Table of Content

21 August 2015, Volume 31 Issue 8

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Review

Response Mechanism of Plant Cuticular Wax Involving in Drought Stress Response

Wei Baiyang, Xu Xiaojing

2015, 31(8):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.001

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The plant cuticle, which connects with the external environment directly, is composed of an intermeshed cutin polyester membrane overlaid with free waxes. Plant cuticle forms specific structure and complex chemical composition in the long-term process of adaptation to outer environment. The most important function of cuticular wax is recognized as involving in blocking water loss through non-stomatal transpiration, thus improving the use efficiency of water in plant, and finally adapting to the drought environment. Drought stress can lead to metabolic changes in cuticular wax, which is eventually conducted through the regulation of gene expression. Recently, many wax metabolism-related genes involving in plant adaptation to drought stress have been discovered. Some genes have been cloned and used to improve the drought tolerance of crops. However, it is still not clear that the molecular mechanisms of these genes involve in drought tolerance as well as the relationship with ABA. Here, we review the changes of metabolism, including the composition and content of wax while plants adapting to water deficit conditions, and the main genes involved and their molecular biology. Understanding the role and molecular mechanism of cuticular wax in the adaptation of plants to drought may provide new molecular markers and important target genes for breeding the drought tolerance of agricultural crops, i.e., better service for agricultural practices.

Research Progress on Regulation of Oil Synthesis in Plants

Zhao Guomiao, Zeng Yanru, Xu Ya’nan, Jia Ning, Tang Yanyao

2015, 31(8):  9-16.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.002

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Plant seeds are a principal source of oils in humans’ daily life. In recent years, researches on plant seeds have illustrated the principle of oil synthesis and mined some key enzymes and genes involved in the regulation of oil synthesis pathway. Based on the previous study, the article added progress in pathways and genes involved in oil synthesis, and transcriptional regulation, as well as summarized the possible influence of carbon supply and transportation, endosperm, etc. on oil synthesis.

Research Progress on Invertebrates DNA Methylation

Liu Ying, Tang Yongzheng, Gao Li

2015, 31(8):  17-23.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.003

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DNA methylation is an important epigentic mechanism which can change organism’s transcriptome and phenotype without altering DNA sequence. In different evolutionary braches, the states of DNA methylation are fundamentally different. In invertebrates, DNA methylation usually occurs in transcription region and is closely related to genetic expression. Research shows that DNA methylation plays an important role in caste formation of social insects. It can improve the phenotypic plasticity of organism by increasing the amount of transcription variants via promoter recognition and exon skipping in highly fluctuating environments. This article reviewed the mechanism and function of DNA methylation in invertebrates to provide reference to related research and make prospects for future research.

Research Progress on Proteins Related to Deer Antler Regeneration

Wang Quanwei, Dong Zhen, Wang Guiwu, Yang Fuhe, Liu Hui, Li Chunyi

2015, 31(8):  24-29.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.004

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Deer antler is an only known mammalian organ that can periodically regenerate, and grow very rapidly without going cancerous. Proteins are the carrier and the function executor of life activities, the research of proteins related to deer antler is an important measure to unlock the secrets of antler’s regeneration. The proteins involved in angiogenesis, cartilage formation, nerve regeneration and other functions during antler regeneration are reviewed which aims at providing basic data for revealing antler regeneration process at the protein level.

Research Advances in the Studies of Plant Entophytic

Chen Long, Liang Zining, Zhu Hua

2015, 31(8):  30-34.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.005

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Entophyte is a kind of micro living organism that lives within a plant for at least a certain period of its life without causing apparent symptoms of disease to the plant. The studies found out that it mainly includes entophytic bacteria, entophytic fungi, and entophytic actinomycetes. Entophyte can produce active substances similar to that of the host plant, which thereby broadens the medicinal resources and correspondingly have a great application value. The authors summarize the following aspects: the distribution patterns and growing characteristics of entophyte, its symbiotic relationship with the host plant, its research fields and current situation of development, together with existing problems. Also, authors forecast the prospects of entophytes utilized in the development of medicinal resources.

The Applicatioin of Stenotrophomonas maltophilia in Environmental Remediation and Agriculture

Li Yulong, Han Zhengmin

2015, 31(8):  35-43.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.006

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Stenotrophomonas maltophilia is a gram-negative bacterium and widely distributed in nature. It has multiple dioxygenase that can biodegrade alkane and polycyclic aromatic hydrocarbons (PAHs), and also adsorb heavy metals. S. maltophilia is a kind of typical plant growth-promoting rhizobacteria. It can secrete various hydrolytic enzymes and secondary metabolites that enhance plant growth, prevent and control fungal diseases along with nematode. Owing to the above features, S. maltophilia possesses the significant application values in environmental remediation, agriculture and forestry. The mechanisms of bioremediation and plant growth-promoting of S. maltophilia are summarized, which is expected to provide a reference for further studies and application of S. maltophilia.

Technique

Research Progress on Metabolic Engineering for Microbial Production of L-serine

Liu Yan, Wang Hui, Shi Jiping, Zhao Zhijun, Cong Li-na

2015, 31(8):  44-49.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.007

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L-serine is an important intermediate metabolite as a metabolic precursor to L-glycine and many other amino acids, nucleotides, choline, and phospholipids in organisms, and it has been widely used in medicine, food, cosmetics and other industries. The methods of producing L-serine include protein hydrolysis, chemical synthesis, enzymatic/microbial conversion and microbial fermentation, among which microbial fermentation has some advantages such as low cost of raw material, less environmental pollution, and high purity of product, etc. The strategies of metabolic engineering for improving L-serine production are reviewed, including the regulatory mechanisms and genetic modification of L-serine biosynthesis, as well the modified measures and subsequent effects. Furthermore, the prospect of L-serine breeding technologies is also discussed.

RNAi Technology and Its Application in Fungal Gene Functional Studies

Su Zijing, Li Qiaoling, Huang Cheng, Xie Chengjian, Yang Xingyong

2015, 31(8):  50-58.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.008

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RNA interference (RNAi) is a phenomenon of post-transcriptional gene silencing triggered by highly conserved exogenous or endogenous double-strand RNA (dsRNA) during the evolution. RNAi, as an efficient and specific gene blocking technology, is widely applied to gene functional studies and the areas of treating infectious diseases and malignant tumors. RNAi has become one of the hotspots in the 21st century. This paper reviewed the several aspects of RNAi, including the history of discovering it, mechanism, characteristics, and the application prospects in fungal gene functional analysis, which may provide the theoretical basis for related researches.

Research Progress on Cellulase Immobilized by Magnetic Nanoparticles as Carriers

Xing Zhaohui, Su Yuelong, Zhang Qi, Ruan Xinyi, Lin Yan, Wang Xinze, Kong Hainan

2015, 31(8):  59-65.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.009

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Conversion of biomass to reducing sugar by cellulase is the foundation of the biomass fuel production. Nowadays researchers have invented the enzyme immobilization technology to avoid the disadvantages of free enzymatic hydrolization. Utilization of magnetic nanoparticles as carriers for the cellulase immobilization can increase the catalytic activities of enzyme and enhance stability of the enzyme. Moreover, replacing conventional mechanical stirring by external magnetic field can give full play to the role of carriers’ magnetic response; with the immobilization on magnetic nanoparticles, the produced cellulase can be separated easily from the mixture in the reaction systems. Researchers have proposed many excellent immobilization methods. Here the different methods of cellulase immobilization by magnetic nanoparticles are reviewed, their applications are explained in detail, and consequently the advantages and disadvantages as well as the development prospect are discussed.

Detection of the RYR1 Gene in Guizhou Indigenous Pigs and Three-crossbred Pigs

Feng Wenwu, Long Weihai, Ding Mei, Chen Xiang, Chen Cunnian

2015, 31(8):  66-70.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.010

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To provide the technical support for the cultivation of "strain of specialization with anti-stress and sensitive gene" of Guizhou local pig genes, the RYR1 gene in 151 samples from 5 local pig species of Baixi, Qianbei Black, Zongdi Hua, Nuogu, Congjiang Xiang and three-crossbred commercial pig were detected by PCR-RFLP technique. The pigs with HalNn of recessive anti-stress and sensitive genotype, HalNN of anti-stress and sensitive genotype and Halnn of stress and sensitive genotype were screened out. The results showed that all of 5 Guizhou indigenous pigs belonged to HalNN , there was no Halnn gene in the three-crossbred pigs, HalNn accounted for 52.63 % of the samples, and Haln allele frequency was 26.32%.

A RT-LAMP Assay for Detection of Novel Duck Reovirus

Yu Kexiang, Ma Xiuli, Han Hongyu, Liu Cunxia, Li Yufeng, Huang Bing, Song Minxun

2015, 31(8):  71-75.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.011

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A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting novel duck reovirus (NDRV) was established with 4 primers based on 6 conserved positions of the S3 gene. The process of assay was completed by using Bst DNA within 45 min at constant 63℃. RT-LAMP assay had solid specificity because no amplification was found with the samples of 6 other common duck diseases. The minimum detection limit of the RT-LAMP assay was 0.1 pg of viral RNA, which was 100 times of RT-PCR. The results of clinical application showed that the coincidence rate between the assay and the method of virus isolation and identification was 98%, and the requirement of instrument for the assay was relatively low. Therefore, the assay is a potential useful technique for NDRV detection in the field.

Research report

Dynamic Expression of miR169o and Its Target Genes OsNF-YAs in the Early Response to Water Deficiency in Rice

Chen Yutong, Chen Huamin, Yu Chao, Amy Thein, Tian Fang, He Chenyang

2015, 31(8):  76-81.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.034

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MicroRNAs（miRNAs）is noncoding RNAs and play important roles in plant development and response to various environment stresses. Recent evidence have indicated that miR169 is upregulated response to drought stress, and overexpression of miR169 contributes to improve the tolerance to drought stress in plant. However, it is still unclear to the expression pattern of miR169 under drought stress and the regulatory mechanism response to drought stress in rice. In this study, the dynamic expression patterns of miR169o and its target gene NF-YAs（Nuclear Factor Y A subunit, NF-YA）in the root, stem and leaf of rice were analysed systematically by qRT-PCR after water deficiency treatment. Generally, miR169o showed up-regulated after water deficiency stress treatment compared to that before treatment, while target gene NF-YAs showed imperfect reverse expression pattern to miR169o. In addition, the expression pattern and abundance of miR169o in rice root, stem and leaf suggested that the expression of miR169o is tissue-specific.

Cloning and Transformation of Gene GmGW2 in Soybean

Xiong Hewen, Wu Zhihui, Duan Siyu, Xie Hao, Qin Yutao, Guo Bei

2015, 31(8):  82-87.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.012

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GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is located in cytoplasm. It is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers. It was reported that the mutation of GW2 lead to change the size of rice seeds. According to rice GW2 gene sequence, blast in the NCBI, we found a soybean GW2 gene which is homologous. We cloned the target gene and carried on bioinformatics analysis, then we constructed a over-expression vector pCAMBIA1300∷GmGW2∷EYFP, and transformed into soybean named zhonghuang10 by Agrobacterium-mediated method. Seeds were germinated on sterile moistened paper towels in Petri dishes for 24 h then infected the immature embryo by Agrobacterium EHA105 which obtained recombining vector. When the explants have 3 compound leaves, we extract the genome DNA and PCR identified the report gene EYFP. The content of EYFP is enhanced in PCR positive plant was observed by LSCM. It is demonstrated the transformation of target gene is successful. The sequence analysis showed that the target gene have higher homology with those of Alfalfa and Gram.

Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana

Hu Tao, An Yan, Lv Qundan, Xu Yingwu

2015, 31(8):  88-93.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.013

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The proteins containing SPX domain exist widely in eukaryotes, and the functions of this kind of proteins are not clear yet, however some of them are involved in phosphorus signaling and some in iron signaling. SPX proteins in Arabidopsis thaliana can be divided into 4 families. AtSPX1 used in this paper belongs to the family containing only SPX domain, while other 3 families containing extra gene sequences. Phylogenetic analysis showed that amino acid encoded by AtSPX1 was close with dicots in sequence, but distant from monocots. In order to reveal the relationship between structural property and biological function, we carried out solubility expression experiments of the proteins in vitro, constructed prokaryotic expression vector in vitro, and had the high soluble expression of the protein in Escherichia coli’s cells. The expressed His-tag proteins allowed the purification more convenient, i.e, the inserted SUMO fusion protein tag could be digested by protease, and the target protein could be purified by ammonium sulfate precipitation. The further purification was completed using size exclusion chromatographic column, which yielded a profile corresponding to a monomeric AtSPX1 in solution. This work provided a strategy for the purification of AtSPX1 protein.

Preliminarily Research on Involvement of Arabidopsis GCR2 Responsing to N-Butyryl-DL-homoserine Lactone

Ai Qiushi, Zhang Zhe, Qu Lingbo, Liu Fang, Zhao Qian, Song Shuishan

2015, 31(8):  94-101.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.014

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N-Butyryl-DL-homoserine lactone（C4-HSL）is a main quorum sensing signal in gram-negative bacteria. It could significantly promote root elongation and activate Ca²⁺ channel at cytomembrane. C4-HSL can regulate metabolization of plant. However, little is known about the molecular mechanism of plants responding to C4-HSL. Arabidopsis thaliana GCR2 is receptor of abscisic acid（ABA）. It is important for metabolization of plant. This research aimed to explore whether the GCR2 is involved in the process of Arabidopsis thaliana reacting to C4-HSL. qRT-PCR showed that C4-HSL could regulate expression of GCR2. Expression of GCR2 was significantly upregulated after 1 h treated by C4-HSL and maximaized at 6 h. ELISA also showed that expression of GCR2 maximaized at 6 h. GCR2 was purified and condensed to 0.6 mg/mL for Microscale Thermophoresis（MST）measurement. MST indicated that dissociation constant（K_d）of GCR2 and C4-HSL was 166 nmol/L, which meant that they had strong binding affinity. Taking BSA as negative control, this certified that binding of GCR2 and C4-HSL had special character. These results showed that Arabidopsis GCR2 maybe involved in the pathway of plant responding to C4-HSL.

Identification and Construction of RCAS RNA Interference Vector for Chicken Vezf1

Zhang Chao, Ge Jun, Yuan Mingjing, Ye Jun, Yuan Li

2015, 31(8):  102-107.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.015

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In order to study the role of Vezf1 during the earlier development of chicken embryo, RNA interference vector for the chicken Vezf1 was constructed based on RCAS viral vector system, and the gene silencing of chicken Vezf1 was carried out in both embryonic cells and chicken embryos, and the mRNA expression was measured by Real-time PCR and in situ hybridization. Results showed that this interference vector from RCAS virus could keep Vezf1 expression silencing successfully in chicken embryo fibroblasts and chick embryos. This study provides a fundamental data for studying the Vezf1’s role during early embryonic development using the chicken model.

Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System

Qiu Tianxiu, Li Changhong, Chen Jiong

2015, 31(8):  108-113.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.016

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The mature peptide of Wap65-2 gene（LcWap65-2m）in large yellow croaker（Larimichthys crocea）was used to construct the yeast two-hybrid bait vector, and its self-activation and toxic effect were measured. LcWap65-2m from the liver cDNA of large yellow croaker was amplified, and cloned into the plasmid pGBKT7. After verification by PCR amplification, enzyme digestion and sequencing, the bait vector pGBKT7-LcWap65-2m was transformed into the yeast strain Y187. The self-activation and toxic effect of the recombinant protein were detected in selective nutrition medium, and the expression of the bait protein LcWap65-2m was analyzed by Western blot. LcWap65-2m was successfully amplified and inserted into the plasmid vector pGBKT7. Western blot analysis showed that the bait protein LcWap65-2m was expressed in yeast cells;phenotypic screening tests revealed that the bait protein LcWap65-2m had no self-activation activity and toxic effect. In conclusion, the yeast two-hybrid bait vector pGBKT7-LcWap65-2m was successfully constructed, which laid the foundation for screening the protein interacted with the bait protein Wap65-2 using the yeast two-hybrid system.

Enzymatic Characterization of NADP-dependent Isocitrate Dehydrogenization in Pinus sylvestris var. mongolica Ectomycorrhiza

Wang Yichao, Yao Qingzhi, Zhu Heping, Chen Lixia, Guo Xin, Yang Qianqian, Yan Wei

2015, 31(8):  114-118.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.017

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The purpose of the work is to purify the isocitrate dehydrogenase（IDH）in mycorrhizal tissue of Suillus luteus-Pinus sylvestris var. mongolica, root tissue of P. sylvestris var. mongolica and cultured fungal mycelia of S. luteus, and identify their enzymatic characterizations. The IDHs of 3 sources were purified by ammonium sulfate precipitation and glucan gel chromatography and tested by SDS-PAGE electrophoresis, and enzymatic characterizations were studied. The K_m for NADP⁺ of mycorrhiza, root and cultured fungal mycelia were 10.7 μmol/L, 11.4 μmol/L and 22.1 μmol/L, respectively;the K_m for isocitrate were 71.7 μmol/L, 79.3 μmol/L and 87.8 μmol/L, respectively. The optimal pH of mycorrhiza, root and cultured fungal mycelia were 8.2, 8.0 and 7.5 respectively;they were all slightly in alkaline. The optimal temperatures of the IDHs were 45℃ for mycorrhiza and root, and 42℃ for the fungus. The activities of 3 IDHs relied on the binding of divalent metal ions, the maximum activities of IDHs were observed when assayed with Mn²⁺ or Mg²⁺ as metal cofactor;however, Ca²⁺, Co²⁺, Cu²⁺ and Zn²⁺ dramatically inhibited the activity of IDHs. Conclusively, protein content and enzyme activity of mycorrhizal IDH have been increased.

Purification and Enzymatic Properties of Laccase from Myrothecium verrucaria GH-01

Wang Zijuan, Zhao Min

2015, 31(8):  119-124.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.018

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Myrothecium verrucaria has the advantages of short growth cycle and high enzyme activity of secreting laccase. In this study, the crude enzyme was produced by the M. verrucaria GH-01 obtained by separation. Further we purified the crude enzyme by fractional precipitation, dialysis and chromatography. The results of SDS-PAGE and Native-PAGE indicated that the monosome protein with laccase activity was obtained by purification. The study of enzymatic properties of laccase showed that the optimal temperature of catalyzed reaction for laccase was 40℃, and the laccase activity against ABTS showed a maximum at pH4.0. It had the favorable stability under the low temperature and alkaline conditions. Besides, studying the laccase decolorizing of 4 representative dyes demonstrated that the laccase decolorizing rate for orange I of azo dyes and alizarin red of anthraquinone class was the highest with decolorizing rate over 80% by 1 h, lower for fuchsin of triphenylmethane class with decolorizing rate only 20% and lowest for methylene blue of heterocyclic class. In 10 U laccase system, the decolorizing degree of the 50mg/L dye concentration was optimal.

Screening of High-yield Strain of Wuyiencin Gene wysR Over-expression and Its Biological Characteristics

Wang Jiawang, Ge Beibei, Liu Yan, Liu Yanyan, Zhang Kecheng

2015, 31(8):  125-131.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.019

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In this research the over-expression strains with positive-regulating gene wysR synthesized from Wuyiencin were utilized as primary materials, 35 relatively optimal strains were selected from 2060 individual colonies of over-expression strains by agar blocks, then 9 high-yield strains were screened out adopting the method of shake flask fermentation. Ultimately, the strain W-273 was determined as the optimal strain by genetic stability analysis. Comparing the physiological and morphological characteristics between the strain of W-273 and original CK-15, W-273 strain grew faster, the production of spore was higher, and the time of spore production was in advance. The time to complete spore production for W-273 strain was 3-4 d, and this was in advance for 3-4 d than the original strain CK-15. Through electron microscope observation, the spores of W-273 were oval or rod, and the CK-15 strains were circular. Shake flask fermentation showed that strain W-273 had a higher metabolism compared to strain CK-15, antibiotic production increased 79%-289%, and the best fermentation time shortened 4-8 h.

Screening the Extracts of Marine Fungi for the Activity of Damaging the DNA of Escherichia coli and Researches on Active Strains

Bao Hanyan, Gu Pengjuan, Zhang Yi, Mu Jun, Feng Yan, Zhao Chenyan

2015, 31(8):  132-139.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.020

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Screening of antimicrobial and antitumor agents from marine microorganisms is one of the hot spots for the research of marine drugs, and screening models play an important role in the study. The authors set up an Escherichia coli differentiated DNA damage repair test model, which can be used for the screening of antimicrobial and potential antitumor substances. This model was further applied to screen the bioactivities of the extracts of marine symbiotic fungi. As the result, five marine fungal strains’ extracts showed strong selective inhibition to E. coli strains with DNA damage repairing genes-deficiency, i.e, the potential of damaging DNA. The five strains were identified by molecular biological methods. Furthermore, analyses by thin layer chromatography（TLC）and high performance liquid chromatography（HPLC）-bioactivity tracing revealed that these strains produced diverse bioactive substances.

Selection and Identification of Oil-producing Energy Microalgae in Northeast Region

Shi Wenjing, Liao Sha, Sun Qimei, Wang Pengxiang, Li Xiaoshu

2015, 31(8):  140-146.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.035

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In the 21st century, the ideal microalgae biodiesel fuel has been widespread concern, but how to filter high in fat content from a lot of microalgae species has become a problem to be addressed. In this study, 93 strains of microalgae were isolated and purified from the water samples of the Northeast region, and 30 strains were screened to obtain eight oil-producing algae by Nile red fluorescence. Oil production capacity of these eight microalgae were evaluated with bubble column photobioreactor. This experiment obtained a higher oil yield algal and its total oil yield reached 133. 9 mg /（L · d）. On this basis an oil-producing energy microalgae was identificated by 18S rRNA. This oil-producing energy microalgae was identificated as Chlorella sp.

Cloning,Expression and Enzymatic Activities of chiA73 Gene from Bacillus thuringiensis

Zhang Yuan, Zhou Guowang, Li Haitao, Liu Rongmei, Zhao Yikui, Gao Jiguo

2015, 31(8):  147-152.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.021

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In order to identify and detect chitinase activities from Bacillus thuringiensis strains, chitinase gene chiA73（GenBank accession number：KJ508093）from genomic DNA of Bt DLD171 producing high-yield chitinase was cloned by PCR. Gene of chitinase chiA73 was expressed and the weight of expression product was approximate 74 kD. The results measured by fluorogenic substrates showed that the expression product of chiA73 hydrolyzed 4-MU-（GlcNAc）₃ only, but not 4-MU-GlcNAc. In conclusion, the work indicated that ChiA73 was one of endochitinase, and had the highest activity at pH8 and 40℃.

A Study of Bacillus thuringiensis Strain LTS290 Inhibiting Fusarium

Zhou Guowang, Li Yuhong, Zhang Yuan, Li Haitao, Liu Rongmei, Gao Jiguo

2015, 31(8):  153-158.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.022

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In oder to expand the biocontrol scope of Bacillus thuringiensis（Bt）, the inhibiting effects of confront culture and sterile fermented liquid of strain BtLTS290 on the growth of Fusarium were investigated. Results showed that BtLTS290 inhibited the growth of 6 strains of Fusarium, and the sterile fermented liquid of Bt had solid antibacterial effect. The fungistatic substances were not sensitive to temperature, and it still had the bacteriostatic activity for 20 min under 121℃. After storing 12 h while pH varied from 3 to 11, the range of antibacterial activity was not significantly changed. The bacteriostatic activity still existed after being treated with proteinase K. Conclusively, the work convinced that the BtLTS290 was a biocontrol strain that had efficient antagonistic activity against Fusarium of causing dry rot of potato.

A Study on Antibacterial Mechanisms of Ethanol-extracts from Scutellaria baicalensis Against Vibrio parahaemolyticus

Xie Liling, Peng Qi, Cai Lianchun, Zhou Liang, Zhu Yankun, Han Guangyao

2015, 31(8):  159-165.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.023

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The goal of this work is to screen one with strong antibacterial activities against Vibrio parahaemolyticus from 4 traditional Chinese medicines（TCMs）, and study the underlying mechanisms. The sensitivity of ethanol-extracts from TCMs to Vibrio parahaemolyticus was determined by Oxford cup assay and liquid double dilution method. The antibacterial mechanism was investigated by measuring growth curve and bactericidal curve, electron microscope examination, and SDS-PAGE analysis. The results showed that different ethanol-extracts from 4 TCMs had different antibacterial activities upon V. parahaemolyticus, among them the ethanol-extract from S. baicalensis had the best antibacterial activity with an inhibition zone diameter of 22.00 ± 1.00 mm. Both the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of the extract against V. parahaemolyticus were 3.91 mg/mL. V. parahaemolyticus treated with Scutellaria extract in MIC still entered a normal log and stationary phase after a longer lag phase. Bacterial surface became rough, showing a sign of cell lysis. Protein concentration in the supernatant increased significantly. However, the concentration of soluble proteins changed lightly after the treatment of Scutellaria extract. Conclusively, Scutellaria extract has a significant inhibition effect against V. parahaemolyticus by destroying the integrity of cell membrane and causing cell lysis.

The Effects of luxR_19 on the Synthesis of Phenzaine-1-carboxamide in Pseudomonas chlororaphis HT66

Chen Yawen, Peng Huasong, Wang Wei, Zhang Xuehong

2015, 31(8):  166-173.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.024

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In order to study the potential effects of regulator gene luxR_19 on the synthesis of antibacterial substance phenazine-1-carboxamide（PCN）and physiological-biochemical characteristics in Pseudomonas chlororaphis HT66, strains of gene luxR_19-knockout, -complementation, -overexpression and -point-mutation were constructed, and then the growth curve, fermentation production of PCN, swimming and swarming activities were tested at the target strains. The results showed that the knockout of luxR_19 lowered the production of PCN and the over-expression of gene luxR_19 increased the double production of PCN compared to the wild type;luxR_19 gene affected the expression of several genes relating to the regulation or synthesis of phenazines. Besides, the knockout of luxR_19 had no effects on the growth and swimming activity, however, promoted the swarming activity of HT66. The production of PCN in point-mutation strain of luxR_19（G85A）was similar to that in wild type. The above results indicated that luxR_19 was the positive regulator of PCN production, and could affect the swarming activity of HT66. Besides, the point-mutation at 254^th site in luxR_19 did not affect the production of PCN.

Construction of a Recombinant Strain Producing High-yield Dihydroxyacetone

Xu Xiaojing, Zhao Qiong, Wang Xianghe

2015, 31(8):  174-179.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.025

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This work aims to construct a recombinant strain containing gene sldAB over-expressing membrane-bound glycerol dehydrogenase for raising the yield of dihydroxyacetone（DHA）. Firstly, using genomic DNA of Gluconobacter oxydans ATCC621H as a template, sldAB amplified by PCR was ligated to the plasmid pBBR1MCS-2 and the expression vector pBBR1MCS-2-sldAB was constructed;subsequently the plasmid pBBR1MCS-2-sldAB was transformed into the G. oxydans ATCC621H by electrotransfer, and the recombinant strain GOX205 was obtained. Results showed that the recombinant strain was constructed successfully, and the activity of glycerol dehydrogenase increased by 26% compared with the original strain. When initial concentration 100 g/L of glycerol as carbon source, the growth of GOX205 was significantly improved compared with the original strain, the final DHA concentration was 94.1 g/L and increased by 19.7%, and the residue of glycerol decreased 15.1 g/L. Therefore, this study provides a basis for further improving the biotransformation process of DHA production.

Optimization of Preparing L-citrulline by Recombinant Arginine Deiminase

Ma Yue, Su Lingqia, Wu Dan, Wu Jing

2015, 31(8):  180-185.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.026

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The arcA gene encoding ADI from Pseudomonas putida ACCC 10185 was cloned into the expression vector pET-24a（+）. The vector was then transformed into Escherichia coli BL21（DE3）for intracellular production of ADI. The crude enzyme was obtained by ultrasonic treatment, and activity in the fermentation broth of recombinant E. coli BL21（DE3）was 26 U/mL. Furthermore, the condition for enzymatic conversion of L-arginine monohydrochloride to L-citrulline by the recombinant ADI was optimized. At 650 g/L of L-arginine monohydrochloride, pH6.0, 37℃, 100-200 r/min, and 24 U ADI per gram substrate incubated for 7 hours, 100% of the L-arginine monohydrochloride was transformed into L-citrulline, which was the highest level of preparing L-citrulline by enzyme method in home and abroad presently.

Process Optimization of Extracting Polysaccharides from Onchidium struma,and Evaluation of Its Antioxidant Activity in Vitro

Cheng Zhiqing, Shen Heding, Yao Lixiang, Diao Ya, Liu Chen

2015, 31(8):  186-192.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.027

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Taking Onchidium struma as the raw material, its polysaccharides was extracted by the traditional method of hot water. Process of extracting polysaccharides from O. struma was optimized by response surface methodology. Through the single factor experiment, the effects of extraction temperature, extraction time, and liquor-to-material ratio to the extraction rate of polysaccharide were investigated. Furthermore, the experiments were done by Box-Behnken, and the data was analyzed by Design-Expert 8.05 software. Antioxidant activity of O. struma polysaccharides was determined by the capacity of scavenging free hydroxyl radical and DPPH radical. On the basis of the analysis of regression equation, determination of factors affecting the extraction of polysaccharide was conducted by the extraction rate of polysaccharide served as response value. Extraction temperature at 92℃, extraction time of 30 h, and liquor-to-material ratio of 1∶29 g/mL, leaching rate of O. struma polysaccharide reached 9.206%. O. struma polysaccharide had ability to clear off free hydroxyl radical and DPPH radical. Conclusively, the extraction technology is reasonable and reliable for the extraction of polysaccharides O. struma, which provides the theoretical support for the future industrial production, and its polysaccharides have significant antioxidation in vitro.

A Study on Transesterification and Acid-resistant Antiprotease of Carboxypeptidase in Porcine Pancreas

Feng Shiyuan, Sun Tongwei, Mou Huiyan, Zhang Huitu, Lu Fuping

2015, 31(8):  193-199.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.028

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Carboxypeptidase is a kind of protease that cleaves the peptide bond of an amino acid residue at the C-terminal end. It can be used in feed additives, and promotes the growth of livestock and poultry. A carboxypeptidase A1（CPA1）was obtained from porcine pancreas, and it can hydrolyze proteins and decompose oils. It showed the specific activity as 53.14 U/mg and 22.4 U/mg when casein and olive oil used as substrate, respectively. The CPA1’s relative activity reached 95.65% and 78.14% respectively after the treatment of pH2.0 acidic and trypsin. The results indicate that the CPA1 has the function of transesterification and the feature of distinct acid-resistant antiprotease.

Preparation and Identification of Polyclonal Antibody Against CD47 Derived from Camel

Jia Erke, Feng Yaning, Li Jiangwei

2015, 31(8):  200-205.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.029

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This work aims to verify whether or not recombinant human CD47 would induce high antibody response in Xinjiang Camelus bactrianus, and provide the experimental evidences for preparing anti-CD47 nanobody of high affinity. The gene segment of CD47 extracellular region was amplified by PCR and ligated into pET30a plasmid, and a prokaryotic expression vector was constructed. The recombinant CD47 protein (rCD4) was expressed by IPTG induction and purified with Ni-affinity chromatography. A male C. bactrian camel was immunized with purified rCD47 antigen. The enzyme-linked immunosorbent (ELISA) and Western blotting were applied to assay the titer of polyclonal antibody and the specific binding to CD47 protein. The purified rCD47 of high purity (90%) from prokaryotic expression vector of CD47 extracellular region bound specifically with B6H12.2 of anti-CD47. The ELISA results revealed that the titer of polyclonal anti-CD47 antibody in camel at least reached 1:200000 after the 7th immunization. The Western blotting results demonstrated that camel antiserum of resisting CD47 specifically bound with rCD47, and CD47 on membrane of Jurkat and Raji cells. In conclusion, the anti-CD47 polyclonal antibodies of high titer were raised in camel and it lay a promising foundation for the future experiment.

Prokaryotic Expression of CP Gene of Yam Chlorotic Necrotic Mosaic Virus and Preparation of Its Antiserum

Zhang Pengyuan, Ren Longhui, Wang Jianguang, Chen Zhuangzhuang, Zhong Yu, Chen Suiyun

2015, 31(8):  206-212.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.030

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Yam chlorotic necrotic mosaic virus（YCNMV）is a new tentative species of genus Macluravirus in the family Potyviridae. Referring to the 3'-terminal sequence amplified with degenerate primers Sprimer/M4 by RT-PCR, we designed the specific primer pair of CP gene of YCNMV. The PCR product of CP gene was inserted into the pET30a prokaryotic expression vector, which was subsequently expressed in BL21（DE）strain by induction. Fusion protein was purified with Ni⁺ -NTA affinity column and was used to immunize New Zealand white rabbits to produce the antiserum. Results showed that the CP gene ligated in the prokaryotic expression vector shared 99.65% homology with the template CP gene in both nucleotide and protein sequence. SDS-PAGE results indicated that the CP fusion protein was about 44 kD in size, which was in accord with the prediction. The antiserum was tested by both ID-ELISA method and Western blotting, which showed that the polyclonal antibody had a relatively high level of titer, and reliably, sensitively and specifically bound with the YCNMV virus particles. These results indicate that the antiserum is suitable for YCNMV detection.

Expression and Purification of Outer Membrane Lipoprotein OprF in Recombinant Pseudomonas aeruginosa,and Preparation and Identification of Polyclonal Antibody

Chen Chunlin, Liu Xiang, Ju Xiong

2015, 31(8):  213-218.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.031

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The outer membrane lipoprotein of Pseudomonas aeruginosa（OprF）could antagonize bacterial infection by efficiently activating body immunologic mechanism, and therefore has an important perspective in vaccine development. The expression strain for OprF protein of P. aeruginosa was obtained by molecular clone;OprF protein was purified by the way of SDS-PAGE gel extraction and urea gradient renaturation, and the purified protein was used to immunize mice to prepare the polyclonal antibody. The titer of OprF antibody was 1∶12 800 according to ELISA, and Western blotting proved that the antiserum had good specificity. Homology analysis of OprF by DNAMAN showed that the C-terminal regions shared high homology in different bacteria. Phylogenetic analysis by MEGA revealed that genetic relationship with Pseudomonas genus bacteria was higher than that with others.

Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human

Su Jialin, Que Biao, Zhang Jiqin, Li Jinhui, Wang Min, Ji Weidong

2015, 31(8):  219-224.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.032

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This work aims to establish the CRISPR/Cas9 system of knocking out gene AIP1 for producing nephocyte cell（293T）of human embryo in which gene AIP1 efficiently, stably and permanently is knocked out. Three 20 bp sgRNAs（sp1, sp2 and sp3）targeting AIP1 exons were designed and inserted in PX458 vector to construct PX458-sgRNA expression vector for knockout. CRISPR/Cas9 efficiency of knockout was assessed using T7E1 assay. sgRNA of the maximum knockout efficiency was inserted into lentiCRISPRv2 vector to construct lentiCRISPRv2-sgRNA expression vector. The correct recombinant plasmid was transfected into 293T cells. The supernatant was collected and filtered, and then infected the 293T cells. The stable 293T cell lines were generated by limiting diluting the cells in which genes AIP1 were knocked out successfully. The expression level of AIP1 in the stable 293T cells were detected by Western blot. Three sgRNAs of AIP1 were correctly inserted into PX458 vector respectively, T7E1 verified that the knockout rate of AIP1sgRNAsp2 was the maximum. LentiCRISPRv2-sgRNAsp2 expression vector of AIP1-knockout was successfully constructed, and infected 293T cells. The stable 293T cell lines with AIP1-expression-deficient were obtained by Western blot. In conclusion, the stable 293T cell lines of AIP1-knockout were successfully generated by CRISPR/Cas9 system, which provides the foundation for further studying the functions of AIP1 gene.

An Analysis of Environmental and Biological Effects of Chlorpyrifos

Yu Kaimin, Feng Weimin, Li Guochao, Zhang Jiayu, Liu Lili, Yan Yanchun

2015, 31(8):  225-230.  doi:10.13560/j.cnki.biotech.bull.1985.2015.08.033

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With the prohibition of high-toxicity organophosphorous pesticides, the chlorpyrifos as their substitute has been used in a large-scale. The residue of chlorpyrifos, degrading slowly in the water, may cause the potential damages to aquatic organisms and others. To investigate the endocrine disrupting effects of low-concentration chlorpyrifos, flow cytometry was used to analyze the growth cycle of endometrial cancer cell HEC-1B of human. Moreover, zebrafish embryos were selected for chlorpyrifos exposing treatment 60 h at various concentrations of 0, 1.0, 2.0, 3.0 and 4.0 ppm. It was found that the chlorpyrifos caused the zebrafish embryos to death and significant embryonic malformation, the survival rate of the embryos was inversely proportional to chlorpyrifos’ concentration, and the malformation rate was proportional to chlorpyrifos’ concentration. Further, the mRNA expression levels of 5 neurodevelopment maker genes in the chlorpyrifos-treated zebrafish embryos were detected. These results showed that chlorpyrifos of low-concentration acted as endocrine disruptor, and chlorpyrifos of high-concentration affected the normal development of nerve system.