Table of Content

27 June 2016, Volume 32 Issue 6

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Review

Research Progress on the Safety Assessment of Stacked Genetically Modified Plants

LIU Xiao-dan, XU Wen-tao, HUANG Kun-lun, MEI Xiao-hong

2016, 32(6):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.002

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With the development of transgenic technologies，genetically modified(GM)crops have been commercialized. Nowadays，the transgenic crops of single-trait are unable to meet the needs of agriculture，while the transgenic strategy of stacked-trait broadens the functions of GM crops and improves the utilization of resources，therefore meets the multiple needs of farmers，owns the broad application prospects，and is a new trend for the development of GM plants. Since transgenic technology may bring certain risks，it is critical to manage the safety of GM crops. For stacked-trait GM plants，different countries have declared different rules and safety assessment regulations to supervise the new-developed plants. In this paper，the safety assessment regulations to stacked-trait GM plants in different countries were reviewed，which may provide the reference for the supervision of the stacked-trait transgenic plants in China.

Research Progress on Plant Rab Family

QI Hui-jie, QIN Xiao-hui, LIU Ling-yun

2016, 32(6):  7-12.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.003

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As a kind of important small G protein，Rab family plays an integral role in various physiological activities of eukaryotic cell. Higher plants have evolved a unique set of Rab GTPases that presumably meet the specific demands of plant cell trafficking. Rab proteins involve in the regulation of plant growth and development，and the response to environmental stresses through the change of own activity by the perception of upstream signals. Here we review the research progresses of Rab family in respects of evolution characteristics，structural features，and functions of members in the signal transduction，growth and development，and stress responses in plant.

Advances on Transcriptional Activator AtWRI1 of Arabidopsis

YANG Xian-you, HUANG You-jun, ZHANG Tong, HUANG Chun-ying

2016, 32(6):  13-18.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.004

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As lipid has high economic value，studying the regulation process in oil biosynthesis is of great significance. WRI1，a transcriptional activator，plays a key role in this process. This paper focuses on its identification and structural characteristics，expression pattern，function and the regulation mechanism of AtWRII. It belongs to AP2 subtribe of AP2/EREB family and has 7 exons. It expresses at different developmental stages of each organ，and the expressional level reaches the maximum at the oil synthesis stage of seeds. Through binding corresponding cis-function components，it activates the transcription of genes involving in glycolysis and fatty acid synthesis，and consequently promotes the oil synthesis. AtWRII is one of the downstream genes of AtLEC1 and AtLEC2. Mediated by CRL3-BPM complex，AtWRI1 is degraded via ubiquitin-26S proteasome pathway.

The Application Progress of Microfluidic Chips in Studying Plant Cells

WANG Wei-xuan, SUN Jing-yi, LIU Wei-na , HOU Ling-yu, YU Wang-ning, HE Xiang-wei, XIE Xiang-ming

2016, 32(6):  19-29.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.005

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Conventional methods of studying plant cells rely on growing plant cells in soil pots or agarose plates，followed by screening the plant phenotypes in traditional greenhouses and growth chambers. These methods usually need a large number of experiments，and suffer from low spatial resolution. While the microfluidic chips have many advantages，such as miniaturization，small volume consumption and high-throughput analysis，etc，moreover，it allows to precisely control the micro-environment of plant cells at micron-level. Therefore，it can reduce the cost and experimental time，and achieve in vitro single cell analysis and characterization. In this paper，the materials and manufacturing methods of the microfluidic chip were firstly introduced，then the recent microfluidic chips for studying plant cells were summarized，further mainly the application progress of microfluidic chips in plant root，pollen tube，protoplasts，and cell wall biomechanics were expounded，and finally the future research directions of microfluidic chips for plant cell were prospected.

Research and Application Progress on Human Glucagon-like Peptide-1

XU Fang-fang, WANG Nan, LI Gang-qiang, LIU De-hu

2016, 32(6):  30-37.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.006

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The cause of Diabetes mellitus is very complex，so far，the effective measures to completely cure diabetes have not been found yet. The chemical drugs for the treatment of diabetes in market continuously emerge，and these drugs may lower the blood sugar，but concurrently，they will inevitably lead to many toxic side effects，and cannot delay the development of diabetes complications. Human glucagon-likepPeptide -1(GLP-1)is a small polypeptide with 30 amino acids，which is potent to reduce blood sugar in a glucose-dependent manner without any adverse reactions，in addition to the multiple functions of GLP-1，it becomes a research hotspot in curing the diabetes II. However，to realize its clinical application，it is urgent to solve the problem of its short half-life in vivo. Hence，the reviews of the latest progress on the research and clinical application of GLP-1 in China and abroad on the basis of previous studies will provide reference for the researchers and medical workers in the development of GLP-1 relevant drugs.

Technique and method

An Overview of High Throughput Biological Screening Methods and Its Application

ZHAI Xu-zhao, WANG Guang-bin, ZHAO Liang-tao, ZENG Hai-juan, LI Jian-wu, DING Cheng-chao, SONG Chun-mei, LIU Qing

2016, 32(6):  38-46.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.007

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The emergence of genomics，proteomics，metabolomics and other molecular biology studies makes the rapid attainment and assessment of large amounts of biological data become extremely important. The traditional detection methods are time-consuming and laborious，and cannot satisfy the needs of contemporary biological science research for massive biological information. High Throughput Screening(HTS)methods are significant means of quick attainment of massive biological information. This paper will make an overview of microarray chip，microfluidic chip，pyrosequencing，fluorescence polarization immunoassay，quantum dot fluorescence immunoassay and multiple PCR，outline research focus and research results of HTS methods in recent years，and briefly introduce its application in food safety，medicine and other fields.

Research Progress on Recombinase Polymerase Amplification

JING Zhi-gang, DONG Hao, DI Dong-dong, TIAN Li-li, FAN Wei-xing

2016, 32(6):  47-53.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.008

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Recombinase Polymerase Amplification(RPA)is a recently developed isothermal amplification method that offers highly sensitive and specific DNA and RNA detection. Using RPA，the initial copy number of target nucleic acid sequence from different samples can be absolutely quantified，and multiple target nucleic acid sequences can also be detected simultaneously. Combining with less complicated device such as lateral flow strips or a sequence-specific fluorescent probe，the results can be observed directly. This article summarized the fundamental principles，continuous development and technical features of RPA. In addition，research and application progress of RPA in the field of in vitro diagnostic，pathogen detection and so forth were reviewed，aiming at providing guidance for further development and application of this method.

Optimization of Sample Pretreatment Method for Metabolomics Studies of Aureobasidium pullulans Producing Polymalic Acid

FAN Xu-jia, BIAN Yan-hui, YIN Hai-song , QIAO Chang-sheng

2016, 32(6):  54-59.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.009

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In order to acquire more accurate and complete intracellular metabolite information，and therefore to more realistically reflect the microbial intracellular metabolism，the systematic optimization of metabolomic sample preparation process was carried out from 3 aspects of quencher，extraction and solvent agent based on the GC-MS platform. The leakage of intracellular metabolites and cell injury with different quenchers was compared，and the results showed that 60% methanol/0.9% NaCl had the least leakage. The effects of extraction and solvent agent on relative abundance of metabolite distribution were investigated，and 60% methanol presented the significant extraction effect，and pyridine had the dissolving effect．The optimal sample pretreatment method for metabolomic analysis of Aureobasidium pullulans was determined as follows：pre-cooled 60% methanol/0.9% NaCl as quencher，ground in liquid nitrogen and repeated freezing and thawing for cell disruption，pre-cooled 60% methanol for the extraction of metabolites，and then the extracts were dissolved by pyridine and derivatized for 90 min under MSTFA at 40℃. By using this optimized protocol，more than 300 kinds of metabolic components were identified by GC-MS，including a large number of amino acids，organic acids and sugars. Thus our established method can be used to effectively analyze the A. pullulans metabolites.

Visual Detection of Vibrio harveyi Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

CHENG Die, CHAI Fang-chao, CAI Yi, ZHOU Qian-jin, CHEN Jiong

2016, 32(6):  60-68.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.010

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Based on nucleotide enrichment by a loop-mediated isothermal amplification(LAMP)and chromatographic visualization by a lateral flow dipstick(LFD)assay，this work aims to develop a novel LAMP-LFD method for the rapid detection of Vibrio harveyi. Three pairs of primers were designed using the hemolysin gene(vhhA)of V. harveyi as detection target，and used in LAMP reaction，among which the forward inner primer vhhA-FIP was biotinylated. Similarly，a fluorescein isothiocyanate(FITC)-labeled probe vhhA-HP was designed to specifically hybridize with LAMP products. And then the hybridized LAMP products were visually detected by LFD. The optimized LAMP was performed at 63℃ for 40 min；and visual detection via LFD took 50 min. The results indicated that LAMP-LFD was able to specifically identify V. harveyi from other 9 pathogenic bacteria commonly existing in the aquatic animals，such as V. vulnificus. The detection limit of LAMP-LFD was 1.0×10² CFU/mL for V. harveyi pure cultures(equivalent to 2 CFU per reaction)，and 5×10² CFU/mL for V. harveyi contaminated tissues of large yellow croaker(equivalent to 20 CFU per reaction)，both of which were 100 times lower than that of the conventional PCR method using both outer primers vhhA-F3/vhhA-B3. Therefore，this rapid and accurate LAMP-LFD method is a promising alternative in the surveillance and point-of-care test of V. harveyi in sea farming.

Research report

Resistance Evaluation of Bt cry1Ah-transgenic Maize to Asian Corn Borer, Cotton Bollworm and Oriental Armyworm

SONG Miao, WANG Hai, ZHANG Jie, HE Kang-lai, LIANG Ge-mei, ZHU Li, HUANG Da-fang, LANG Zhi-hong

2016, 32(6):  69-75.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.011

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Bt cry1Ah gene was transferred into inbred line maize Zong31 via Agrobacterium-mediated method，and transgenic maize HGK60 with significant resistance to corn borer was acquired. In order to investigate its insecticidal activity to Lepidoptera pests，we evaluated the insecticidal effects of HGK60 to Asian corn boner(Ostrinia furnacalis)，cotton bollworm(Mythimna separate(Walker))and oriental armyworm(Helicoverpa armigera Hubne)through laboratory and field bioassay. The results of laboratory bioassay indicated that no Asian corn boner feeding on HGK60 leaves survived. HGK60 presented the toxic effect on neonate of cotton bollworm，and different tissues of it had different insecticidal effects. Compared to the non-transgenic maize，the body-weight of armyworm neonate was significantly inhibited after a week of feeding HGK 60 leaves. The results of field bioassay showed that HGK60 had solid insecticidal effects to O. furnacalis and H. armigera in high resistance level，while the efficacy to M. separate was in resistance level.

Cloning and Phylogenetic Analysis of cDNA Sequences Coding for ARC1 and Exo70A1 in Brassica oleraces var. capitata L.

YANG Dan, LIAN Xiao-ping, ZHOU Yan, ZHANG He-cui, DU Dan, GAO Qi-guo, REN Xue-song, ZHU Li-quan

2016, 32(6):  76-88.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.012

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Interaction between ARC1 and Exo70A1 in Brassica is a key point of self-incompatibility(SI)signal transduction. In order to study the evolutionary relationship of ARC1 and Exo70A1 from 10 cultivars of SI head cabbages developed in recent years，we successfully cloned and sequenced cDNAs of ARC1 and Exo70A1. Furthermore，by bioinformatics，we had mutation and evolutionary analysis of those coding region sequences，including interaction region and non-interaction regions of ARC1-Exo70A1 from the 10 cabbages and prior published 83 ARC1 and 73 Exo70A1 sequences. The results revealed that：1)The coding nucleic acid sequences of ARC1 and Exo70A1 from the 10 cabbage materials presented parallel evolutionary relationships，and belonged to the Brassica branch. 2)The evolution rate of coding sequence of ARC1 was faster than Exo70A1. 3)The interaction coding region of ARC1-Exo70A1 was obviously in the progress of co-evolution，while the non-interaction coding area of ARC1-Exo70A1 showed significant differentiation rate in evolution. 4)The evolutionary rate of interaction coding area was faster than non-interaction coding region.

Responses of Overexpressed Arabidopsis VHA-c3 to Dark，ABA and Sugar

SU Jie, GUO Rong-qi, LI Guo-jing, WANG Rui-gang

2016, 32(6):  89-95.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.013

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For studying the role of vacuole H⁺-ATPase subunit c gene(VHA-c3)in plant growth and abiotic stress response，an over-expression vector of VHA-c3 was constructed and introduced into wild-type Arabidopsis thaliana. The expression levels of VHA-c3 in the transgenic homozygote’s plants were detected by semi-quantitative RT-PCR，then the transgenic homozygote were treated by dark，ABA and sugar. The results indicated that 6 lines of homozygous T2 transgenic plants were obtained，and their corresponding transcript levels were increased. The root lengths of 5 lines were reduced among the dark-grown seedlings. Among the ABA-treated seedlings，3 lines were insensitive in primary root growth and the extent of cotyledon unfolding，and 5 lines were insensitive in seed germination. When treated by glucose and sucrose，5 lines and 6 lines reduced the sensitivity in seed germination，respectively. These results indicate that VHA-c3 may affect the cell expansion of root，and be involved in the signal transduction pathways mediated by ABA and sugar.

Identification of a New Anthracnose Pathogen Colletotrichum camelliae and Its Pathogenicity Test on Camellia oleifera

LI Yang, LI He, ZHOU Guo-ying, LIU Jun-ang

2016, 32(6):  96-102.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.014

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Anthracnose is one of the most critical diseases of Camellia oleifera in China because it infects the leaves，flowers and fruits of C. oleifera，leading to severe defoliation and pre-mature drop of fruits. Pathogens from anthracnose-diseased C. oleifera leaves collected in Hunan，Jiangxi and Hainan provinces were identified based on morphological observation and multiple-gene sequences，and pathogenicity test was conducted on various cultivars of C. oleifera. The results revealed that isolated pathogens were Colletotrichum camelliae，which is the first report on this pathogen in China. Pathogenicity tests on various cultivars showed that C. camelliae infected the leaves of different C. oleifera cultivars，such as Xianglin-1，Xianglin-69，Xianglin-89，Ganwu-1，Ganwu-16，Guangxi red flower，Changlin-3，Changlin-4，Changlin-23，Changlin-53，Changlin-148，and Changlin-190，however，spot appearances were different on the leaves of different C. oleifera cultivars.

Orthogonal Test of EMS Mutation and NaCl Stress and Screening of Salt-tolerant Fiber Flax Germplasm in Vitro

WANG Wei-guo, DENG Qian, JI Qiao-ling, WANG Wei

2016, 32(6):  103-110.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.015

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This work aims to lay the primary foundation for breeding salt-tolerant fiber flax plant induced from the callus. The hypocotyl segments from sterile seedlings of 3 kinds of fiber flax were treated in the orthogonal test with EMS induction and salt stress，and cultured through bud and callus induction. Further，the physicochemical characteristics of the buds and callus were explored. The optimal treatment combination for adventitious buds derived from “Fanny”，“Tianxin 3” and “Shuangya 5” all were 0.025% EMS for 2 h or 4 h，and then 150 mmol/L NaCl for 12 h. Total bud yield from high to low were the hypocotyls segments of “Fanny”，“Tianxin 3” and “Shuangya 5”. The order of factors affecting the emergence and growth of the adventitious buds was concentration of NaCl>concentration of EMS>treating time with EMS. The optimal medium for the induction of adventitious buds from 3 species fiber flaxes was MS+0.000 2 mg/L TDZ+0.02 mg/L KT+0.005 mg/L NAA+0.3% sucrose+0.1 g/L inositol+3.3 g/L phytogel. The callus was treated with 0.025% EMS for 4 h then 150 mmol/L NaCl for 12 h and screened through salt and salt-free medium twice alternately，and the physicochemical properties of which were determined. The results showed that proline content，soluble sugar content，soluble protein，SOD activity and POD activity in the callus of treated group of 3 species fiber flax all increased remarkably(P<0.05)compared with their control group. Conclusively，the callus of treated group with 0.025% EMS for 4 h then 150 mmol/L NaCl for 12 h possessed solid salt tolerance.

Isolation，Identification and Phylogenetic Analysis of Endophytic Diazotrophs in Tengxian Medical-use Oryza officinalis

HU Wen-zhe, TAN Ze-wen, WANG Yong, XU Xian-wei, TAN Zhi-yuan

2016, 32(6):  111-119.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.016

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This research aims to select the endophytic diazotrophs from wild medical-use Oryza officinalis growing on Teng County of Guangxi for collecting the strain resources contributing to the production of microbial fertilizer. Endophytic diazotrophs were isolated from O. officinalis using three selective mediums of free nitrogen and plate streaking method. Acetylene reduction assay was used to determine the activity of nitrogenase in the endophytic diazotrophs. The SDS-PAGE(dodecyl sulfate sodium salt-polyacrylamide gel electrophoresis)patterns of whole-cell proteins and IS-PCR DNA fingerprinting patterns were employed for the clustering analysis of the isolated endophytic diazotrophs. The 16S rRNA gene sequences of the representative strains of each group were analyzed to construct the phylogenetic tree. The abilities of producing auxin，producing siderophores，dissolving phosphorus and the activity of ACC deaminase were tested by colorimetry of spectrophotometer method. Thirty-four strains of endophytic diazotrophs were isolated from O. officinalis and assigned to 5 groups and their nitrogenase activity was between 5.0 to 1 036.7 nmol/(mL·h). Group I and II each had 8 strains. Group III，IV，V had 2，5，11 strains respectively. The homology of 16S rRNA gene was 97.13% between Group I and Azospirillum amazonense ATCC 35119^T，99.71% between Group II and Klebsiella variicola DSM 15968^T，99.86% between Group III and Pseudomonas monteilii ATCC 700476^T，100% between Group IV and Xanthobacter flavus ATCC 35867^T，and 99.86% between Group V and Burkholderia vietnamiensis LMG 10929^T. In conclusion，some strains have the ability of producing auxin，producing siderophores，ACC deaminase activity，and dissolving phosphorus. The results suggest that endophytic diazotrophs in medical-use Oryza officinalis present genetic diversity and potential applications in agricultural practices；and group I probably is new.

The Correlation Between Soluble Carbohydrate Metabolism and Lipid Accumulation in Castor Seeds

ZHANG Yang, LIU Ai-zhong

2016, 32(6):  120-129.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.017

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The relationship between soluble carbohydrates metabolism and lipid accumulation in the development of castor seeds was studied by HPLC，RNA-seq sequencing and radioactive carbon isotope tracing method. Soluble carbohydrates in developing castor seeds were mainly composed of glucose，fructose and sucrose，and decreased obviously with seed development and oil accumulation. The significant negative correlation between sucrose content and lipid accumulation(r=0.980)was observed. Hexose-to-sucrose ratio was much higher and the genes related to carbohydrates metabolism were highly expressed in the early stage of seed development. Especially，sucrose synthase played the key role in carbohydrates metabolism. Hexose-to-sucrose ratio was decreased and the genes involved in carbohydrates metabolism were down regulated with the rapid accumulation of seed oil during the middle and late developmental stages，while the expressions of genes related to fatty acid synthesis and lipid accumulation increased significantly. Confirmed by ¹⁴C-sucrose isotope tracing experiments，the conversion of carbohydrates to oil was significantly inhibited by reducing the sucrose intake；consequently lipid accumulation was limited in developing castor seeds. Therefore，soluble carbohydrates metabolism(mainly sucrose)may play an important role in the process of lipid accumulation in castor seeds.

Replacing Xylene with n-heptane for Paraffin Section of Arabidopsis thaliana Anther

TU Lei, ZHANG Li-yao

2016, 32(6):  130-134.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.018

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Xylene is widely used as clearing agent for dewaxing and clearing in paraffin section. However，due to its severe harm to human health，researchers have been seeking adequate substitutes. In this study，Arabidopsis thaliana anthers in various periods were processed with n-heptane replacing xylene as a novel clearing and deparaffinizing agent in paraffin section，and the rest of steps were the same as the conventional paraffin section. The results showed that n-heptane with strong and rapid transparency was able to be mixed with ethanol and gum and dissolve paraffin，thus could be utilized as clearing and dewaxing agent. The tissues processed with n-heptane was in fine physic-chemical property and saturated with paraffin；due to the proper hardness of the section，continuously slicing was easy and slice thickness was uniform，moreover，the section was expanded naturally in the water of 42℃ and was flat and no folds. In addition，the cells stained with toluidine blue were in fine quality of staining，and had a clear definition of the nucleus and the cytoplasm，as well as the morphology and structure of the cells were maintained well，meaning that there were no significant differences from the section with xylene as clearing agent. In conclusion，n-heptane is a novel，safe and efficient substitute replacing xylene in the histological techniques for plants.

Cloning and Expression Pattern of Ribosomal Protein S18 Gene in Musca domestica

HU Ya, LU Cheng, WEI Chuan-chuan, XIU Jiang-fan, WU Jian-wei

2016, 32(6):  135-142.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.019

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This research is to confirm the expression stability of ribosomal protein S18（RPS18）gene in Musca domestica. The complete coding sequence（Registration number in NCBI：KT006855）of RPS18 gene was obtained via PCR amplification using the cDNA of RPS18 gene from the cDNA library of M. domestica larva as a template，further the gene and its encoding protein were predicted and analyzed by bioinformatics method. The constructed recombinant plasmid of pET28a/RPS18 was transferred into Escherichia coli Transetta（DE3）for the induced expression and protein purification，and the polyclonal antibody was prepared as well as the specificity of antiserum was analyzed. The transcription and translation expressions of RPS18 gene at different growth stages of the M. domestica larva and in the different tissues of 3-year larva were analyzed by RT-PCR，qPCR and Western blot. The results showed that the RPS18 ORF was 459 bp in length encoding 152 amino acids with a predicted molecular mass of 17 590.5 Da and pI of 10.48. The recombinant plasmid was transferred into E. coli Transetta（DE3），and the purified protein RPS18 was acquired. The purified protein was immunized to rabbits，then the polyclonal antibody was harvested，and the single band was visualized by both His single resistance identification and antiserum specificity analysis. The analysis by RT-PCR，qPCR and Western blot indicated that RPS18 genes expressed stably in different growth stages and different tissues of the larva. In conclusion，comprehensive analysis suggests that the gene may maintain solid stability in different growth stages of M. domestica and different tissues of larva.

Expression of diptericin Gene from Musca domestica in Escherichia coli

LU Min, BAI Jie, WEI Feng-xian, WANG Lin-yi, XU Bin, YIN Qing-qiang, LI Shao-yu

2016, 32(6):  143-147.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.020

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In order to efficiently acquire the antibacterial peptide by prokaryotic expression，diptericin gene for the antibacterial peptide in housefly was reversely synthesized by RT-PCR method. Then，diptericin gene was cloned to pGEX-4T-1 expression vector and transformed into Escherichia coli BL21 for protein expression. Sequencing result showed that housefly’s diptericin gene was 345 nucleotides in length. Recombinant pGEX-4T-1 was induced by IPTG，and the expression of diptericin fusion protein was detected by SDS-PAGE electrophoresis and Western blot. The result showed that diptericin fusion protein with GST-tag was about 37 kD，and the target protein was purified by GST purification column.

Preparation and Identification of Monoclonal Antibody Specifically Against Human NT-proBNP

LIU Peng-zhan, HUANG Qi-ling, HE Xiao-wei, LI Wen-mei, ZHANG Wen-qi, ZHANG Sai

2016, 32(6):  148-154.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.021

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This study aims to prepare and identify monoclonal antibodies specifically against human NT-proBNP. Balb/c mice was immunized with labeled recombinant protein NT-proBNP，then the spleen cells with high titer of the mice’s serum were fused with SP2/0 myeloma cells. Further，the positive hybridoma cell lines were established after HAT selection and subcloning by limiting dilution，ascites were prepared by injecting cells to mice’s abdomen，and monoclonal antibodies were purified by caprylic acid-ammonium sulfate precipitation method. Finally，basic functions of these monoclonal antibodies such as subclasses，titers，specificity，affinity constants were analyzed. Four positive hybridoma cell lines stably secreting human monoclonal antibodies of anti-NT-proBNP were screened out，and named as 1G12，1B2，3D5，and 2D11；1G12 and 1B2 belonged to IgG2a，2D11 to IgG1，and 3D5 to IgG2b. The titers of 1G12，1B2 and 3D5 reached 10⁶，while the titer of 2D11 only was 10⁴ . The affinity constants of 1G12，1B2，3D5 and 2D11 were 9.32×10¹¹，1.07×10¹²，2.31×10¹²，and 6.33×10¹⁰，respectively. In conclusion，the monoclonal antibody of specifically against human NT-proBNP was prepared successfully.

The Expression of Transcription Factor GATA4 Gene in Takifugu rubripes

HAO Wei-wei, GAO Chang-fu, QIU Xue-mei, JIANG Zhi-qiang, WANG Xiu-li

2016, 32(6):  155-160.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.022

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In this study，GATA4 expression were analyzed in kidney，muscle，brain，spleen，liver，testis and ovary of mature Takifugu rubripes，and were analyzed at individual level during different times in young T. rubripes by flurescence quantitative Real-time Polymerase Chain Reaction(qRT-PCR). The results showed that no statistically significant difference was observed of expression level among muscle，spleen，kidney and brain. There was the significantly higher expressionof GATA4 in testis ，liver and ovary，than that in other organs(P<0.05). The GATA4 expression level of testis and liver were significantly higher(P<0.05)，than that in ovary. During the different periods(23，30，40，60，80 days of age(d)) of T. rubripes，the GATA4 expression level in males increased gradually from 23 d to 30 d and subsequently decreased at 40 d，and the 30 d expression level of GATA4 was higher than that in 30 d female individuals significantly(P<0.01). While from 60 d to 80 d，the GATA4 expression level went steadily up. In females，as the same as the expression tend in males，the GATA4 expression level increased gradually from 23 d to 40 d and subsequently decreased at 60 d，and the 40 d GATA4 gene expressed higher than that in the same time male individuals significantly(P<0.01). Nevertheless from 80 d，the GATA4 expression level stared to rise. Apart from 40 d individuals，the GATA4 expression level were higher in males than in female in every period.

Identification and Expression Analysis of emx1 from Paralichthys olivaceus

SUN Wen-hui, ZHANG Jun-ling, SHI Zhi-yi, SUN Jin-jin, XIANG Yu-ting, CHEN Xiao-wu

2016, 32(6):  161-167.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.023

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To explore the function of the empty spiracles homeobox 1(emx1)gene in the development of Paralichthys olivaceus，the emx1 was cloned by PCR，the sequence feature was analyzed by BioEdit and MEGA software，and the expression in early development and adult tissues was detected by real-time quantitative PCR. The obtained emx1 cDNA was 1 127 bp，including a 708 bp open reading frame(ORF)，encoding a polypeptide of 235 amino acids. The alignment analysis of amino acid sequence discovered that the emx1 from P. olivaceus showed the highest 95% homology with that of Stegastes partitus and Poecilia formosa，the sequence similarities between P. olivaceus and Danio rerio, Astyanax mexicanus, Xenopus tropicalis, Gallus gallus, Mus musculus and Homo sapiens，were 87%，84%，83%，80%，72% and 72%，respectively. The Emx1 protein was highly homologous with that in other fish through phylogenic analysis. Real-time quantitative PCR revealed that the emx1 mRNA was expressed in all detected tissues of P. olivaceus. The expression of emx1 was the highest in the ovary and secondly highest in the testis，while relatively low in other tissues. In early development stage，i.e.，from gastrula to 10 d after hatching，a relatively high expression of the emx1 mRNA was also observed，and the highest expression occurred in the blastopore stage and heart-beating stage. The findings indicated that the emx1 played an important role in early development and gonad development stages of P. olivaceus.

Isolation and Identification of an Antagonistic Bacterial Strain Against Rice Blast Fungus

GUAN Ling-li, REN Zuo-hua, LI Jun-jun, LIU Er-ming

2016, 32(6):  168-173.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.024

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It is quite important to develop novel and environment-safe strategies to control rice blast caused by Magnaporthe oryzae，and screening effective microorganism resources is primary basis for the bio-control of blast. Total 817 strains were isolated from healthy ones among diseased rice plants of the susceptible cultivar Xiangzaoxian 24，a single-season cropping rice，in the mountainous area of Taojiang，Hunan，in 2014. One strain JN005 with efficient antagonism to M. oryzae among 817 strains was screened by the plate confrontation method. The inhibition rate of metabolite from active bacterium to the rice blast reached 84% by measuring the growth rate of mycelium of the blast fungus. Further，antimicrobial spectrum of JN005 against 5 plant pathogenic fungi，4 plant pathogenic bacteria，and also Escherichia coli and Staphylococcus aureus sub. sp. aureus，was tested. The result revealed that JN005 also presented high inhibitory effect to Fusarium graminearum and Ustilaginoidea virens，and the inhibition rates to two fungi were 70.1% and 73.6%，respectively. Based on analysis of the morphological，physiological，and biochemical characteristics and the 16S rDNA phylogeny，strain JN005 was identified as Bacillus subtilis.

Isolation of a Chitinase-producing Strain Brevibacillus laterosporus and Its Enzymatic Properties

LIU Pu-lin, CHENG De-yong, MIAO Li-hong

2016, 32(6):  174-180.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.025

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This study aims to screen and identify Bacillus strain producing high yield of chitinase and to study its enzymatic properties for laying groundwork in the efficient utilization of chitinase resources. Firstly，the Bacillus producing chitinase was isolated and purified，and then chitinase gene in the target strain was heterologously expressed，and enzymatic parameters of the purified chitinase were assayed at optimal conditions. Considering the bio-safety of the strain，Brevibacillus laterosporus CDY64 was selected as study object，and its chitinase(Chi72A)was expressed in Escherichia coli BL21(DE3). Enzymatic studies revealed that the purified Chi72A by Ni-NTA affinity presented solid activity at pH range 6.0-8.0，and the optimal temperature of its activity was 60℃；and the Km and k cat of Chi72A were 5.85 μmol/L and 29.27 S^-1，respectively while using colloidal chitin as substrate. In conclusion，Chi72A from CDY64 had high catalytic ability at high temperature and exhibited high antifungal activity against many phytopathogenic fungi in vitro.

Cloning and Function of Gene for Cytochrome P450 from a Chlorogenic Acid-producing Endophytic Bacterium

WANG Chuan, LI Li, WEI Pi-wei, HUANG Fei

2016, 32(6):  181-186.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.026

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Based on a chlorogenic acid-producing endophytic Bacillus subtilis，cloning and function of a key gene in the synthetic pathway of chlorogenic acid was studied. The cytochrome P450 gene of the endophyte was cloned and expressed in prokaryotic vector，and the cinnamate 4-hydroxylase(C4H)activity of the recombinant proteins were determined. Four cytochrome P450 genes were cloned and expressed in prokaryotic vector，and the recombinant protein P-4 presented C4H activity. The coumarate produced by the C4H activity was the same as the result measured by LC-MS. The activity of P-4 existed in a broad optimal temperature and was inhibited by product coumarate. It is supposed that，taking cytochrome P450 as isozyme of C4H，the endophyte led the product coumarate to the synthetic pathway of chlorogenic acid.

Colorimetric Method in the Determination of 1-Deoxynojirimycin Isolated from the Mutant Strain of Bacillus subtilis

LONG Ling, YANG Yan, ZHU Nai-shuo

2016, 32(6):  187-192.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.027

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This work is to seek a novel method to detect 1-Deoxynojirimycin(DNJ)in the culture broth of Bacillus subtilis var. niger-231(B-231)and to improve DNJ production. DNJ reacts rapidly with carbon disulfide while Cu²⁺ existing，and the final product extracted by organic solvent was measured by spectrophotometer at 440 nm，thus DNJ content was determined. Then mutant strain B-231 was induced with ultraviolet. The standard curve for measuring standard DNJ was drawn，and the precision，sensitivity，accuracy and stability of the products while using the novel method of measuring DNJ were studied. Orthogonal test was designed to obtain the optimal reaction condition. The novel method was applied to measure the content of DNJ in the culture broth of bacteria. A high-yield DNJ strain was screened and named as B. subtilis var. niger- 431(B-431)by mutagenizing the B-231. The content of DNJ by B-431 was up to 87.3 mg/mL，and increased 4.2 times compared to B-231. This study established a novel method to determine 1-deoxynojirimycin in the culture broth of DNJ-producing strain for the first time. The induction by ultraviolet increased the capacity of this bacterium producing DNJ.

Colonization of Double-resistance Strain of Marine Bacterium L1-9 and Its Biocontrol Effect on Fusarium Wilt of Cucumber

WANG Jun-qiang, WANG Jing-jing, WANG Qi, MA Gui-zhen, BAO Zeng-hai, WANG Shu-fang, ZHOU Xiang-hong

2016, 32(6):  193-198.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.028

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The Paenibacillus polymyxa strain L₁-9 of marine bacterium was labeled by antibiotic marker，and strain L₁-9^Str，rif showed the resistance to rifampicin and streptomycin at the concentrations of 160 μg/mL and 20 μg/mL，respectively. The antibacterial characters of double-resistance strain L₁-9^Str，rif and its resistance to rifampicin and streptomycin were still stable after 10 times subculturing. The pot experiment showed that L₁-9^Str，rif successfully colonized in cucumber rhizosphere soil，root tissue，stem base，cotyledon，and true leaves. The colonized dynamics of L₁-9^Str，rif in cucumber ecto-rhizosphere soil，rhizosphere and rhizoplane soil，and cucumber tissues were similar；the amount of L₁-9^Str，rif colonized in cucumber tissue was little in the early stage，but increased gradually along with the cucumber growth，and reached a peak，then decreased gradually. The amount of L₁-9^Str，rif was the most in rhizoplane soil(1.76×10⁹ CFU/g)，the followed in the rhizosphere，while the least in ecto-rhizosphere soil. The detection of L₁-9^Str，rif in cucumber tissues showed that it was the most in cotyledons(5.63×10⁴ CFU/g)，followed by its number in roots and stem bases(0 - 2 cm). The amount of bacteria in the rhizosphere soil was still in a stable level at 26 d，and the quantity from rhizoplane soil was the highest with 2.41×10⁷ CFU/g. The quantity of bacteria in the cotyledons got the highest of 4.15×10⁴ CFU/g among cucumber tissue samples. The greenhouse test showed that both strain L₁-9 and strain L₁-9^Str，rif had favorable control effects on fusarium wilt of cucumber with the control efficacy over 70% at different stages. The above results indicated that strain P. polymyxa strain L₁-9 may colonize in cucumber rhizosphere soil and tissues，and present a great potential for the bio-control of fusarium wilt of cucumber.

Optimization of γ-aminobutyric Preparation by Recombinant Glutamate Decarboxylase

HUANG Yan, SU Ling-qia, WU Jing

2016, 32(6):  199-204.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.029

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Glutamate decarboxylase(GAD)，a pyridoxal 5'-phosphate(PLP)-dependent enzyme，irreversibly catalyzes the decarboxylation of L-glutamate to be the valuable food additive γ-aminobutyric acid(GABA). In this study，a recombinant Escherichia coli BL21(DE3)/pET-24a-gad producing Lactobacillus brevis WJH3 GAD was constructed as strain in the flask culturing of fermentation and induction. The activity of GAD produced in the supernatant of culturing for 24 h medium supplemented one-time with 0.05 mmol/L PLP was 81.7 U/mL，and this was 1.8-fold of that without PLP supplementation. Furthermore，the condition for GABA preparation by enzymatic conversion was optimized；under the condition of 250 g/L monosodium glutamate(MSG)，pH5.0，37℃，60 U GAD per gram substrate incubated for 18 hours，and rotation rate 200 r/min，100% of the MSG was transformed into GABA. These results establish the utility of PLP supplementation and lay the foundation for large-scale enzymatic production of GABA.

Isolation and Characterization of the Chlorpyrifos-degrading Trichoderma Strains from the Vegetable Soil in Greenhouse

ZHANG Guang-zhi, ZHANG Xin-jian, LI Hong-mei, GUO Kai, YANG He-tong

2016, 32(6):  205-210.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.030

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To explore and protect the Trichoderma resources that can degrade chlorpyrifos，six different types of Trichoderma strains with high activity of degrading chlorpyrifos were screened from the long-term organophosphorus pesticide contaminated soil. Analyses of morphological characteristics combined with internal transcribed spacer(ITS)rDNA sequences were used to identify them as T. harzianum, T. viride, T. koningii, T. koningiopsis, T. longibrachiatum，and T. brevicompactum，respectively. T. longibrachiatum TC5 with the highest degradation activity was selected to investigate the characterization of degrading chlorpyrifos under the culture condition including extra carbon source，pesticide concentration，inoculum density and pH. Under neutral or alkaline conditions，the strains TC5 had solid degradation activity to chlorpyrifos. Either adding carbon or increasing the inoculum concentration promoted the degradation rate. Degradation activity gradually increased with the increasing of chlorpyrifos concentration in the range of 50-300 mg/L. In the pot experiment，Trichoderma TC5 remained the high degradation activity to chlorpyrifos，but the degradation activity in the natural soil was significantly lower than in sterilized soil. The Trichoderma strains have great potential application in remedying the pesticide-contaminated soil.

Identification of Two Novel Phosphorylated Sites of Homeoprotein Msx1

CHENG Qing-ling, WANG Jing-qiang

2016, 32(6):  211-218.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.031

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Some phosphorylated sites in the homolog Msx2 of the homeoprotein Msx1 has been identified，which plays a crucial biological role. However，the phosphorylated sites in Msx1 are not known yet，thus in order to examine the Msx1 phosphorylated status，we applied bioinformatics analysis to predict phosphorylation sites in Msx1，further used immunoprecipitation and mass spectrometry to experimentally verify the phosphorylation sites in Msx1. Tryptic digests of Msx1 protein complexes purified from C2C12 cells were separated and analyzed by nLC-MS-MS. Two novel phosphorylation sites(Ser152 and Ser160)were identified，moreover，these two sites were highly conserved in mouse and human. Furthermore，the specifically-phosphorylated antibody was prepared targeting these two phosphorylation sites.

Renaturation Technology of Recombinant Fusion Protein Trx-IFN-CSP

HUANG Yan-ting, LU Xue-mei, YANG Xiao-rong, JIN Xiao-bao, ZHU Jia-yong

2016, 32(6):  219-225.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.032

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This work is to establish and optimize the method of refolding fusion protein Trx-IFN-CSP. The refolding of recombinant protein in vitro was studied，i.e.，investigating the effects of pH，temperature，protein concentration，redox systems and auxiliary refolding molecules on the refolding process of fusion protein. The results were as below. The proper measure to refold the Trx-IFN-CSP was to obtain the inclusion bodies by repeated combined freeze-thaw with ultrasonic to crack bacteria. The inclusion bodies were preliminarily purified using scrub solution of 1％ TritonX-100，2 mol/L urea，and 2% DOC，and then denatured in 6 mol/L guanidine hydrochloride. After the pulse dilution，the renaturation was conducted under 4℃ by gradient dialysis with the assistance of L-Arg. After the Trx-tag was removed by recombinant enterokinase digestion，approximately 110-130 mg of the pure recombinant liver-targeted interferon was obtained from 1 L Escherichia coli culture. Each batch of protein had a purity of over 95% and antibacterial activities were about 1.9-2.4×10⁸ U/mg，thus the technology of preparation was stable.

Study on the Highly Secreted Expression of the Recombinant Insulin in Pichia pastoris

LIANG Guo-yi, GAN Yi-di, LIU Xiao-hang, CAI Xiang-sheng

2016, 32(6):  226-230.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.033

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In order to improve the efficiency of insulin secretion expression in yeast，firstly the pro-insulin gene was synthesized，and a recombinant secreted expression vector(pPICZα-A-Pro-INS)was constructed by inserting a leader peptide to N-end of the gene’s sequence. Linearized expression vector was transformed into the competent cells of Pichia pastoris GSll5. A strain with highly secreted expression of the insulin was screened，by which the secreted recombinant protein expression level was 300 mg/L，accounting for about 40% of the total secreted protein. This result revealed that the leader peptide is crucial to the gene of highly expressing insulin in P. pastoris.

Evaluation on Growth and Biochemical Properties of Eight Strains of Marine Microalgae Nannochloris sp.

CHEN Cheng-hao, WU Jia-yi, TANG Ming-xing, LI Tao, WU Hua-lian, WANG Guang-hua, DAI Shi-kun, XIANG Wen-zhou

2016, 32(6):  231-237.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.034

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This work is to evaluate the productive potential of Nannochloris sp. by analyzing the growth and biochemical characteristics of 8 strains screened from the South China Sea region. Using dry weight determination，soxhlet extraction，kjeldahl nitrogen method，phenol sulfuric acid determination combined with gas chromatography-mass spectrometry technology，the microalgal growth，the content of lipid，protein and polysaccharide and the composition of fatty acids were analyzed correspondingly. The maximum dry weight of eight strains was 0.39 - 0.91 g/L and the SCS-761 strain grew the fastest. The total lipid content ranged in 19.06% - 33.67%，and that of SCS-249，SCS-589，and SCS-655 was over 25%，and the lipid productivity of SCS-655 strain was as high as 22.23 ± 0.71 mg/(L·d). The protein contents of SCS-249，SCS-325，SCS-355，SCS-640，and SCS-769 were over 30%，of which the highest for SCS-640 strain(36.70 ± 2.20)%. Moreover，the polysaccharide content of each strain was between 19.23% to 29.34%，of which the highest was SCS-761 strain and the least was SCS-655 strain. In addition，the analysis of fatty acid composition showed that the structures of fatty acid in all microalgae strains were similar，mainly composed of C16 saturated fatty acids and C18 series of fatty acids. Linoleic acid was the highest content among unsaturated fatty acids，and the linoleic acid contents of SCS-249，SCS-355，and SCS-761 were over 40%. In conclusion，the results indicates that marine microalgae Nannochloris sp. has relatively abundant germplasm resources including lipid，protein and linoleic acid，and owns considerable application potential；thus it is necessary to have more in-depth research.

Lipid Production from the Photoautotrophy of Three Strains of Scenedesmus Species

CHEN Chuan-hong, WU Hong, LI Qing, YIN Shun-ji, LUO Shao-jing, CUI Chun-li

2016, 32(6):  238-243.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.035

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The growth and the lipid content of 3 strains of Scenedesmus were compared by column photobioreactor，and it was concluded that the strain of producing the highest biomass was Scenedesmus deserticola with 0.48 g/(L·d)，followed by Scenedesmus sp(S. sp)0.41 g/(L·d)，and the lowest by S. dimorphus with 0.35 g/(L·d). In the late culture stage，the lipid distribution was clearly visible by Nile red staining. The total lipid content and the lipid productivity of S. deserticola were 55.3 % of DCW and 0.29 g/(L·d)，respectively，followed by S. dimorphus in 46.7 % and 0.18 g/(L·d)，S. sp in 43.6 % and 0.20 g/(L·d). The main fatty acid composition of 3 Scenedesmus were C18 and C16，accounting for over 85% of total fatty acids，satisfying the requirements of biodiesel production. Comprehensive comparison revealed that the S. deserticola was a lipid-producing strain with high performance.

Differential Expression Profile Analysis of MicroRNAs in Doxorubicin-induced Hepatoma Cell Line HepG2

YANG Ya-lan, GUO Zhi-yun, DING Ruo-fan, MAO Can-quan, GUO Jian-xiu, XIONG Li-li

2016, 32(6):  244-249.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.036

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The aims of this work are to study the miRNAs of hepatoma cell HepG2 involving in the response to doxorubicin-induced DNA damages，and to analyze the these miRNAs target genes and the biological processes and pathways related to the response to the hepatoma DNA damages. Small RNA sequencing was used to detect the differentially expressed miRNAs between doxorubicin-treated hepatocellular carcinoma cell line HepG2 and untreated one，and the functions of target genes were analyzed by GO(gene ontology)and KEGG pathway enrichment. Remarkably，68 significantly differentially-expressed miRNAs were identified，13 miRNAs were up-regulated and 55 were down-regulated. After the functional analysis of miRNA targets，the 53 miRNAs were enriched in the biological processes of cell proliferation，cell apoptosis，cell invasion and cell cycle arrest，as well as the pathways of p53 signal，cancer，Wnt signal and MPK，etc. The results demonstrate that these differentially-expressed miRNAs are correlated with DNA damage response-related biological processes and signaling pathways，indicating the prominent importance of these miRNAs in the doxorubicin-induced DNA damage response in hepatocellular carcinoma.

Preparation of Porous Biological Carrier with DHP-galactose Complex and Its Application in Culture of Human Hepatocytes

WU Chen-xi, XIE Yi-min, YE Zhe-zi, WANG Peng, LE Xi

2016, 32(6):  250-257.  doi:10.13560/j.cnki.biotech.bull.1985.2016.06.037

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In order to enhance the biocompatibility of dehydrogenation polymer(DHP)with human hepatocytes，the DHP was copolymerized with D-galactose，by which a porous biological carrier for medical use was prepared using the method of hydrogel. Fourier transform infrared spectrometer(FT-IR)，nuclear magnetic resonance(NMR)spectroscopy，BET specific surface area determination，and scanning electron microscope(SEM)were applied to elucidate the main composition，chemical and physical structure，and morphology of the porous biological carrier. Inverted microscope and albumin and glucose kits were used to check the morphology and metabolic activity of cultured hepatocytes. The results indicated that the porous biological carrier with excellent performance was prepared with co-polymer of DHP and D-galactose as raw material，and the specific surface area was 6.013 m²/g. The human hepatocytes healthily adhered on the porous biological carrier to grow and proliferate while the carriers were used to culture human hepatocytes. The maximum values of albumin secretion and glucose consumption using biological carrier containing galactose were 9.85 g/(d·L)and 16.134 mmol/(d·L)，respectively，demonstrating that the hepatocytes presented high metabolic activity during culturing，and the porous biological carrier prepared with DHP-galactose complex had a great biocompatibility.

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2016, 32(6):  300-300. 

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Table of content

2016, 32(6):  400-400. 

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Copyright

2016, 32(6):  500-500. 

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