6.98; the FeSOD protein located in the mitochondria with a probability of 73.9%; the FeSOD protein had no signal peptide; and the secondary structure prediction results showed that a-helix and random coil in FeSOD accounted for 53.39% and 39.41%, respectively. Then, the transcriptional expression of Fesod at different concentrations of salinity and salicylic acid was detected by real-time fluorescent quantitative PCR. The results showed that Fesod expression increased with the salinity varying from 15‰ to 45‰, and the expression quantity was 3.61 times under 45‰ salinity culture than that under 15‰ salinity. Moreover, salicylic acid could inhibit the mRNA accumulation of Fesod to a certain extent under 45‰ salinity, and its accumulation firstly increased and then decreased with the salicylic acid concentration from 0.1 to 2.0 mmol/L. In conclusion, the expression of Fesod was induced by salinity, while salicylic acid showed no significant effect on its expression.

Cloning and Tissue Expression Analysis of C-type Lectin Gene from Humphead Snapper（Lutjanus sanguineus）

Cai Jia, Zhang Xueli, Wu Zaohe, Jian Jichang, Lu Yishan, Feng Dongyue, Jiao Maoxing

2015, 31(5):  179-185.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.028

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According to conservative region of known C-type lectin（CTL）gene, a pair of degenerate primers were designed, and full length cDNA sequence of C-type lectin gene was amplified by RT-PCR and RACE PCR from spleen of humphead snapper, Lutjanus sanguineus（GenBank accession number: AGT37609）for the first time. The total cDNA sequence of the gene was 828 bp, consisting of a 663 bp open reading frame（ORF）and encoding 220 amino acids. The deduced amino acid sequence of humphead snapper CTL shared 30%-68% identities with other species CTLs. Phylogenetic analysis showed that humphead snapper CTL was clustered closely with mummichog, rock bream, and orange-spotted grouper CTL. Moreover, the mRNA expression levels in different tissues were analyzed by real time quantitative PCR. The result showed that humphead snapper CTL gene expressed in all tested tissues with highest level in liver, spleen and skin, moderate level in head kidney, kidney, stomach, intestine and muscle, and lowest level in heart and brain.

Expression of a Xylanase Gene Originated from Rumen Anaerobic Fungi Neocallimastix frontalis in Pichia pastoris

Wang Yan, Li Xiao, Chen Yong, Wu Yun

2015, 31(5):  186-193.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.029

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The anaerobic fungus Neocallimastix frontalis is one of main microorganisms in the rumen degrading xylan and cellulose and its xylanase has the potential application value. In this study, a xylanase gene Xyn11B originated from N. frontalis was codon optimized, and the optimized gene Xyn11Bm was synthesized. Based on the gene engineering technology, the yeast expression vector pPIC9K-Xyn11Bm was constructed, and the xylanase was induced and expressed in Pichia pastoris GS115. In shake flask level, enzyme activity of the recombinant Xyn11Bm reached the maximum up to 4874.8 U/mL. In 10 L fermentor, at 96 h after induction, the activity of recombinant enzyme was 5139.7 U/mL, cell wet weight and dry weight were 216.7 g/L and 117.3 g/L. Enzymatic properties analysis showed that the optimum reaction temperature and pH of Xyn11Bm were 50℃ and 5.0. In pH5.0-8.0, the enzyme had sound stability, but poor temperature stability. Substrate specificity analysis showed that recombinant Xyn11Bm could hydrolyze oat spelt xylan, birch xylan and soluble xylan 4-O-Me-D-glucurono-D-xylan, but not degrade lichenin and barley β-glucan. This indicated that the recombinant Xyn11Bm had potential application value.

Cloning of CAV-3 in Yak and Analysis Its Expression in Yak and Cattle

Zhao Juanhua, Pei Jie, Liang Chunnian, Guo Xian, Wu Xiaoyun, Zhang Liangbin, Yan Ping

2015, 31(5):  194-199.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.030

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This study aims to clone, analyze bioinformatics and determine the expression pattern of CAV-3 gene in yak. A pair of special primers were designed according to released mRNA sequence of bovine CAV-3 in GenBank. A coding region sequence of yak CAV-3 was amplified by RT-PCR; the general physical and chemical properties, hydrophobicity, protein domains and protein secondary structures were systemically analyzed and predicted by bioinformatics techniques. The expression levels of CAV-3 mRNA in some organs of yak and cattle were detected by semi-quantitative PCR. Real-time PCR was employed to examine the expression levels of CAV-3 mRNA in yak and cattle muscles. The coding region sequence of CAV-3 gene in yak contains a complete ORF（631 bp）which encoded 151 amino acids. CAV-3 mRNA expression in the heart and muscle was detected, but not in any other examined tissues, and its expression level in the heart was higher than in the muscle. The result in yak was consistent with that in cattle. Expression of CAV-3 gene in the yak muscle was less than in the cattle muscle, but not significantly（P>0.05）. The cloning and analysis of CAV-3 provided scientific basis for further study the function of CAV-3 gene in muscle development and muscle physiological process in the future.

Cloning，Expression and Sequence Analysis of Musca domestica Antifungal Peptide-1 and Musca domestica Lysozyme

Peng Chuanlin , Wei Chuanchuan, Wu Jianwei, Wang Yu, Xiu Jiangfan, Shang Xiaoli, Zhao Xuejun

2015, 31(5):  200-205.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.031

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The aim of this study is to have bioinformatics analysis of Musca domesitca antifungal peptide-1（MAF-1）and lysozymeb（LMZ）, also clone fused MAF-1-LMZ gene and have expression analysis of it. The encoding sequences of MAF-1 and LMZ were fetched from GenBank, and the structures and functions of 2 proteins were analyzed and predicted. The gene MAF-1-LMZ was amplified by polymerase chain reaction（PCR）, then was ligated into pET 28a and transformed into E. coli Origmi（DE3）competent cell, and induced with IPTG. The fusion protein MAF-1-LMZ in the expression vector was analyzed by SDS-PAGE. The activity of the protein was validated by methods of small test tube and doubling dilution. The open reading frame of the MAF-1-LMZ was 969 bp, encoding a putative protein consisting of 322 amino acids with a predicted molecular weight of 35 468.6 Da and pI of 8.31, indicating the gene expressed in E. coli Origmi（DE3）successfully. The purified target protein had the antifungal activity.

Study on Effects of Acetic Acid on Enzyme Production of the Recombinant Escherichia coli BL21 and Its Action Mechanism

Dai Kun, Wang Tengfei, Hao Zhaocheng, Tang Dandan, Liu Hongjuan, Wang Ruiming

2015, 31(5):  206-213.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.032

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Our objective is to study the effects of acetic acid from fermentationon the growth of the recombinant Escherichia coli BL21（DE3）/pET15b-TreS as well as the expression of the recombinant trehalose synthase gene, and the action mechanism was also discussed. Firstly, we studied the effect of acetic acidon the growth of recombinant Escherichia coli by experiments with adding acetic acid（sodium acetate）, and observed the morphological changes of recombinant bacteria by using environmental scanning electron microscope. Then, the effects of acetic acid（sodium acetate）on the permeability and the integrity of cell membrane were studied through the determination of electrical conductivityof the brothand the changes of OD_{260 of the supernatant. The activity of β-galactosidase in the supernatant was measured to determine the effect of sodium acetate on intracellular membrane, and then the influence of acetic acidon the conformation of membrane protein by using fluorescence analysis were detected. Finally, the effect of sodium acetate on the expression of the recombinant trehalose synthase gene by SDS-PAGE was studied. Results showed that the acetic acid（sodium acetate）could have certain inhibition effects on the recombinant bacteria growth, and caused cell surface to be depressed and shrinkage. The permeability and integrity of cellmembranes were affected by the acetic acid（sodium acetate）, leading some substances in cells to leak out. And experiments also revealed that the acetic acid could make effects on the conformation of the membrane proteinin the cell membrane,which caused a certain degree of damages to the membrane structure. SDS-PAGE analysis suggested that the sodium acetate had impacts on the expression of the recombinant trehalose synthase gene. The acetic acid（sodium acetate）was confirmed to affect the cell growth and the expression of the recombinant trehalose synthase gene, and the cell membrane was a target of its function.}

Mutagenesis of Trichoderma harzianum with Pesticide Mutiple-resistance by Atmospheric and Room Temperature Plasma

Zhou Wenchen, Zhan Xiaobei, Zhu Li, Zheng Zhiyong, Wu Jianrong

2015, 31(5):  214-223.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.033

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This study aimed to improve the adaptability of biocontrol Trichoderma in the complicated agricultural environment in where herbicides, insecticides, and fungicides co-exist. Trichoderma harzianum was used as initial strain and induced by atmospheric and room temperature plasma（ARTP）. The Trichoderma harzianum mutants resistant to both fungicides mancozeb and insecticide dinotefuran were obtained and one with the most double-resistance was named as Th-36. The mutant was subject to further mutation by ARTP and screened on the medium with mancozeb, dinotefuran and herbicide oxyfluorfen. Two mutants with triple-resistance were obtained and named as Th-3-36 and Th-4-B. The toxicity resistance test showed that EC₅₀s of mancozeb for Th-3-36 and Th-4-B were up to 5089.2 μg a.i.·mL^-1 and 5498.2 μg a.i.·mL^-1；for dinotefuran up to 758.5 μg a.i.·mL^-1 and 785.9 μg a.i.·mL^-1；for oxyfluorfen up to 198.2 μg a.i.·mL^-1 and 200.3 μg a.i.·mL^-1 respectively. The above values were all higher than the average commercially recommended doses of these pesticides. The screened mutants showed the significant increasing resistance to pesticide, inherited parental broad-spectrum antibacterial property, and presented genetic stability. Furthermore growth capacity and enzyme characteristics of the mutants were better than those of the parental strains.

The Study of Genetic Diversity in Tibetan Pig of Tibet

Guo Yongbo, Cai Yuan

2015, 31(5):  224-230.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.034

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mtDNA D-loop was selected as a marker to determine genetic diversity of the 4 Tibetan pig populations（Nyingchi, Shannan, Chamdo and Shigatse）in Tibet. The results showed A + T content（62.90%）was significantly higher than the G + C content（37.1%）, indicating that Tibetan pigs’ mtDNA D-loop was rich in A and T; there was the presence of a base bias. A total of 20 variable sites and 26 haplotypes were identified in the 435 bp nucleotide sequence of mtDNA D-loop, haplotype diversity（Hd）was 0.705 ± 0.021, the average number of nucleotide differences（k）was 1.231, and nucleotide diversity（Pi）was 0.00283. Among them, Hd, k and Pi were the highest in Chamdo Tibetan pig population, and lowest in Shigatse. In addition, Hap1 and Hap3 were 4 shared haplotypes, revealing that there were 2 common maternal ancestor haplotypes in 4 Tibetan pig populations.

Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7

Wen Xianchun, Han Cuicui, Zhao Yuesheng, Yu Haitao, Li Chengchong, Yue Liling

2015, 31(5):  231-236.  doi:10.13560/j.cnki.biotech.bull.1985.2015.05.035

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This study aimed to construct the lentiviral RNA interference（RNAi）vector of human FUT8 gene and determine its effect on proliferation of human breast cancer cells MCF-7. Designing three short hairpin RNA（shRNA）sequence targeting the FUT8 gene, and then synthesizing the complementary DNA chains containing the target sequence, miRNA lentiviral vector plasmid with linear pGC-LV-GFP carriers were constructed and then transformed to DH5α cells. After identified by sequencing, then packaging lentiviral vectors and measuring the virus titer were done. The recombinant lentiviral vector pGC-shFUT8 was transfected into human MCF-7 cells, and then the expression of FUT8 in transfected MCF-7 cells was detected by real time-PCR and Western Blotting. The effect of shFUT8 on MCL-7 cell proliferation was measured by MTT assay and colony formation experiment. Gene sequencing confirmed the successful construction of RNAi lentiviral vector targeting the FUT8 gene. The lentiviral vectors were packed successfully by 293T cells and the virus titer was more than 5×10⁸ TU/mL. Expressed GFP in transfected cells were observed under the fluorescence microscope, and the transfection efficiency was over 90%. Real-time PCR and Western Blotting analysis indicated that the expression levels of FUT8 mRNA and protein in the transfected group significantly reduced when comparing with the negative control group. Particularly the pGC-shFUT8-2 sequence could interfere 80 % of FUT8 gene expression. The proliferation of MCF-7 decreased after FUT8 was inactive.