Biotechnology Bulletin

Study Progress on Gibberellin Metabolism and Signaling Transduction Pathway in Fruits Trees

WANG Wen-ran, FAN Xiu-cai, ZHANG Wen-ying, LIU Chong-huai, FANG Jing-gui, WANG Chen

2017, 33(11): 1-7. doi:10.13560/j.cnki.biotech.bull.1985.2017-0650

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Gibberellin,as one of five major plant hormones,plays a key role in flower bud differentiation,inflorescence development,flowering and fruit setting,fruit growth and plant morphogenesis,etc. However,the research on molecular biology of gibberellin in fruit crops compared with field crops differs largely. In order to more effectively and reasonably use gibberellin to regulate fruit and flower development of fruit trees in fruit crops production,it is necessary to study the synthesis of gibberellin and the molecular regulation mechanism of signal transduction pathways. Here,two aspects,the synthesis of gibberellin of fruit crops and gibberellin signaling pathways,are reviewed,focusing on the key enzyme genes in the process of gibberellin synthesis and their localizations,as well as important components of gibberellin signaling pathway such as gibberellin receptor GID1,DELLA protein and so on. The results showed that the expressions of KO,GA2ox and GA20ox in GA synthesis were negatively correlated with the dwarf of fruit trees,while the content of KS was positively correlated with plant height. The male sterility of chestnut was also closely related to the expression of KO and KAO. GAMYB gene and LFY gene play an important role in the reproductive growth of fruit crops,such as flower formation and stamen development. DELLA protein,as a negative regulator in the GA signaling pathway in fruit trees,lead to the formation of dwarf plants,and DELLA protein ubiquitination degradation plays a vital role in the cell cycle,transcription regulation,flower formation,cell signal transduction,and many physiological processes of the fruit crops. This paper aims at providing an important theoretical reference for the modulation of fruit crop growth and development by effectively utilizing gibberellin.

Research Advances on Induced Mutation Breeding of Sesame

YU Mu, ZHOU Qiu-feng, ZHAO Jian-guo, ZHANG Guo-guo

2017, 33(11): 8-12. doi:10.13560/j.cnki.biotech.bull.1985.2017-0219

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In recent years,the use of space environment mutagenesis,ion beam irradiation and nuclear radiation mutagenesis has become increasingly active in improving crop and exploring new genes in China. Induced mutagenesis technology used in sesame new material creation and new cultivar breeding began in 1950. According to incomplete statistics of the FAO/IAEA database,up to March 2017,there are officially released 3 246 mutant cultivars from 217 crop species in more than 60 countries of the world;25 sesame mutants were released by 8 countries,China released 5 sesame mutants,ranking in the second while accounting for 20% of the total. In this paper,the results of mutagenesis of sesame and the mutations of agronomic traits and disease resistance of sesame are reviewed,and the objectives and methods of future sesame mutation breeding are also discussed. This paper aims at providing an important theoretical reference for the study of sesame functional genomics to provide rich and diverse materials and sesame germplasm innovation.

Promoting Mechanism of Plant Growth-promoting Rhizobacteria in Medicinal Plants

ZENG Mei-juan, ZHONG Yong-jia, DIAO Yong

2017, 33(11): 13-18. doi:10.13560/j.cnki.biotech.bull.1985.2017-0359

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Medicinal plants are the most important sources of Chinese traditional medicine system,and are the natural treasure for preventing and treating diseases. Rhizosphere is the main location where exchanging the substance and energy between plant and environment happens. Plant growth promoting rhizobacteria can change physicochemical properties of rhizosphere soils in medicinal plants,accelerate transformation of available nutrient and produce corresponding specific substances,subsequently affecting its growth,development,metabolic activities,and so on. These bacteria play an extremely important role in plant growth. We here summarize the recent domestic and overseas research progress on nitrogen fixation,phosphate solubilization,producing hormone and biological control of plant growth promoting rhizobacteria in medicinal plants. We aim to provide a theoretical basis for medicinal plants research and new ideas for the cultivation,production and germplasm innovation of medicinal plants.

Research Advances of NDPKs in Plants

ZHU Xin-ni, WANG Shan-shan, ZHOU Jia-qin, ZHU Shi-hua

2017, 33(11): 19-28. doi:10.13560/j.cnki.biotech.bull.1985.2017-0261

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Nucleoside diphosphate kinases（NDPKs）are a class of highly conserved proteins with a size between 70 to 100 kD. They exist as hexamers in most organisms while as tetramers only in few prokaryotes. NDPKs are mainly responsible for keeping the in vivo cellular balance between nucleoside diphosphates and nucleoside triphosphates. Four types of NDPKs,i.e.,NDPKⅠ,NDPKⅡ,NDPKⅢ and NDPKⅣ,are found in plants,however,till now researches have been mainly focused on the first three. NDPKIs are found to be involved in plant growth and development,and response to abiotic stress,pathogens and hormones. NDPKⅡs take part in photosynthesis and removal of reactive oxygen species. NDPKIIIs are involved in energy metabolism and programmed cell death. NDPKIVs are only found in Arabidopsis and rice genomes and predicted to be located in endoplasmic reticulum with unknown function. Besides,NDPKs also display special functions in certain plants,such as DNA replication,starch and cellulose synthesis,auxin signaling and ribozyme activities;however,whether these functions are species-specific needs further study. This article summarizes the phylogenetic analysis and the most recent research progress on biological functions of NDPKs. Finally,the article discusses the prospect of NDPKs research,thus it will facilitate the in-depth and comprehensive study of their function in plants in the future.

Response Mechanisms of Elevated CO₂ Concentration on Endogenous Hormones of Plants

WANG Qi, XU Meng, SHI Xin, GAO Jia, MA Lian-ju, WANG Lan-lan

2017, 33(11): 29-34. doi:10.13560/j.cnki.biotech.bull.1985.2017-0433

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With the development of industrialization,especially the rapidly increasing use of fossil fuels,the atmospheric carbon dioxide concentration is rising. The continuous increase of CO₂ concentration will impact the growth and development of plants largely,and the coordination of various hormones in plants is key to regulate plant growth and development. Therefore,it is significant and promising to study the change of endogenous hormone content and the intrinsic response mechanism of plant after elevated atmospheric CO₂ concentration. At this stage,the research on plant root morphology,growth and development under high CO₂ concentration is widespread,but few studies have been done with plant endogenous hormones. This paper reviewed the research achievements of other scholars,and found that elevated CO₂accelerated the net assimilation rate,improved the net photosynthesis,concurrently accumulated growth-promoting hormones,reduced the growth-inhibiting hormone,which subsequently regulated the allocation of assimilates and promoted the growth of plants. The paper also summarized the effects of elevated CO₂ concentration on IAA,GA₃,ABA,CK and ET,and analyzed the effects of CO₂ on related hormone synthesis and gene expression of signal transduction pathway,including the changes of the contents and the internal mechanism of the endogenous hormones in different plants. The future research directions in this field were also discussed.

Latest Research Progress on RNA Interference Technology

WANG Wei-wei, LIU Ni, LU Qin, LING Xiao-fei, CHEN Hang

2017, 33(11): 35-40. doi:10.13560/j.cnki.biotech.bull.1985.2017-0455

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RNA interference（RNAi）is a phenomenon in which the target gene is silenced by double-stranded RNA（dsRNA）inducing homologous mRNA degradation in the eukaryotic cells. At present,RNAi technology,as an effective tool for gene regulation,is widely used in pest control,cure of tumor and cancer duo to its high specificity,high efficiency,transmission and other characteristics. Especially in the medical application,compared with the traditional vaccine prevention and drug therapy,RNAi technology has unparalleled advantages of target medication,reducing side effects and inhibiting the expression of virulence genes,thus it plays an increasing significant role in the new treatment methods of cancer and other diseases. The mechanism,introducing methods of RNAi and its latest application in plant improvement,biological control,insect resistance,disease prevention and treatment are synthetically analyzed and evaluated in this paper,for providing the reference for researchers in related fields.

Research Progress and Prospects on Anticancer Peptides

YUE Shuo-hao, TIAN Chi, HU Yuan-zhao, ZHANG Jun-lin

2017, 33(11): 41-47. doi:10.13560/j.cnki.biotech.bull.1985.2017-0424

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With the increase of cancer global incidence in each year,cancer has become a major cause of death in global and public health problem. Anticancer peptides,which could directly kill tumor cells or inhibit the growth and metastasis of cancer cells as well as the formation of tumor blood vessels,almost showing no hemolysis and no damages to normal human cells,has become a focus in new antitumor drug research. The main sources of anticancer peptides can be divided into four categories. Antimicrobial peptides such as Lactoferricin B（Lfcinb）and Cecropin,feature the broad-spectrum and highly effective inhibitory activities to bacteria and other microorganisms,and simultaneously inhibit the proliferation and growth of a variety of cancer cells,thus induce the apoptosis of cancer cells. Peptide hormones such as atrial natriuretic peptide（ANP）and other peptides in the formation process of ANP,having the ability of increasing vascular permeability,antihypertensive,natriuretic,diuresis,vasodilation,regulating water and salt balance and inhibiting cell proliferation in clinic,were recently discovered that they can directly kill tumor cells or inhibit the synthesis of cancer cell DNA,leading to the growth inhibition of cancer cells. Peptide toxins such as bee venom,snake venom,scorpion venom and other strong toxins all showed inhibitory effects on multiple tumors. Endostatin also may prevent the growth and metastasis of cancer cells by suppressing the formation of tumor blood vessels. Anticancer peptides inhibit the proliferation of cancer cells by inducing apoptosis,destroying the membrane structure of cells,altering the pH of cells around or intracellular and enhancing immune response. Therefore,this paper reviewed the sources of anticancer peptides,the mechanism of inhibiting cancer,and analyzed the application prospect of anticancer peptides,providing a reference for the further research and development of anticancer peptides.

Application of Aptamers Conjugated Inorganic Nanomaterial in Tumor Research

PENG Li, ZHANG Xing-mei

2017, 33(11): 48-53. doi:10.13560/j.cnki.biotech.bull.1985.2017-0301

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At nowadays in the promotion of precise medical treatment,how to achieve tumor targeted therapy and reduce the side-effects of radiotherapy is particularly critical after we understood the mechanism of the key molecular targets closely related to tumorigenesis. Aptamers are single-stranded DNA or RNA oligonucleotides which can bind with various targets at high affinity and specificity. Inorganic nanomaterial is an important part of nanomaterial as a medical diagnosis and treatment preparation. Taking the advantage of the properties of aptamers binding with their targets at high affinity,the system of their binding with inorganic nanomaterial can be applied in the tumor research in an integrated approach of targeted binding,biological imaging and drug delivery,concurrently which efficiently promotes the development of nucleic acid nanotechnology. In this review,we discuss the application of varied inorganic nanomaterial binding with the aptamers in tumor research and the safety of inorganic nanomaterial in medical applications,aiming at earlier achieving the development of new targeted therapy strategies to tumor.

Advances on the Proteomics of Bovine Oocyte and Preimplantation Embryo Development

PU Li-ping, CHEN Fu-mei, ZHAO Xiu-ling, WANG Huan-jing, HOU Zhen XU, Zhuang-zhuang, ZHANG Peng-fei, ZHANG Ming

2017, 33(11): 54-59. doi:10.13560/j.cnki.biotech.bull.1985.2017-0404

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Proteomics,as a high-throughput method for displaying protein expression profiles of specific biological processes in a panoramic view,is being applied in more and more research fields. Bovine oogenesis and development of preimplantation embryo are inseparable from a series of changes in protein,thus the molecular mechanism of bovine oocyte maturation and embryo development can be investigated by proteomics. By establishing the proteomic expression profiles of oocytes before and after maturation as well as preimplantation embryos in different stages,we obtained marker proteins related to oocyte maturation and further validated and analyzed these markers,benefiting to understand the molecular mechanism of bovine oocyte maturation,in vitro fertilization and preimplantation embryo development. It lays a foundation for improving the mature rate of bovine oocyte,the rate of embryonic development and the rate of bovine reproduction. In this paper,the progress of proteomics in the bovine oocytes and development of preimplantation embryos was reviewed from the methods of studying proteomics,such as two-dimensional polyacrylamide gel electrophoresis,chromatography and mass spectrometry. To provide reference for further study of bovine oocyte and preimplantation embrv.

A Method for Extracting Total RNA of Taxus chinensis at Large-scale

CHEN Xiao-feng, ZENG Qi-feng, DENG Xu, ZENG Guang-yao, ZHOU Ying-jun

2017, 33(11): 60-66. doi:10.13560/j.cnki.biotech.bull.1985.2017-0319

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This study was directed to develop the optimal method for the extraction and purification of total RNA in Taxus chinensis R. First,we investigated the effects of extraction reagent,time,and method on the yield of total RNA from T. chinensis R. using Trizol and CATB extraction methods in respect of RNA concentration and the ratios of OD₂₆₀/OD₂₈₀ and OD₂₆₀/OD₂₃₀. Furthermore,we compared the effects of PVP,β-mercaptoethanol,high salt precipitation,low concentration ethanol precipitation and gel column chromatography on the purification of total RNA. The optimal extraction method was determined as follows：treatment of taxus leaves with Trizol by the action volume of 3：40 in 5 minutes using chloroform and water（1：5）as solvent. It’s also found that column chromatography significantly improved the purity of total RNA of taxus and obtained small molecular RNA with rich miRNA. Finally,the high-quality RNA in concentration of 1000µg/mL with 2.00 - 2.20 of OD₂₆₀/OD₂₈₀ and 1.7 of OD₂₆₀/OD₂₃₀was acquired. Compared with previous methods,this method provides taxus RNA in large-scale with high quality and purity as well as with low cost,which guarantees sufficient materials.

Improvement and Optimization of Aptamer Selection

LI Shi-yu, FU Qiang, YAN Ya-xian

2017, 33(11): 67-75. doi:10.13560/j.cnki.biotech.bull.1985.2017-0469

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Aptamers have gained extensive attentions due to their similar function as antibody and many superior characteristics,how to use systematic evolution of ligands by exponential enrichment（SELEX）technology to efficiently screen aptamers is the key to its application. Combined with the research progress of aptamers in recent years,the article introduces optimization strategies for increasing the screening efficiency in three different stages of the screening process,including the design and modification of the initial random oligonucleotide library before SELEX,the selection method and generation of ssDNA library during SELEX,and the optimization of aptamer performance after SELEX（Post-SELEX）. Especially,the article compares many screening methods including capillary electrophoresis-SELEX,cell-SELEX,magnetic beads based-SELEX,tailored SELEX,microfluidic SELEX,microarray SELEX,and high-throughput SELE,as well as the methods for the generation of ssDNA such as asymmetric PCR,λ exonuclease digestion,magnetic beads-based separation,unequal length PCR,and some comprehensive methods. Also we explore the issues and prospects of the development of aptamers,providing references for related researchers.

Effects of ABA and Its Synthesis Inhibitor Sodium Tungstate on Carotenoid Associated Genes and Enzymes of Cassava Tuber Root

DENG Chang-zhe, AN Fei-fei, LI Kai-mian, CHEN Song-bi

2017, 33(11): 76-83. doi:10.13560/j.cnki.biotech.bull.1985.2017-0349

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This work aims to analyze the effect of the exogenous ABA and its inhibitor sodium tungstate（Na₂WO₄）on carotenoid associated genes and protein of cassava tuberous root. Manihot esculenta Crantz SC9 was chosen as research subject and the β-carotene of cassava tuber root was detected by HPLC,and Western blot was used to analyze the carotenoid associated protein expression and QPCR to analyze the genes expression. The results showed that exogenous ABA and sodium tungstate increased the β-carotene content in the tuber root. Both exogenous ABA and sodium tungstate increased carotenoid biosynthesis key genes PSY2 and LCYB expressions,meanwhile they restrained the degradation genes ZEP and NCED3expressions,which explained the change of β-carotene content at the transcription level. The LCYB and ZEP transcriptions were consistent with translations. Plastid associated gene PAP1 and enzyme FBN1a were up-regulated,which increased the number of plastidglobules. Exogenous ABA and sodium tungstate accelerated the reactive oxygen（ROS）related superoxide dismutase（Fe-SOD and Cu/Zn-SOD）expressions,keeping the carotenoid content stable. In conclusion,these three factors jointly increase the β-carotene content of tuber root.

Clone and Expression Characteristics of MeTPS9 Gene in Cassava

DING Ze-hong, FU Li-li, TIE Wei-wei, YAN Yan, HU Wei

2017, 33(11): 84-91. doi:10.13560/j.cnki.biotech.bull.1985.2017-0416

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This work aims to reveal the function of MeTPS9 gene in abiotic stresses（e.g.,drought,cold,and shade）in cassava. RT-PCR method was applied to clone MeTPS9 gene（GenBank accession number：MF276889）from cassava leaves,MEGA software to construct its phylogenetic tree,DnaSP software to analysze its structural variation,PlantCARE software to analyze its promoter cis-element,and quantitative RT-PCR（qRT-PCR）to explore its expression patterns in different tissues and in response to different abiotic stresses. Results showed that a TPS（trehalose-6-phosphate synthase）gene,MeTPS9,was cloned from cassava. This gene had a 2598-bp open reading frame,encoded 865 amino acids,and contained a TPS conserved domain. Phylogenetic analysis revealed that MeTPS9 had close genetic relationship with its homologs from Capsella grandiflora and Arabidopsis thaliana,and the sequence similarity was up to 76.2% and 75.6%,respectively. Gene structural variation demonstrated that a total of 66 mutation sites were identified between wild and cultivated cassava species,of which 11 were mis-sensed. Promoter assay showed that MeTPS9 contained drought-related motif MBS,cold-related motif LTR,heat-related motif HSE,and ABA-related motif ABRE. qRT-PCR analysis revealed that MeTPS9 expressed the highest in fibrous root but the lowest in leaf. In addition,the expression of MeTPS9 was significantly induced by drought,cold,and shade stress. MeTPS9 played a regulatory role in abiotic stress such as drought,cold,and shade treatment at the transcriptional level and it could be served as a candidate to further study its functions in abiotic stress defense in cassava.

Research On the Induced Resistance of Protein Elicitor PeaT1 with Jinggangmycin in Tobacco Against TMV

PENG Zhi-chao, TONG Zan-hua, QIU De-wen

2017, 33(11): 92-95. doi:10.13560/j.cnki.biotech.bull.1985.2017-0454

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Tobacco virus infects tobacco,tomatoes,peppers,potatoes,and other crops,causing a variety of crop diseases that are difficult to control during production. To explore the optimal mixed proportion of protein elicitor PeaT1 and Jinggangmycin to defense tobacco virus disease,this study chosen Nicotiana benthamiana cv. Huangmiaoyu having 3 to 7 leaves,respectively by spraying water,Jinggangmycin,and combination of PeaT1 and PeaT1-Jinggangmycin. Virus TMV-GFP was inoculated by the way of friction,and viral infection situation was observed under UV light. The results showed that the combination of Jinggangmycin mixed with PeaT1 had better synergism than using single one;PeaT1：Jinggangmycin at 3：4 mixed ratio had the most cost-efficient,and cotoxicity coefficient reached 157.64,the relative control effect reached 73.39%. Therefore,we thought that Jinggangmycin and PeaT1 both generated tobacco resistance against TMV,and their combination had synergetic effect.

Cloning and Expression Analysis of Cinnamate 4-hydroxylase Gene from Glycyrrhiza uralensis Suspension Cultured Cells

ZOU Guang-ping, LI Ya-li, FENG Meng-wei, YANG Xiu, XU Zhi-wei

2017, 33(11): 96-100. doi:10.13560/j.cnki.biotech.bull.1985.2017-0466

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According to the reported C4H（cinnamate 4-hydroxylase）gene sequence（GenBank accession ID：D87520.1）from Glycyrrhiza echinata,the coding sequence of C4H gene from Glycyrrhiza uralensis Fisch suspension cultured cells was obtained by RT-PCR cloning technology,and the bioinformatics analysis on it was carried out. In addition,the expression patterns of C4H gene induced by methyl jasmonate（MeJA）were analyzed by q-PCR. The length of the cloned C4H’s open reading frame（ORF）was 1 515 bp,encoding 504 amino acids. The q-PCR experiments showed that C4H gene was induced by MeJA and reached the highest expression level at 12 h after MeJA added into the cell suspension system.

Extraction of Volatile Oil from Zanthoxylum and Determination of Its Antibacterial Activity and Insect Resistance

WANG Shan-shan, WU Hao, LI Liang

2017, 33(11): 101-105. doi:10.13560/j.cnki.biotech.bull.1985.2017-0412

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Extraction effects of volatile oil from Zanthoxylum by different solvents were studied,then the experiment of its antibacterial activity was conducted,and the antifeeding and repellency of cotton bollworms to volatile oil from Zanthoxylum were investigated. The results of the experiment were as follows,the best extraction rate was 8.77% while using ether as solvent,and there was no significant difference on antibacterial activity between strains. The extracted volatile oil from Zanthoxylum by ether showed priority on antifeeding and repellency of cotton bollworm larvae than those by other solvents,thus it should have better inhibitory effects on the larvae.

Identification and Resistance of an Endophytic Fungus YD02 Strain in Wild Glycine soja

ZHOU Zhen-yu, HU Jin-li, SU Xin, LIU Dong-xue, BU Ning, MA Lian-ju

2017, 33(11): 106-111. doi:10.13560/j.cnki.biotech.bull.1985.2017-0403

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The endophytic fungus YD02 was isolated from a wild Glycine soja and identified using morphological feature and molecular biology methods. The single factor experiment and the orthogonal test were applied for determining the optimal fermentation conditions of the strain. Further,dry weight method was used to determine the resistance of the strain on heavy metal Cd stress,and the confrontation culture method was to value its effect on the pathogenic bacteria of plant. The results showed that YD02 colony was black or dark green,hyphae branched and spores produced,and it belonged to Alternaria Nees. The optimal fermentation condition was 4% sucrose,0.6% ammonium chloride,0.4% potassium hydrogen phosphate,0.05% magnesium sulfate,pH 5 or pH 9,culture temperature 28℃ with 60% fluid amount,120 r/min and 60 h fermentation on the base of PDB culture medium. When cadmium concentration reached 140 mg/L,YD02’s growth stopped. YD02 strain presented obvious antagonistic effects on the Pythium aphanidermatum and apple moldy core pathogens,and the bacteriostatic rate was 64.15% and 71.68%,respectively.

PCR-based Method for the Rapid Detection of ACC Deaminase-producing Rhizosphere Bacteria

QIN Yuan, PAN Xue-yu, YUAN Zhi-lin

2017, 33(11): 112-122. doi:10.13560/j.cnki.biotech.bull.1985.2017-0370

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Producing ACC（1-aminocyclopropane-1-carboxylate）deaminase to reduce ethylene levels and eliminate salt stress is the key mechanism of plant-growth-promoting rhizobacteria（PGPR）in improving plant growth and stress tolerance. This work aims to establish an expeditious approach to screen bacteria containing ACC deaminase based on polymerase chain reaction（PCR）. We set the acdS gene encoding the ACC deaminase as the probe,then selected three pairs of primers（acdSf3/acdSr4,DegACCf/DegACCr and F1936f/F1938r）respectively,and detected the various bacterial strains isolated from the root and rhizosphere soil of diverse halophytes and Populus deltoids. The results showed that single specific amplified bands were produced and the rate of amplification was high when using the primers acdSf3/acdSr4 combined with touch-down PCR methods;however,DegACCf/DegACCr or F1936f/F1938r was in poor specificity. Finally,we acquired 25 strains containing acds gene from 257 bacterial isolates,aiming at laying a foundation for reserving rich and functional microbial resources and further studying the genetic diversity and function of acdS in PGPR.

Screening of Probiotics for Inhibiting Pathogens and Preliminary Determination of Antimicrobial Substances

WANG Li-jie, YU Xiao-bin, FANG Yin-bing, GU Qiu-ya

2017, 33(11): 123-129. doi:10.13560/j.cnki.biotech.bull.1985.2017-0411

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In order to obtain probiotics that inhibit pathogens and to determine their antimicrobial substances,strains that can produce inhibitory zones were isolated from the soil,feed additives and fertilizer additives. Strains isolated and preserved in the laboratory were re-screened with the Oxford Cup method. Then the unknown strains were identified with morphological observation,16S rRNA -based phylogeny and physiological and biochemical reaction. The antimicrobial activity of fermentation supernatant was determined after treated by acid-base reaction,high temperature,protease decomposition,salting-out and dialysis. Results showed that 8 strains presented the strong inhibitory effect on Escherichia coli,Staphylococcus aureus and Micrococcus luteus,and 4 strains were known as Lactobacillus bulgaricus,Lactobacillus casei,Lactobacillus rhamnosus and Lactobacillus plantarum. Three of the 4 unknown strains were identified as Bacillus licheniformis and one was identified as Pediococcus lactis. Bacillus licheniformis can produce a variety of antimicrobial substances with different molecular weights and high temperature resistance,which can or cannot pass through 8-14 kD dialysis bags,and the antimicrobial substances produced by Bacillus licheniformis are preliminarily determined as polypeptide,and antimicrobial substances from 5 acid-producing strains are mainly acidic.

The Screening of β-Glycosidase-producing Strain and the Transforming of Resveratrol

FENG Wei, HU Xiao-yan, MA Ming-na, GUO Meng, LU Fu-ping, LI Yu

2017, 33(11): 130-135. doi:10.13560/j.cnki.biotech.bull.1985.2017-0439

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Resveratrol has a lot of benefits such as anti-cancer and anti-oxidation,and thus is widely applied in cosmetics,medicine and so on. This study aims to screen bacteria which can secrete β-glycosidase and study it’s ability of transforming polydatin. Through the geniposide plate screening and polydatin shake flask,a strain secreting β-glucosidase and transforming polydatin to resveratrol was acquired. It was identified as Bacillus safensis by 16S rDNA sequence analysis,named as CGMCC 13129. Under these conditions of 37℃,with 7 percent of inoculation,pH7,and conversing for 8 h,the conversion rate of polydatin was up to 90%. HPLC,HPLC-MS and ¹H-NMR were used to test and identify the product as resveratrol,and the purity of the product was up to 99.3% by one-time methanol extraction.

Biocatalysis of Phenolic Glycosides Natural Products in Escherichia coli Strain Using UGT73B6

HE Qing-lin, BAI Yan-fen, ZHOU Wei, YIN Hua, CHEN Ning, ZHUANG Yi-bin, LIU Tao

2017, 33(11): 136-142. doi:10.13560/j.cnki.biotech.bull.1985.2017-0363

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The glycosyltransferase UGT73B6 from Rhodiola sachalinensis with substrate flexibility can glycosylate many natural products catalyzed by UDP-glycosyltransferases（UGTs）. To achieve the biosynthesis of phenolic compounds in Escherichia coli,in this study,we utilized the catalytic activity of the glycosyltransferase UGT73B6 and fed 7 different phenolic compounds in Escherichia coli BL21（DE3）. By using biotransformation,we successfully achieved the biocatalysis of 8 glycoside compounds for the first time in Escherichia coli with UGT73B6,and further we explored the substrate flexibility of UGT73B6. On the basis of this study,we provided an effective and economical method to produce phenolic glycosides by microbial transformation,which has a great application value.

Aspartokinase G359D from Corynebacterium glutamicum Relieves the Synergistic Inhibition of Lysine and Threonine

XU De-yu, ZHENG Xiao-mei, ZHAO Jing, ZHENG Ping, ZHAO Shu-xin

2017, 33(11): 143-152. doi:10.13560/j.cnki.biotech.bull.1985.2017-0387

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Aspartokinase（AK）is a key enzyme involved in the lysine biosynthesis,but its activity is synergistically inhibited by end-products such as lysine and threonine. This study focuses on finding new mutation to relieve the synergistic inhibition and unveiling this molecular mechanism form protein structure analysis. The amino acid sequences of AK from high lysine-producing strain Corynebacterium glutamicum ZL5 and wild-type（WT）C. glutamicum ATCC 13032 were aligned,and it was shown that G359D mutation was detected in aspartokinase from C. glutamicum ZL5. To discovery the biological function of this mutation,these WT and G359D Aks were expressed in Escherichia coli and were purified by the affinity chromatography;then,the purified recombined proteins were used for enzymatic activity detection when added the inhibitors lysine and threonine. The G359D protein displayed high resistance against the synergistic inhibition of lysine and threonine. The enzymatic activity of G359D remained at 76.94%±1.61% when the concentration of lysine and threonine reached 10 mmol/L,but the WT protein only was in 4.38%±1.28%. The similar result was also verified by the reverse mutation of G359D in the genome of C. glutamicum ZL5 producing high lysine yield,and the lysine yield decreased by 15.57%. From the further homology modeling and protein structure analysis,the G359D still interacted with lysine and threonine,but it enabled to relieve the allosteric effect of lysine,resulting from that the Arg¹⁵¹and Glu⁷⁴ in the catalytic active site of G359D could not form the ionic bond,thus allow the substrate into the active site. The aspartokinases G359D enabled to relieve the synergistic inhibition of lysine and threonine by blocking the allosteric effect of lysine.

Screening and Identification of a Marine Bacterium Producing Chitin Deacetylase and Optimization of Its Fermentation Condition

LAI Jiang-li, LIU Shu, HU Sheng-yuan, GU Zhang-hui, WANG Shu-jun, FANG Yao-wei

2017, 33(11): 153-159. doi:10.13560/j.cnki.biotech.bull.1985.2017-0413

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A strain MCDA02 producing chitin deacetylase was screened with the media added nitro-N-acetyl aniline from the marine mud of Haizhou bay. Then,it was identified as Microbacterium keratanolyticum through morphological,biochemical characteristics,and 16S rDNA sequence analysis. With single-factor strategy and orthogonal experiments,the fermentation condition was optimized as followings：fermentation period of 48 h,initial pH of 7.0,culture temperature of 30℃,50 mL of liquid medium in a 250 mL flask,and 3% chitin. Under the optimized conditions,the highest chitin deacetylase activity of strain MCDA02 reached 158.47 U/mL,which was about 3.2 times of that before optimization. The results laid a foundation for further study of the chitin deacetylase from the strain MCDA02.

Effects of Nitrogen Stress on Growth and Oil Accumulation of Chlorella emersionii

CHENG Wei-lan, SHAO Xue-mei, SONG Cheng-fei, SHI Fei-fei, JI Chun-li, LI Run-zhi

2017, 33(11): 160-165. doi:10.13560/j.cnki.biotech.bull.1985.2017-0519

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Fast-growing Chlorella emersionii strain SXND-25 with great ability of purifying waste water was used as the target microalgae to investigate the effects of culture condition under varied nitrogen（N）concentration on microalgae biomass and oil yield,aiming at establishing an optimized culture system of such valued chlorella for commercial production of high quality biofuels. BG11 medium was used as the standard medium containing NaNO₃ as nitrogen（N）source（1 N）. This new strain of C. emersionii was continuously cultured in the medium containing different nitrogen concentrations in the medium set as 0 g/L（0 N）,0.375 g/L（1/4 N）,0.75 g/L（1/2 N）,1.125 g/L（3/4 N）,and 1.5 g/L（1 N）,respectively. The biomass,oil content and fatty acid composition of the microalgae were analyzed by means of optical density measurement,Nile red staining,direct ester extraction of oil and GC analysis. When cultured in 1 N-medium for 8 d,the C.emersionii increased its biomass up to the maximum level with 3.4 g/L dry weight（DW）,whereas oil content was only 28.24% of DW,with 0.96 g/L oil yield on average. Culture of 8 days at 0 N-medium led to the minimum biomass of the C.emersionii with 0.49 g/L DW,but the highest level of oil accumulation（44.57% of DW）and small average oil yield（0.22 g/L）. Notably,cultivation of 8 d in 1/2 N-medium resulted in the C. emersionii to achieve high biomass（3.2 g/L DW）and moderate oil contents（40.36% of DW）,but the highest oil yield of 1.3 mg/L on average. which was 1.4 folds of oil yield cultured in the standard medium（1 N）. Moreover,the fatty acid profile in the Chlorella cultured in 1/2 N-medium for 8 d was detected to be more suitable for production of high-quality biodiesel. In conclusion,comprehensive analysis of various indexes including biomass,oil content and oil yield per unit demonstrates that 0.75 g/L nitrogen concentration can be used as an optimized parameter for developing an effective cultivation system of this special Chlorella strain in producing high-quality biodiesel at large scale.

Cloning and Expression Profiles of Lc-UBE2D4 Gene from Large Yellow Croaker Larimichthys crocea

HAN Kun-huang, ZHOU Peng, ZOU Zhi-hua, ZHANG Zi-ping, WANG Yi-lei

2017, 33(11): 166-173. doi:10.13560/j.cnki.biotech.bull.1985.2017-0451

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Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diverse biological processes,including cell-cycle progression,cell apoptosis,gene transcriptional regulation and expression,etc. In these processes of ubiquitin-proteasome pathway,ubiquitin-conjugating enzyme E2 is of pivotal importance that mediates more than 80 percent of protein degradation of eukaryotic cells,and it plays a key role on the specific selection of degraded of target protein. In this study,a homologous gene fragment of ubiquitin-conjugating enzymes E2 D4（Lc-UBE2D4）was screened from previous constructed normalized gonad cDNA library of large yellow croaker Larimichthys crocea was obtained,and its full length cDNA was cloned by using SMART-RACE technology,and then the differential expression profiles in different tissues and different gonadal development stages were analyzed using quantitative real time PCR（qRT-PCR）. The full length cDNA of Lc-UBE2D4 gene was of 798 bp,with 441 bp of ORF encoding a protein of 147 amino acids,and it contained the highly conserved ubiquitin-conjugating enzymes UBC domain and an active site cysteine. The qRT-PCR results revealed that the highest expression level of Lc-UBE2D4 presented in spleen and testis which were extremely higher than those in other tissues（P < 0.01）,and its expression level in ovary was significantly higher than other organs（excluding spleen and testis）（P < 0.05）. Moreover,its expression levels in three stages of testis were higher than in the same growth stages of ovary（P < 0.05）. These results suggested that Lc-UBE2D4 played essential roles in the gonad development,especially in testis development. Meanwhile,Lc-UBE2D4 may be involved in immune function due to highest expression in spleen.

Screening of Reference Genes for Quantitative Real-time PCR Analysis in Asarum sieboldii

ZHAO Xiao-bing, PAN Lei, LIU Wei, LIN Mao-yi, LI Hong-qing, LIU Zhong

2017, 33(11): 174-179. doi:10.13560/j.cnki.biotech.bull.1985.2017-0352

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Selecting appropriate reference gene is key step for the accurate analysis of the expression variations of target genes. Using several common-used reference genes of SAND-1,ACT,18S rRNA,CYP2,GAPDH and TUB as candidate genes and quantitative real-time PCR analysis of gene expression,the expression stabilities of these 6 candidate genes in various samples from roots,rhizomes,leaves,petioles and flowers in different growth stages of nutrition growth,flowering and post-flowering were assessed,with a set of approaches such as geNorm,NormFinder,BestKeeper and Delta CT,representatively. It is for screening the appropriate reference genes in analysing gene expressions of Asarum sieboldiiMiq. in varied tissues at different growth stage. The results demonstrated that 18S rRNA was the optimal object to act as a proper reference gene in this species because it displayed the most stable expression in almost all samples. This study will provide a feasible and valid calibrate not only for gene function characterization concerning chemical compound biosynthesis but also for gene differential expression analysis in A. sieboldii,ensuring the accuracy and reliability of results.

Knockdown of Long Non-coding RNA HOTAIR by CRISPR/Cas9 System

LIU Hong-bo, ZHENG Peng, YIN Ze, ZHANG Tong-cun

2017, 33(11): 180-187. doi:10.13560/j.cnki.biotech.bull.1985.2017-0394

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Recently,as a mature gene editing technique,the CRISPR/Cas9 system has been widely applied. However,it is rarely used in the studies of long non-coding RNAs（lncRNAs）,owe to their specific secondary structure. The lncRNA HOTAIR was called oncogenic lncRNA overexpressing in the vast majority of cancer types,regulating various biological processes such as cell growth,apoptosis,proliferation and metastasis. Our study performed that HOTAIR was knocked out using the Cas9 system based on the stably genetic cell lines HeLa-cas9 and a pgRNA library was built. The qRT-PCR analysis suggested that the RNA expression of HOTAIR decreased by 60% with pgRNA/Cas9. MTT proliferation detection and cell scratch test revealed that HeLa cell proliferation and migration were significantly inhibited after the knockout of HOTAIR. Meanwhile,results indicated that it was associated between HOTAIR and some tumor suppressors by measuring the RNA level of p21,p53,pRB and DLC1 after the knockout of HOTAIR. Thus,it provided a novel insight to comprehensively understand the function of HOTAIR in cancer cells.

Prokaryotic Expression of Human GPC3 and Preparation of Polyclonal Antibody

TENG Qiao, XIA Li-jie, ZHANG Fu-chun

2017, 33(11): 188-193. doi:10.13560/j.cnki.biotech.bull.1985.2017-0379

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This work aims to construct the prokaryotic protein of glypican3（GPC3）and to prepare its anti-serum to mouse. The complete open reading frame（ORF）of human GPC3’s cDNA was amplified by PCR,and then cloned into prokaryotic expression vector of pET28a. Further,the GPC3 fusion protein was expressed in Escherichia coli BL21,the fusion his-protein was purified by affinity chromatography and the fusion protein of purity was measured by SDS-PAGE electrophoresis. Finally,the protein content was also measured. The Balb/c mice were immunized by the purified protein for preparing the anti-serum,and the anti-serum was identified with ELISA analysis. Results showed that the polyclonal antibody against GPC3 protein was successfully prepared and the titer of antisera was 1：51200;moreover,the specificity was validated to be fine by Western blot. Conclusively,recombinant human GPC3 protein expressed in the prokaryotic system has strong immunogenicity.

Screening of Anti-Tuberculosis Marine Actinomycetes and the Bioactivity of Strain HY286

QU Jia, ZHAO Ling-xia, CHEN Rui, LU Peng-peng, SUN Xiao-yu, SHEN Wei-rong

2017, 33(11): 194-199. doi:10.13560/j.cnki.biotech.bull.1985.2017-0388

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Marine actinomycetes are recognized as an important resource of screening new anti-tuberculosis drugs and their precursor compounds. Nine marine actinomycetes strains with efficient antagonism to Mycobacterium smegmatis（Msm）were screened by plate confrontation method from 77 actinomycetes in shallow soil,Zhangzhou. One strain HY286 with the highest inhibitory effect was identified as Actinomadura genus by colony morphological,physiological and biochemical characteristics,and 16S rDNA sequence analysis. The MIC of fermentation crude extract of strain HY286 was 200 μg/mL,indicating that it had a significant inhibitory effect on Msm. The fermentation crude extract of strain HY286 also showed inhibitory effects on Bacillus subtilis and Bacillus pumilus,and the inhibition rate to HeLa and HepG2 was 89.3% and 94.2%,respectively.

Study on the Molecular Mechanism of ANGPTL8 in Regulating the Glucose Metabolism

MA Shi-nan, LI Zhong-yao, GUO Xing-rong

2017, 33(11): 200-206. doi:10.13560/j.cnki.biotech.bull.1985.2017-0353

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This work aims to study the effect and molecular mechanism of ANGPTL8 on glucose metabolism by insulin. By tail vein hydrodynamic transfection we confirmed that over-expression of ANGPTL8 in the liver of mice improved glucose tolerance in feeding period. Detecting insulin and glucose effect on ANGPTL8 expression by qPCR in HepG2 cell,we found that ANGPTL8 was dramatically up-regulated only when insulin was added together with glucose into the medium;neither of these two reagents alone affected ANGPTL8 expression. Western blotting was employed to analyze the total proteins and their phosphorylation expressions of insulin-mediated PI3K/AKT signaling pathways in ANGPTL8 knockout（ANGPTL8^-/-）and ANGPTL8 over-expression（ANGPTL8⁺⁺）HepG2 cells. The results demonstrated that the over-expressed ANGPTL8 promoted insulin-mediated AKT,GSK3β or FoxO1 phosphorylation in PI3K/AKT signaling pathway in HepG2 cells. PAS staining results suggested that ANGPTL8 promoted glycogen synthesis.

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