Table of Content

26 March 2017, Volume 33 Issue 3

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Global Status on Low Level Presence of Genetically Modified Crops

LIU Fang-fang, DONG Mei, LI Kai, WAN Yu-song, JIN Wu-jun, LI Liang

2017, 33(3):  1-5.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.001

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Over the last two decades,the land area under cultivation with genetically modified organisms（GMOs）has been growing steadily,and the amount and speed of circulation of many GM products in international trade tend to be rising. The issue that low content of GMO in non-GM product occurred in different countries and regions,i.e.,low level presence（LLP）of transgenic components has been increasingly serious. Considering this issue,this article expounds the definitions of LLP in seven different countries and regions（organizations）,and analyzes the common characteristics;then summarizes the current policies in every country,the threshold of LLP,and the effects of LLP on import and export trade. This study would provide the basic information for the relevant policies and detection standards for China.

Research Progress of the Molecular Biology in Heavy Metal Tolerance of Plants

CHI Chun-ning, DING Guo-hua

2017, 33(3):  6-11.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.002

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Heavy metal pollution has caused great negative impact on China’s economic development and human health,which has became a hotspot of study. This paper reviewed the research progresses on toxicity of heavy metal on plants and mechanism of plants tolerance,including the effect of metal on plants growth and development,the ways of plant resistance on toxicity of heavy metal,the tolerance gene and protein of plants to heavy metal,and so on,existing problem and major topics of future research were also disussed.

Research Progress on Tumor Markers by Secretomics

YU Le-zheng, LIU Feng-juan, WU Zheng-yu, RAN Xiao-qiang

2017, 33(3):  12-21.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.003

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In the occurrence and development processes of tumor,a large number of proteins are secreted by tumor cells,and some of them are considered as tumor markers and used for clinical detection and prognosis. With the rapid development of proteomics techniques,secretomics is born at the right moment and provides new ideas and methods for tumor research. Now the strategy and the advance in the study of tumor markers by secretomics are reviewed,which may provide reference for the researchers in the discovery and screening of tumor markers.

Research Progress on Small Non-coding RNA of the Xanthomonas spp.

YAN Yu-ping, ZHONG Xi, WANG Xue-feng

2017, 33(3):  22-28.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.004

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The gram-negative plant-pathogenic bacterium of the genus Xanthomonas infects a variety of monocotyledonous plants and dicotyledonous plants,such as rice,cabbage,tomato,citrus and other economic crops. Type III secretion system and other virulence determinants contribute to the infection and proliferation of Xanthomonas. The majority of bacterial small non-coding RNAs（sRNAs）modulates the expression of target mRNAs by base pairing mechanisms,or affects various physiological functions of the cell by binding directly with the protein. This review summarizes the recent study advances in the classification and the effects of sRNA on bacterial protein regulation,metabolism,transcription and virulence regulation,focusing on the identified sRNAs of Xanthomonas and their biological functions,which aims at providing new ideas for controlling crop’s diseases caused by Xanthomonas.

Research Progress of miR-1246 in Diseases

HU Yu, WANG Ling, BI Wu, TAN Lin, XING Dan, ZUO Fu-yuan

2017, 33(3):  29-36.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.005

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miRNAs are a class of small non-coding RNAs,involve in tissues metabolism,cells proliferation,differentiation,apoptosis and target genes expression,thus play an critical role in physiological and pathological processes. Nowadays,inventing effective miRNAs activators or inhibitors to treat and diagnose disease has become a new direction or entry point in the field of medical research. The newly discovered miR-1246 is regulated by P53,and is closely related to molecular mechanism of diseases. In this article,we summarize the miR-1246’s sequence structural features,molecular functional mechanism,review application potentials of miR-1246 in treating cancer,Ebola virus infection,and Down’s syndrome,and finally discuss its prospects,aiming at providing a theoretical reference for medical research.

Research Advance on the Red Stele Root Rot of Strawberry

CHEN Zhe, HUANG Jing, ZHAO Jia, LIANG Hong

2017, 33(3):  37-44.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.006

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The red stele root rot of strawberry is caused by infection of Phytophthora fragaria with strong host specificity. Its incidence is increasing year by year,especially the strawberries in the whole continuous cropping field might be destroyed if serious red stele root rot would erupt,which has become one of the main challenges in strawberry production. This article reviews the recent knowledge of red stele root rot,including its discovery,hazards,biology and molecular detection methods of pathogens,the characteristics,and control methods. Finally,the prospects are forecasted to provide some references and theoretical basis for further study.

Research Advances on Induced Mutation Breeding Techniques of Wheat

YU Mu, ZHOU Qiu-feng

2017, 33(3):  45-51.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.007

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Induced mutation breeding is a technology that uses physical radiation factors of ray,ion and neutron as well as the spaceflight environment to induce the seeds or in vitro tissues of plants,thus the advantageous mutants are obtained,and the breeding cycle is shortened. According to incomplete statistics of the FAO/IAEA database,up to May 2016,more than 3200 officially mutant cultivars in 214 crop species are released in more than 60 countries of the world. And 254 wheat mutants have been released by 21 countries,and 164 wheat mutants of which are in China and accounts for 64% of total,which is the top one in the world. This article summarizes the domestic achievements of inducing techniques for wheat breeding,outlines the mutations of traits such as wheat agronomic traits,yield and quality traits,and forecasts the goals and methods of induced mutation in wheat breeding. Those aim to provide reference for the further development of wheat breeding by induction,and to promote the application and development of modern physical agriculture.

Progress on Detection Technology of Programmed Cell Death in Plant

DENG Yu-qing, LI Ping, ZHOU Yan, XIONG Ke-cai, LI Zhong-an

2017, 33(3):  52-57.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.008

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Programmed cell death (PCD) is an active response in cell biology, which widely exists in plant growth and development. It has become a hot research for life science in recent years. In this paper, progress in the detection of PCD by morphology, biochemistry and molecular biology were reviewed. Furthermore, the disadvantages for the existing detection methods of PCD were also discussed in order to lay a sound foundation for better study and use.

CRISPR/Cas Genome Editing Technology and Its Application in Fungi

YIN Chao-min, FAN Xiu-zhi, SHI De-fang, GAO Hong

2017, 33(3):  58-65.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.009

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CRISPR/Cas system is a self-defense system against exogenous virus or plasmid in bacteria and archaea. CRISPR/Cas9,an expeditious genome editing technology,was developed according to the type II CRISPR/Cas system. Nowadays,the CRISPR/Cas9 genome editing technology has broadly utilized in gene function and genetic modification research in animal,plant and microorganism. Here,we briefly introduce the structure,mechanism and classification of CRISPR/Cas system,describe the application of CRISPR/Cas9 genome editing technology in fungi,and analyze the advantages,disadvantages as well as the prospect and application values of this technology. It is aimed at providing a useful reference for researchers in genetic modification of fungi.

Changes of Endogenous Hormones During the Culture of Callus from Sopatholobus suberechtus

ZHANG Ya-fang, HE Gang, RONG Guang-tian, LIU Xian-gui, NI Shang-ge, ZHANG Shi-liang

2017, 33(3):  66-70.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.010

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This work is to establish a regenerative system of Sopatholobus suberechtus tissue and determine the dynamic changes of endogenous hormones during the regeneration process. The enzyme-linked immunosorbent assay（ELISA）was used to determine and analyze the dynamic changes of four endogenous hormones（IAA,ABA,ZT,and GA₃）contents during the callus induction and differentiation. As results,the content of IAA always kept at a high level and reached maximum when the callus growth was the most exuberant in the process of callus induction;while the contents of ABA,ZT and GA₃ steadily remained at low levels;and IAA/ABA presented an important regulatory role on callus induction and growth. Further,the content of IAA decreased at first and rose then,reached the highest with 1.15-1.87 μg/g while differentiated to bud,and higher than that in callus induction period. ZT and ABA showed rising trend at first and then down later,while GA₃ tended to rise continuously. Also IAA/ABA decreased at first and rose then,playing an important role in promoting bud differentiation and growth. GA₃/ABA and ZT/ABA decreased at first and rose then,and promoted the successful differentiation of callus tissue to the adventitious bud in synergistic effects. In conclusion,the four kinds of endogenous hormones have important influences on callus induction and adventitious bud differentiation,the appropriate concentration and ratio of them may effectively promote the explants dedifferentiation and re-differentiation,and they synergistically promote cell division,differentiation and growth.

Suspensor PCD Induced by GA in Nicotiana tabacum Under In Vitro Culture

LUO An, CHEN Ke-qiang

2017, 33(3):  71-77.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.011

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Programmed cell death（PCD）is ubiquitous in the entire developmental process of plant,and hormones are a key regulating factor to PCD in plant. To investigate the role of GA in the suspensor PCD,systems of in vitro culture of the ovule and embryo were well established. By setting different concentrations of GA and culture time,100 μmol/L GA was appropriate and embryos may not be cultured for more than 3 days. The suspensor PCD was efficiently induced by exogenous GA during in vitro culture of embryo. As the concentration of GA increased,the proportion of PCD in suspensor cells gradually raised. Howerver,excessively high concentration of GA and long culture time would also induce abnormal PCD in porper cells.

Proteomic Analysis on Tuberous Roots of Cassava Cultivar ZM-Seaside and Mosaic-leaf Mutation

SONG Yan-chao, An Fei-fei, Xue Jing-jing, Qin Yu-ling, Li Kai-mian, CHEN Song-bi

2017, 33(3):  78-85.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.012

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In order to study yield differences between cassava cultivar ZM-Seaside（high yield）and mosaic-leaf mutation（low yield）,the tuberous roots were analyzed from the aspects of agronomic traits and proteomics in the present study. The results will provide a theoretical basis for the selection of high yield cassava varieties. Starch content was determined by polarimetry,and the hydrocyanic acid concentration was measured by silver nitrate titration method. Protein extract were performed by phenol extraction and protein was separated using two-dimensional electrophoresis. Delta 2D software was used to determine the differentially expressed proteins. The differentially expressed proteins were identified using mass spectrometry in combination with the KEGG database to classify proteins according to their functions. Western blot was used to analyze the protein expression level. String online software was used to construct protein-protein interaction network. Results showed that the starch content of ZM-Seaside（29.18%）was significantly higher than that of mosaic-leaf mutation（25.83%）. Hydrocyanic acid contents of two cassava genotypes in flesh were less than 50 mg/kg,and both of them are in the range of edible cassava. The dry matter rate of ZM-Seaside was 40.28%,significant higher that of mosaic-leaf mutation（37.16%）. 39 differentially expressed protein spots were detected in the tuberous root of ZM-Seaside compared with mosaic-leaf mutation,of which 23 were up-regulated,16 were down-regulated. 28 protein spots were successfully identified,in which their functions related to carbohydrate and energy metabolism（7）,chaperones（8）,detoxifying and antioxidant（2）,protein biosynthesis（1）,structure（3）and function unknown proteins（7）. STRING metabolic network showed that Heat shock protein and Molecular chaperone Hsp90-1 were the hub proteins,which probably are the key of the whole regulatory network. They would be the key proteins affecting the yields between mosaic-leaf mutation and ZM-Seaside,suggesting they may use as the marked proteins to select high-yield cassava varieties.

Cloning and Expression Analysis of Cystatin（HbCYS2）from Para Rubber Tree（Hevea brasiliensis）

LONG Xiang-yu, LIANG Qi-fu, QI Ji-yan, Fang Yong-jun, TANG Chao-rong

2017, 33(3):  86-92.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.013

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In plant,the cystatin（CYS）is widely involved in growth and development,and responses to biotic and abiotic stresses. In this study,the HbCYS2 is isolated from rubber tree,and has open reading frame（687 bp）encoding a protein of 288 aa. The HbCYS2 is a secreted protein with signal peptide,and has two CYS domains. The bioinformatics analysis indicates that the HbCYS2 is belonged to CYS II（GeneBank：KX161925）. The expression analysis indicated that the HbCYS2 has high level expression in latex,and similar expression pattern responding to tapping and ET treatment. In addition,the expression of HbCYS2 is apparently regulated by Corynespora inoculation in leaf. It is speculated that the HbCYS2 plays an important role in latex regeneration,tapping treatment and disease resistance in rubber tree.

Expression Analysis of Proteasome Subunit PSMD7 After Flagellar Disassembly of Dunaliella salina

SHI Ke, LIANG Rui-feng, YANG Liang

2017, 33(3):  93-99.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.014

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To study the relationship between the 19S proteasome subunit PSMD7 and flagellar disassembly in Dunaliella salina,the flagellar disassembly was induced by 3-isobutyl-1-methylxanthine（IBMX）and the mRNA expression of PSMD7 was detected by real-time PCR. The high-purity PSMD7 protein of D. salina was obtained by prokaryotic expression and protein purification,followed by the preparation of goat anti-PSMD7 polyclonal antibody. The expression of PSMD7 protein after flagellar disassembly was investigated by Western blot. Results showed that the mRNA expression of PSMD7 increased after flagellar disassembly induced by IBMX and reached the highest at 30 min after disassembly. The goat anti-PSMD7 polyclonal antibody was successfully prepared. Results of Western blot showed that the protein level of PSMD7 rose after flagellar disassembly,indicating that the proteasome subunit PSMD7 was involved in flagellar disassembly of D. salina,thus this provides the basis for further study of the molecular mechanism of ubiquitin-proteasome system in flagellar disassembly.

Labeling and Tracing of Green Fluorescent Protein in Fungal Endophyte with Growth-promoting Activity to Rice Seedlings

CHEN Nan, YU Fei, HE Yan-liu, BU Ning

2017, 33(3):  100-105.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.015

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This work is to transform the gene of green fluorescent protein（gfp）into the fungal endophyte JP4-1 of Suaeda salsa,and to detect the colonization of the fungi in rice seedlings. The plasmid pCT74 with gfp was transformed into the protoplasts with PEG-CaCl₂ mediated method and then integrated with the strain for transformants. Further,the transformants was used to infect rice seedlings,and finally the tracer to JP4-1 and its infection characteristics were detected with fluorescence microscope. Results showed that the transformants were quite stable with promising green fluorescence intensity after six successive subcultures. PCR amplification showed that the gfp was successfully transformed into strain JP4-1 and was expressed stably in rice seedlings. In conclusion,the JP4-1 transformants with stably-expressed gfp was acquired via transformation,the JP4-1 was colonized in the roots,shoots and leaves of rice seedlings,the location of colonization was intercellular,and the growth-promoting activity showed no difference with the wild type.

The Expression of Trehalose Synthase Gene in Bacillus subtilis WB800n Induced by Maltose

LIU Qiang, WANG Teng-fei, WANG Jun-qing, WANG Xi-hui, WANG Rui-ming

2017, 33(3):  106-115.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.016

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This work aims to construct a safe expression system of maltose-induced trehalose synthase while applying the maltose inducible promoter in Bacillus subtilis,and by which the prepared trehalose can be widely used in food and medical industry. The trehalose synthase gene TreS originated from the Pseudomonas putida KT2440 was selected as a reporter gene. The maltose-induced promoter Pglv from B. subtilis was utilized as a regulatory element after its cre sequence was optimized by site directed mutagenesis（AT base instead of CG base）. The shuttle plasmid PHT01 between Escherichia coli and B. subtilis was as the skeleton of the vector. By digestion and replacement of restriction enzyme BamH I and AatII,the highly efficient expression vector Pglv-PHT01-TreS of the trehalose synthase was constructed and transformed into B. subtilis WB800n,and the expression result was verified. The results from the optimization of fermentation conditions using the basic fermentation medium showed that the enzyme activity of trehalose synthase reached 18.9 U/mL when adding maltose with the final mass fraction of 4.5% while the OD₆₀₀ of the fermentation broth reached 1.2,and inducing 18 h at 37℃. In order to increase the expression of trehalose synthase,this experiment also constructed recombinant strain B. subtilis WB800n（ΔamyE）by single crossover method of knock the gene of α-amylase（amyE）,reducing the degradation of maltose into glucose by extracellular α-amylase and improving the maltose-induced expression and reducing the feedback inhibition of glucose. Therefore,the enzymatic activity of trehalose synthase rose to 29.2 U/mL when the expression plasmid was induced to express in this strain. This work is the first report that the highly efficient expression of trehalose synthase in B. subtilis was achieved,and lays the foundation for the safe and efficient expression system of trehalose synthase.

Screening and Identification of Two Aerobic Denitrifying Phosphorus-accumulating Strains,and Denitrifying Biological Phosphorus Removal

NIE Yi-lei, JIA Wei, ZENG Yan-bing, LUO Li-jing, CHEN Xing-wei, ZHUANG Hong, CHEN Hong

2017, 33(3):  116-121.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.017

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It is aimed to screen highly-efficient aerobic denitrification bacterium simultaneously removing nitrogen and phosphorus（P）from the piggery wastewater sample in a pig farm of Fuzhou. Two effective strains,named as N1 and N2,were acquired by culturing with medium for denitrification strain,then isolating with the BTB streak plate,further screening with the blue- and white-colored spot,and finally dyeing with metachromatic granules and PHB（poly-β-hydroxybutyrate）granule. By 16S rRNA gene sequence analyses,two strains were identified as Achromobacter sp. and Brevundimonas sp.,respectively. Combined strain FIM-1 was formulated with N1 and N2. The effectiveness of their removing the nitrogen and phosphorus in artificial wastewater and eutrophic water was investigated. The results showed that the removal rate of nitrogen by N1,N2,and FIM-1 at 40 h was 9.20%,17.16%,and 32.84% respectively,and the removal rate of total P at 18 h was 14.04%,8.77%,and 28.95%,respectively,indicating that FIM-1 performed better than the single ones.

Isolation,Identification and Antimicrobial Activity of Mycoparasites（Pestalotiopsis）from Aecidium pourthiaea

LI Jing, XIE Jin, LI Xiang-nan, ZHOU Zhi-fan, LIU Feng-lu, CHEN Yu-hui

2017, 33(3):  122-127.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.018

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Mycoparasite Pestalotiopsis produces the second metabolites different from endophytic or pathogenic Pestalotiopsis,as an important fungal resource,is yet to be developed and used. In order to obtain highly active mycoparasites ofAecidium pourthiaea,tissue separation method was used to isolate the mycoparasites,and 3 strains were isolated from the leaves of Photinia prionophylla with rust and suspected to be Pestalotiopsis;moreover,they were confirmed to be mycoparasites of A. pourthiaea via the tie back experiments though their morphologies varied obviously. Cultural characteristics on different medium and microscopic features of these three strains were described;also ITS sequences were amplified and analyzed. The morphology and molecular identification confirmed that all 3 strains were Pestalotiopsis. The results of anti-fungus experiment showed that these three strains not only destroyed the aecidiospores of A. pourthiaea,but also inhibited the growth of other 10 plant pathogenic fungi.

Microbial Communities on the Wine Grape Surfaces of Different Cultivars

ZHANG Shi-wei, CHEN Xi, ZHONG Qi-ding, HUANG Zhan-bin, MENG Zhen, LUO Jin-xue, SHI Ling, BAI Zhi-hui

2017, 33(3):  128-137.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.019

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Brewing grape wine is a process involved by many microorganisms that generally come from the grape berries if without exogenous inoculation. The high-throughput sequencing technology was used to analyze the microbial communities on the surface of different cultivars of wine grapes in Shacheng,China,which aims to understand the quality of the wine grapes,and provide the theoretical evidences for investigating the influences of microorganisms on the quality of brewing. The results showed that the richest microbial communities among these wine grapes were Riesling no matter of bacteria or fungi. The bacteria mainly were distributed in 8 phyla of Proteobacteria,Actinobacteria,Firmicutes,etc.,and 6 dominant genera of Erwinia,Pseudomonas,Bacillus,and so on. The 3 phyla of the fungi were Ascomycota,Basidiomycota,and Zygomycota;and the 9 genera of fungi were Alternaria,Cladosporium,Phoma,Fusarium,etc. The most similar species of the bacterial core OTUs were Arthrobacter sp. and Pseudomonas sp.,as well as Cladosporium sp.,Phoma sp.,Alternaria sp. and a lot of unknown ones for the fungal core OTUs. This study illustrates that the cultivar is the most important factor affecting the microbial communities. And from the results,it is inferred that the microorganisms on the surfaces of the wine grapes may be beneficial or adverse to the health of the grape vine,the quality of the grape berries and the process of wine brewing.

Effect of Endophytic Fungi on Iits Essential Oil Accumulation and Physiological-biochemical Characteristic of Cinnamomum longepaniculatum

YAN Kuan, CHEN Fang, WEI Qin, FENG Rui-zhang, ZHOU Wan-hai, ZHOU Min

2017, 33(3):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.020

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The aim of this study was to examine the effects of endophytic fungi on the accumulation and physiological-biochemical characteristics of essential oil in Cinnamomum longepaniculatum. The essential oil of C. longepaniculatum was extracted after spraying four endophytic fungi spore suspension（2J1、3J1、5J2、YB）on the leaves separately,and the content of 1,8-cineole and α-terpineol was determined by GC-MS. The results showed that the content of essential oil reached a peak at 21 d after treatment with 2J1 and 3J1,and the contents of 1,8-cineole increased by 201.53% and 174.47% compared with controls,and α-terpineol increased by 364.14% and 307.29% as well,respectively. Furthermore,the peroxidase（POD）content,catalase（CAT）activity,and malondialdehyde（MOD）content of suspension cells increased significantly. No significant difference was detected between C. longepaniculatum treated with 5J2 and the control group in terms of essential oil accumulation,CAT and POD activity,and MOD contents,nor did YB. Consequently,treatment with 2J1 and 3J1 could significantly increase the accumulation of essential oil in C. longepaniculatum.

Employing Poplar Wood Hydrolysate to Prepare Bacterial Cellulose

CHEN Hui-hui, LIU Yu, WANG Hui-mei

2017, 33(3):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.021

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Selecting the hydrolysate of poplar wood sawdust for fermentation substrate,we used Acetobacter xylinum to prepare bacterial cellulosee,and further studied the effect of reducing sugar concentration,the initial pH,inoculum size,fermentation temperature,and fermentation time on production,water holding capacity,and rehydration rate of bacterial cellulose. The results showed that the production of bacterial cellulose was 3.14 g/L,water holding capacity of bacterial cellulose was 98.72%,and bacterial cellulose rehydration was 86.69% when reducing sugar concentration of hydrolyzed poplar wood sawdust was 2.5%,the initial pH was 6.0,inoculum size was 6%,fermentation time was 6 d,and fermentation temperature was 30℃.The detection results of scanning electron microscope,infrared spectroscopy,and thermo-gravimetric analysis indicated that the structure of synthetic product was super fine mesh and the groups of the product corresponded to the structure of cellulose.

The Extraction Process and Molecular Characterization of Hyaluronic Acid in the Plateau Zokor Tissues

WEI Lin-na, WANG Yang, WEI Deng-bang, WEI Lian, WANG Ning, SUO You-rui

2017, 33(3):  151-161.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.022

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We aim to establish an extraction process of hyaluronic acid（HA）from the tissues of plateau zokor（Myospalax baileyi）. One-factor experiments were used to determine the optimal conditions of crude extraction,enzymolysis,and edulcoration process,and response surface method was applied to further optimize the crude extraction and enzymolysis process. The molecular structure of the extracted HA was determined by infrared spectrophotometry,the content of glucuronic acid by the Bitter-Muir method,and the molecular weight by the method of viscosity and size exclusion chromatography. As results,the optimal conditions of crude extraction was: liquid/material ratio 4 mL/g（water/tissues,V/W）,extraction temperature 26℃,and extraction time 1.6 h. The optimal enzymolysis process was: temperature 37℃,pH 8.7,and 0.7% added enzyme（1:250 trypsasy/tissue,W/W）. The optimal edulcoration process was: 20% chlorobenzene（chlorobenzene/HA solution,V/V）,95% alcohol precipitation ratio in 3 times volume,7% activated carbon（activated carbon/HA solution,W/V）to do edulcoration,and dialysis for 3 h. Under above optimal conditions,144 ± 3.61 mg HA powder was obtained from 500 g tissues of plateau zokor,and the average recovery rate was 72.73%. Infrared spectrum displayed the same absorption peak of HA by standard samples from Sigma. The mass fraction of glucuronic acid was 45.60%,and the protein content was 0.11%. The molecular weight of hyaluronic acid reached 3×106 Da. Conclusively,the HA from plateau zokor’s tissues by this extraction method is of good recovery rate and high purity,and HA in the tissues of plateau zokor is of high molecular weight and high content.

Characteristics of Gene CDH5 in Schizothorax prenanti and Its Response to Aeromomas sobria Infection

DUAN Hui-qin, WANG Li

2017, 33(3):  162-168.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.023

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This work is to characterize cadherin 5（CDH5）gene of Schizothorax prenanti. Bioinformatics tools were used to analyze the gene sequence of CDH5,and real-time PCR to detect the expression variation of CDH5 gene at 0 h,24 h,and 48 h of S. prenanti infected with Aeromomas sobria. The full-length cDNA of CDH5 was 4 825 bp,ORF was 2 313 bp,and encoded 770 amino acids. Its molecular weight was 85.19 kD and pI was 5.01. The CDH5 protein had a signal peptide sequence,and its secondary structure was mainly composed of random coil,extend strand,and α-helix. Multiple alignment analysis revealed that CDH5 shared 74% homology with Danio rerio,low similarity with Homo sapiens（43%）. CDH5 were expressed in all detected tissues after A. sobria infection,the expression of CDH5 in spleen at 24 h was significantly higher than at 0 h and 48 h（P < 0.05）,also the expression of CDH5 in the liver,kidney,and muscle at 48 h higher than at 24 h（P < 0.05）,and significantly higher expression of CDH5 at 48 h in heart and intestines than at 0 h（P < 0.05）. It indicated that CDH5 of S. prenanti might be involved in immune response to A. sobria infection,laying a foundation for the further study on the functions of S. prenanti.

Prokaryotic Expression of Gene VP2 of Porcine Parvovirus Type 1 and the Reactinogenicity Analysis of the Expressed Protein

OU Yun-wen, MA Xiao-yuan, ZHANG Jie, DING Yao-zhong, ZHANG Yong-guang, JIA Ning

2017, 33(3):  169-174.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.024

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This experiment is aimed to study the antigenicity of VP2 protein（amino acid 155-439）of porcine parvovirus type 1（PPV1）for laying a base for the development of detecting PPV1. The 849 bp target fragment was amplified using the DNA of strain AV31of PPV1 as the template. The product was cloned into pET30a（+）vector,and recombinant plasmid pET30a-PPV1-VP2（amino acid 155-439）was constructed,then transferred into Escherichia coli BL21（DE3）for the 6 h induced expression by IPTG. The recombinant protein was purified by Ni-NTA,and refolded by different concentration of urea. The results of SDS-PAGE showed that the gene VP2 was successfully expressed in E. coli BL21（DE3）with a relative molecular weight of 39 kD. The results of Western blot showed that this recombined protein specifically reacted with PPV1 positive serum,while no cross reaction with NA-PRRSV and PCV2 positive serum. This study achieved the aims：the recombinant vector pET30a-PPV1-VP2 was successfully constructed,and the gene was successfully expressed in E. coli,and the purified and re-folded protein demonstrated promising reactinogenicity .

miR-124 Regulates Porcine Adipocyte Differentiation Trough Targeting TNF-α

LI Hong-yi, LI Jia-biao, ZHENG Yi-lin, JI Lu-lin, ZHANG Mao, ZHENG En-qin

2017, 33(3):  175-179.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.025

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In the previous research,porcine TNF-α was found to be the target of miR-124 by dual luciferase assay. Here we verify whether miR-124 has the effect on the differentiation of porcine adipocyte. Porcine pre-adipocyte was transfected with miR-124 mimics or inhibitor to over-express or to repress miR-124,and then induced into mature adipocytes. The accumulation of lipid droplets was observed using Oil Red O staining and the amount of glycerol and triglycerides（TG）were detected by TG assay and glycerol assay. The expressions of PPARγ（proliferator-activated receptor-γ）,FASN（fatty acid synthase）,and HSL（hormone-sensitive lipase）were detected by real time fluorescence quantitative PCR. The result showed that overexpression of miR-124 repressed the expression of TNF-α protein,therefore,lipid droplets were more than that in the control,TG increased significantly（P < 0.01）,also the same for glycerol（P < 0.05）,and the expressions of PPARγ,FASNT,and HSL significantly up-regulated（P < 0.01）. Repressing miR-124 resulted in the reduction of lipid droplets and the significant decrease of TG,and the expressions of PPARγ and FASNT significantly down-regulated（P < 0.05）. In conclusion,miR-124 might regulate the differentiation of porcine adipocyte by suppressing TNF-α,laying a foundation for future studies on the mechanism of miR-124 regulating lipid metabolism.

Over-expression of MicroRNA-199a Intensifies the TNFα-induced Apoptosis of Adipocytes

QI Ren-li, WANG Qi, WU Yong-jiang, WANG Jing, HUANG Jin-xiu, YANG Fei-yun

2017, 33(3):  180-185.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.026

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The aim of this study is to explore the effects of MicroRNA-199a（miR-199a）on adipocytes apoptosis and the role of nuclear factor κB（NF-κB）. Tumor necrosis factor α（TNFα）was used to induce the apoptosis of cultured 3T3-L1 adipocytes. Based on this work,adding PDTC of NF-κB blocker and miR-199a mimic,the effect of overexpression of miR-199a on TNFα-induced apoptosis and the role of NF-κB in it were determined. The flow cytometry was used to analyze the cell apoptosis,kit Caspase3/7 to detect the enzymatic activities,dual luciferase reporter assay to detect the activity of NF-κB,qRT-PCR to detect the expression of miR-199a,and Western blotting to detect the changes of relevant proteins. As results showed,TNFα induced the apoptosis of differentiated adipocytes,concurrently activated NF-κB. The activated NF-κB played a negative regulator role in the apoptosis of adipocytes mediated by TNFα. Over-expressed miR-199a significantly suppressed the activation of NF-κB,and then remarkably promoted the apoptosis induced by TNFα. Conclusively,miR-199a involves in the TNFα-induced apoptosis of adipocytes through regulating NF-κB activation.

Rapid Diagnosis of Brucella by Loop-mediated Isothermal Amplification

XIE Wen-ping, ZHAO Hui-jun, ZHANG Lin, JIANG Hong-xu, WU Bin, SUN Hao

2017, 33(3):  186-192.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.027

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This study aims to apply the loop-mediated isothermal amplification（LAMP）in detecting Brucella. A set of six specific primers specific to regions of 16S rDNA gene were designed,and the turbidity technique was employed to optimize the reaction conditions,by which the specificity and sensitivity of the method were evaluated. Results are：（1）the optimized temperature was 62℃ at constant 60 min,and the concentration of inner primer was 1.50 µmol/µL,0.20 µmol/µL outer primers,and 1.0 µmol/µL loop primers;（2）furthermore,3 strains of Brucella occurred LAMP amplification reaction and achieved the positive turbidity value,but there was no amplification with other control group including Yersinia enterocolitica ATCC9510,Escherichia coil ATCC25922,Salmonella typhimurium ATCC10708,and Staphylococcus aureus ACTT33591;（3）the minimum thresholds for turbidity technique and real-time fluorescence were 4.36 fg/µL and 436 fg/µL respectively,their sensitivities were both higher than the value by conventional PCR,4.36 pg/µL. Obviously,it is fast,efficient,and convenient while LAMP is applied in the clinic diagnosis of Brucella,therefore,it is practically applicable for the pathogenic diagnosis of Brucella in grassroots communities.

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Human Antizyme Inhibition Factor-1

LÜ Ya-feng, YANG Jian-lin, QIN Yu, WANG Yan-lin

2017, 33(3):  193-198.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.028

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We aim to carry out prokaryotic expression and purification of human antizyme inhibition factor-1（AZIN1）,and to prepare as well as identify polyclonal antibody against AZIN1. Prokaryotic expression vector pET-28a/AZIN1 was transformed into Escherichia coli BL21（DE3）,in which the recombinant AZIN1 was expressed by IPTG induction and purified by affinity chromatography with Ni-NTA resin under denaturing condition. The purified recombinant AZIN1 was used as the antigen to prepare the antibody in the BALB/c mice. The titer and specificity of anti-sera were detected by ELISA,Western blot,cell immunofluorescence,and immunochemistry. As results,restriction analysis and DNA sequencing proved that the plasmid pET-28a/AZIN1 was constructed successfully. In E. coli,recombinant AZIN1 protein was expressed by IPTG induction and mainly existed in a form of inclusion body. The AZIN1 protein expressed in the E. coli was effectively purified using affinity chromatography. When used as the antigen to immune mice,this protein induced the production of specific antibody against AZIN1 with serum titer of 1:640 000. The prepared antiserum specifically recognized and bound to the AZIN1 protein expressed in human or mouse tumor cells. And this antiserum was efficiently used in the analysis for AZIN1 by Western blot,cell immunofluorescence,and cell immunochemistry. In conclusion,the prokaryotic expression and purification of human AZIN1 protein and preparation of polyclonal antibody against AZIN1 were successfully performed in this study. These results provide a basis for further research on the roles of AZIN1 in regulating cell proliferation and in disease prevention and treatment.

Establishment and Performance Evaluation of a Quantitative Detection Method for Microalbuminuria Based on Fluorescence Immunochromatography

ZHANG Sai, HONG Yu-hao, LI Kai, HE Xiao-wei, LI Wen-mei

2017, 33(3):  199-204.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.029

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This work aims to develop a rapid quantitative detecting method of fluorescence immunochromatographic assay（FICA）for human urinary microalbuminuria（MAU）. The fluorescence immunochromatographic strips were successfully prepared with anti-human albumin monoclonal antibody and goat anti-rabbit IgG marked by carboxyl fluorescent microspheres as the detection antibody,human albumin as the test line,and the rabbit IgG as the control line. As the results showed,the linear range of prepared kit（i.e.,fluorescence immunochromatographic strips）was in 5 mg/L-300 mg/L with a detection limit of 2.3 mg/L. The intra- and inter-assay coefficient of variation was 6.4%-9.3% and 6.5%-12.3%,respectively. The average recovery percent was 102.3%. The kit showed solid specificity as there was no cross-reactivity with 20 kinds of interfering substances. The shelf time of the kit by real-time stability test was > 12 months. Test with clinical urinary samples by the kit showed high correlation（r = 0.993,P < 0.01）with the control Orion QuikRead U-ALB kit,and Bland-Altman analysis showed that the diagnostic results of the two methods presented promising consistency. In conclusions,the prepared fluorescence immunochromatographic kit provides a simple,rapid and accurate quantitative detection method for microalbuminuria.

Preparation of Magnetic Fe₃O₄/Polyaniline Nanofiber for Laccase Immobilization

LIU You-xun, WANG Yao-kun, YAN Ming-yang, HUANG Juan

2017, 33(3):  205-212.  doi:10.13560/j.cnki.biotech.bull.1985.2017.03.030

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It is the key to prepare a perfect carrier for enzyme immobilization for increasing enzyme loading,efficient use and reducing the cost of enzyme application. Magnetical polyaniline nanocomposite was synthesized via mixing aniline solution and Fe₃O₄ nanoparticles and used as laccase immobilization carrier. The successful synthesis was confirmed by the results of TEM and FT-IR. Different proportions of Fe₃O₄ and aniline had no significant effect on the structure of the carrier,but the enzyme loading was affected. The maximum loading of the synthetic carrier was 210 mg/g,and the conductive properties of carrier were changed after the laccase was immobilized on it. The optimum reaction pH and temperature for the immobilized laccase were 3.5 and 60℃,respectively. A slight shift of optimal pH was observed. Immobilized laccase kept 50% its activity after incubation at 50℃ for 240 min,and 60% of its activity after incubation at 4℃ for 30 d. Over a period of repeated operation,immobilized laccase still remained about 70% after 8 cycles. With the increase of the proportion of Fe₃O₄,the loading of the carrier reduced,and there was a certain electron exchange between laccase and the carrier. The immobilized laccase can be simply recovered from the reaction solution by a magnet. Immobilized laccase onto Fe₃O₄/polyaniline nanofiber shows high stabilities,easy recovery,and reuse capabilities,which demonstrated that Fe₃O₄/ polyaniline nanofiber is a kind of perfect carrier for enzyme immobilization.