Table of Content

25 September 2016, Volume 32 Issue 9

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The Responses of Chloroplasts of Plant Cells to Cold

LI Xian-wen¹, LI Ling², LIN Yang-yang¹, ZHOU Qi-ying¹, LI Xian-wei³, YAO Tian-ze¹, CHEN Hong-min¹

2016, 32(9):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.001

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Freezing is a major environmental stress to the plants growing from subtropical regions to frigid areas,and the plants have successfully evolved the strategy to vurvive the low temperature encountered. In recent years,the cold-adaptation strategy of plants has been widely studied at physiological and molecular levels i.e.,there have been certain understandings on the cold signal transduction network,types and fucntions of cold-induced genes. But the physiological characteristics and survival strategies of chloroplasts in overwintering plant during cold stress,still largely remain unknown. In the current review,we collected and compiled the reported information on the cold damages,cold-signal transduction and cold-adaptation strategy of plant chloroplasts,aiming at being conducive to the researches of this field.

Research Progress on GPX in Plants

QIAO Xin-rong, ZHANG Ji-ying

2016, 32(9):  7-13.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.002

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Adverse stress induces plants to produce excessive reactive oxygen species（ROS）,which can cause oxidative stress,and seriously affect their growth and development. Accordingly,plants produce a variety of antioxidants and antioxidant enzymes adjusting redox balance in order to adapt to many adverse environments. Glutathione peroxidase（GPX）is one of the most important antioxidant enzymes in plant. In this paper,recent research progresses on structure characteristics,subcellular localization,substrate specificity and functions of GPX are mainly reviewed,and finally the future research directions are prospected.

Research Progress on the Family of TCP Genes

LIU li-juan, GAO Hui

2016, 32(9):  14-22.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.003

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The TCP genes encode plant specific transcription factors,containing a bHLH motif,which binds with DNA or produces the interactions between protein and protein. In the family of TPC genes there are 5 members in monocotyledons and over 20 members in dicotyledons. The duplication and diversification of genes evolve two types of TCP gene families with slightly different TCP domains. Here,we just briefly summarize the evolution on the family,their regulations,the biochemical properties of their proteins,and the biochemical functions of some members,especially on cell proliferation controlling developmental tissue. Growing studies on the functions of TCP genes make it possible to adjust plants growth patterns and create new agricultural science character by regarding TCP gene as a tool,to provide ideas for better regulation of plant growth patterns and regulation of physiological characteristics of plants.

Research Advances on Long Non-coding RNA

SONG Na-na, CHAI Zhi-xin, ZHONG Jin-cheng

2016, 32(9):  23-31.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.004

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The completion of the Human Genome Project confirmed that there is a total of 30-35 thousand coding genes,the encoding information of these genes covers only 1.5% of human 3 billion base pairs carrying the genetic information,and over 98 percent of genetic information does not directly encode the proteins. In recent years,with the rapid development of sequencing technology,it is found that genetic information of non-protein-coding genes is closely related to regulation,slicing,transcription and other biological processes. Long non-coding RNA（lncRNA）regulates the critical life processes such as epigenetic regulation,transcriptional regulation,disease control,cell differentiation,and individual development,thus it become an extremely crucial issue that how to seek the RNA functional units and predict the novel long non-coding RNA. In this review,we summarize the origin and evolution of lncRNA,the functioning in cancer biology,and some common database of lncRNA,as well as using the latest bioinformatics tools and related technologies to predict lncRNAs for further functional studies.

Review on Effects of Disulfide Bond and Its Connection on Antimicrobial Activity in Defensins

CHEN Hui-xian^{1, 2, 3}, MAO Ruo-yu^{1, 3}, TENG Da^{1, 3}, WANG Xiu-min^{1, 3}, FENG Xing-jun², WANG Jian-hua^{1, 3}

2016, 32(9):  32-37.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.005

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Defensins are a class of endogenous,highly stable and cysteine-rich antimicrobial peptides,which play an important role in anti-microbial infection and the regulation of host immune. Six to eight conserved cysteines in the sequence form 3 to 4 pairs of disulfide bonds. The number of disulfide bond and its connection play an crucial role in maintaining the structure and antibacterial functions of defensins. In addition,mutants with high activity and low disulfide bond content also play an key role in reducing production costs and simplifying the production processes. The molecular characteristics,structure and function of defensins and their effects of redox status,number and connection mode of disulfide bonds on the antimicrobial activity are reviewed.

Investigation of Autophagy Induced by Amino Acid Deprivation and the Regulatory Mechanisms of miRNA in Autophagy

WANG Qi, QI Ren-li, WANG Jing, HUANG Jin-xiu

2016, 32(9):  38-43.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.006

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Amino acid is a kind of indispensable nutriment and basic material for life activities,and it is critical for animal to maintain physiological function. Autophagy is a pathway to accomplish metabolism and regenerate some organelles by the turnover and recycling of intracellular macromolecules and damaged organelles. The studies confirmed that amino acid deprivation induced the cell autophagy through the mTORC1 signal pathway. However,the molecular mechanism and level of autophagy differ significantly between total and individual amino acid,moreover,the molecular regulatory mechanism is still unclear,and further revealing is necessary. miRNA is non-coding nucleotide with 18-24 nt involving in cell proliferation,differentiation,autophagy,and apoptosis. Recent researches show that miRNA plays an important role in regulating autophagy induced by amino acid deprivation. This paper reviewed the regulatory mechanisms of autophagy by different amino acid deprivation and the key effects of miRNA on autophagy,aim to provide new approach to treat related metablic disease.

Histone Deacetylases and Its Roles in Plants

DONG Ya-ru, DU Jian-xun, CHEN Chuan-jie, ZHAO Dong-xiao, WANG Zhao-hong

2016, 32(9):  44-49.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.007

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Histone deacetylases（HDACs）are a supergene family widely distributed in eukaryotes（including yeast,mammals and plants）. HDACs act in concert with histone acetyltransferases（HATs）to regulate the status of histone acetylation,thus affecting the structure and function of the chromosome and regulating the gene transcription and a variety of functions of the cells. The researches on plant HDACs are gradually increasing,and many HDAC genes have been identified and characterized in different plants. This paper reviews the classification and the functions of HDACs in development and stress responses in plant,to provide a theoretical basis for further study of epigenetic regulation mechanism of HDAC in plant and cultivate new varieties of resilience.

Research Progress on Biocatalytic Oxidation of 5-hydroxymethylfurfural

WU Shu-li^{1, 2}, LIU Qi-shun², TAN Hai-dong², ZHANG Fu-yun¹, YIN Heng²

2016, 32(9):  50-58.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.008

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5-hydroxymethylfurfural（HMF）is an important bio-based platform chemical. Oxidation of HMF to furan chemicals is a crucial developing orientation of biomass conversion. In recent years,with the advantages of high selectivity,mild reaction conditions,and environment friendly,the biocatalytic oxidation of HMF has attracted extensive attentions. In this paper,we reviewed the discovery of biotransformation process,microbial metabolism,whole-cell biotransformation and the enzymatic catalytic oxidation of HMF. Finally,we provided the development prospect of bio-catalytic oxidation of HMF in the future,aiming at providing the theoretical foundation for the green and efficient conversion of HMF to furan bulk chemicals.

Research Progress on the Correlation Analysis Between Diabetes and Gut Microbiota Using High Throughput Sequencing

LIU Dong-lian, LIAO Meng-ling, ZHOU Huan

2016, 32(9):  59-64.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.009

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There is a metabolic link between gut microbiota and Diabetes mellitus. With the development of high throughput technologies, more and more studies show that the community structure of the gut microbiota has changed in the diabetic state. This paper reviewed the latest progress on the formation of the differences of gut microbiota in the diabetic state by the application of high throughput technologies,and also the regulatory mechanisms of gut microbiota on D. mellitus were explained from the aspects of food,drugs,prebiotics and fecal transplants.This paper provided a more methodical idea for prevention and treatment of D. mellitus.

Optimization of ISSR-PCR Reaction System on Ranunculus nephelogenes var. nephelogenes and Primer Selection

SHI Lin^{1, 2}, HU Yan-ping¹, WANG Jian-ke^{1, 2}, WANG Jun^{1, 2}, XU Xiao-ning³, LI Yi¹, WANG Li¹

2016, 32(9):  65-71.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.010

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This work is aimed to establish a stable ISSR-PCR system for Ranunculus nephelogenes var. nephelogenes. A L₁₆（4⁵）orthogonal design was used to screen 5 main parameters（Mg²⁺,dNTP,Taq DNA polymerase,primer,and template DNA）;then the process of ISSR-PCR reaction was optimized,and consequently the optimal reaction system and amplifying procedure for R. nephelogenes var. nephelogenes was established;further,the optimized reaction system and amplifying procedure was verified. Results showed that the optimal concentrations in 20 µL reaction mixture were template DNA 30 ng,Mg²⁺ 1.95 mmol/L,Taq DNA polymerase 0.04 U/µL,dNTP 0.150 mmol/L, and primer 0.5 µmol/L. The optimal reaction procedure was：pre-denaturalization for 5 mins at 94℃,denaturalization for 20 s at 94℃,renaturation for 1 min at 49.6-60.6℃,followed by 38 cycles of extension for 100 s at 72℃,and final extension for 6 min at 72℃. Under the above optimized reaction system and procedure,16 amplified primers were screened from 100 ISSR primers,and the optimal annealing temperature for each primer was determined. Verification in R. nephelogenes var. nephelogenes of different populations with the optimized ISSR system confirmed that bands amplified were clear and steady,thus the established ISSR-PCR could favor further studies on the genetic diversity of R. nephelogenes var. nephelogenes.

Establishment of Membrane Proteomics Platform with Two-dimensional Electrophoresis for Preparing Identifying Plasma Membrane Proteins from Atractylodes lancea

CHEN Fei, ZHOU Tong, WEI Yu-jia, YANG Jing, DAI Chuan-chao

2016, 32(9):  72-82.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.011

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In order to understand related metabolic pathways of secondary metabolites and carry on metabolic regulation of active medical ingredients in endangered medicinal plant Atractylodes lancea,the method of plasma membrane proteomics was studied in this paper. Purified plasma membrane proteins were respectively extracted and purified from root,stem,and leaf of A. lancea using ultracentrifugation combined with aqueous two-phase partitioning. Moreover,the conditions for two-dimensional electrophoresis（2DE）analysis were optimized,and partial proteins in the latter were identified. The results showed that treating the ultra-centrifuged crude membrane compositions with the aqueous two-phase system of 6.4%,6.3%,and 6.1% Dextran T-500/PEG 3350（w/w）respectively,the high- purity plasma membrane proteins of 92.1%（root）,91.5%（stem）and 90.8%（leaf）were obtained. The plasma membrane proteins were decomposed by 2% CHAPS and 2% Triton X-100 as the comprehensive detergents,loaded in the amount of 100 μg and resolved by IEF of 80 000 Vhs and 12.5%-15% SDS-PAGE gradient gel,and total 267,297 and 248 protein spots in the plasma membrane protein profiles of root,stem and leave respectively were detected by the Image Master 2D Platinum 7.0 software. And similarities and differences of 3 tissue proteins were further comparatively analyzed. At last,5 proteins were selected and successfully identified by MALDI-TOF-MS. Conclusively,a complete proteomics technology platform for preparing high-purity plasma membrane proteins from varied tissues of root,stem and leave of A. lancea and identifying the mass spectrometry of membrane protein is established.

Research on Population Structures and Distribution Characteristics of Indigenous Microorganisms in Bohai Oil Field N

WANG Da-wei¹, ZHANG Jian¹, HE Chun-bai¹, MA Ting², LÜ Xin¹, LI Qiang¹

2016, 32(9):  83-92.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.012

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Denaturing gradient gel electrophoresis（DGGE）method was used to analyze the structures of microorganism population in water injection wells and no-water injection wells of an oil reservoir in Bohai oil field N,and investigate the process effect of water injection to the abundance and population of microbial communities in the reservoir,aiming at providing technical support for the field test of indigenous microorganism enhanced oil recovery. The results showed that bacterial abundance and species in water injection wells were significantly higher than that in no-water injection wells,main bacteria in water injection well were Azospira sp.,Pseudomonas sp.,Thauera sp.. The species and abundance of archaea in water injection wells also presented differences from no-water injection wells,main archaea were Methanothermobacter thermautotrophicus,Methanosaeta,and Crenarchaeote. Species distribution of bacteria and archaea in water injection wells and no-water injection wells were limited,but their abundances were high,and which were mainly beneficial bacteria increasing oil recovery,indicating that this block possesses the condition to implement the microbial enhanced oil recovery technology by indigenous microorganisms.

Bioinformatics Analysis of Genomic Structure and Introns of AmCIP Gene from Ammopiptanthus mongolicus

LEI Chen, LIU Sheng-li, YU Ting-qiao, CHEN Yu-zhen, LU Cun-fu

2016, 32(9):  93-99.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.013

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According to the full length cDNA of the AmCIP gene we designed primers, amplified its DNA sequence by PCR, and analyzed the gene structure of AmCIP by using bioinformatics methods. Sequence analysis revealed that the DNA sequence of the AmCIP gene is 1 548 bp, containing two exons and one intron, GenBank accession number KU744005. One 78 bp intron is within the ORF of AmCIP gene, and the intron contains enhancer-like element involved in anoxic specific inducibility（GC-motif）and part of a light responsive element（TCCC-motif）. The 3'-UTR contains more cis-acting elements, which involve in light responsiveness, stress responsiveness, hormone responsiveness and so on; the 5'-UTR region contains only a gibberellin responsive element. The cis-acting elements may play a role in transcription and expression regulation of AmCIP gene. Furthermore, the adenine and thymine（A+T）content of AmCIP gene is 64.7% of total bases. The intron of AmCIP gene is 5'-GT......TG-3', not the GT-AG intron standard form. We suggested the mRNA of the AmCIP gene may have unique splice mechanism.

Effect of Exogenous Abscisic Acid on Chilling Tolerance and Glycine Betaine Accumulation in Jatropha curcas Seedlings Under Chilling Stress

YANG Shuang-long, DENG Feng-fei, DU Chao-kun, CHENG Yun-xiu, GONG Ming

2016, 32(9):  100-106.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.014

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The objective is to investigate the effect of exogenous abscisic acid（ABA）on chilling tolerance and glycine betaine accumulation of Jatropha curcas seedlings under chilling stress. Seedlings of J. curcas under chilling stress were treated with 150 μmol/LABA,and the survival rate,root vitality,electrolyte leakage,the content of malondialdehyde（MDA）,the content of glycine betaine,and the activity of the key enzyme betaine aldehyde dehydrogenase（BADH）of glycinebataine biosynthesis as well as the expression of JcBADH were measured. The results showed that treatment with 150 μmol/L ABA significantly increased survival rate and root vitality,decreased MDA content and electrolyte leakage in J. curcas seedlings under chilling stress. ABA also enhanced the accumulation of glycine betaine,increased the activity of BADH,and improved the expression level of the JcBADH. The results suggested that exogenous ABA treatment may enhance chilling tolerance in J. curcas seedlings,and glycine betaine might play a key role in the increasing of chilling tolerance.

Isolation and Identification of Antagonistic Streptomyces spp. Against Fusarium Wilt of Watermelon and Primarily Potted Inhibition Test

REN Qian-qi, WU Hong-sheng, LI Ji, CHEN Su-yun, XU Ya, YAO Dong-liang, ZHAO Yue, CHEN Jian, LI Mei-ying, SONG Xing-lin

2016, 32(9):  107-113.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.015

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The objective is to select a natural antagonistic bacterium efficiently controlling Fusarium oxysporum f. sp. niveum that is the pathogenic bacterium of the fusarium wilt of watermelon. Using the method of gradient dilution to isolate actinomycetes from rhizosphere soil of continuous cropping pepper,it was purified and the plate confrontation method was applied to select an antagonistic strain. Then the method of basin nursery substrates mixed with bacteria was for inoculation,a potted plant disease-resistant experiment was carried out,and the strains with the optimal antagonistic effects were identified. The results showed that 10 strains of actinomycetes with the antagonistic effects to fusarium wilt of watermelon were isolated from the growing pepper rhizosphere soil,and 3 of them（F22,F23 and F32）presented the significantly antagonistic effects. After 96 hours the diameter of the inhibition zone was 1.70 cm,1.17 cm and 1.47 cm respectively. The result of the potted plant experiments demonstrated that the control effect of the antagonistic strain F32 to the fusarium wilt of watermelon reached maximum with 66.7% at 30 d,moreover,it also had a wide antibacterial spectrum. Based on the phenotypic characteristics,physiological-biochemical characteristics,and the results of 16S rDNA molecular analysis,the F32 was identified as Streptomyces sp.,and F32 presented solid control effects on the fusarium wilt of watermelon,thus it is biocontrol strain with potential application value.

Isolation,Identification and Degradative Properties of Cyfluthrin-degrading Bacterial Strain

WEI Zheng¹, FENG Wei-min¹, SHI Yan-hua², REN Lei¹, YAN Yan-chun¹

2016, 32(9):  114-122.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.016

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Cyfluthrin-degrading strain YC-WZ5 was isolated from the long-term cyfluthrin-contaminated sludge near a pesticide factory by enriched culture. We used physiological properties and 16S rDNA sequence analysis to identify the isolated strain from enriched culture,and used GC to detect the cyfluthrin concentration of the culture in different circumstances. The degrading capacities of the strain under different conditions were studied,and response surface method was used to optimize the conditions for cyfluthrin degradation. The strain YC-WZ5 was identified to be Ochrobactrum intermedium. The 50 mg/L cyfluthrin was fully degraded by YC-WZ5 in 5 days. The conditions for cyfluthrin degradation was optimized as 30.9℃,pH7.16 and 10.9% inoculum size. The degradation rate with different initial concentration of cyfluthrin（100 mg/L,200 mg/L,300mg/L,and 400 mg/L）was 84.5%,54.7%,40% and 30.5% respectively. The degradation rate of cypermethrin,cyhalothrin,deltamethrin and bifenthrin by YC-WZ5 reached 99.83%,91.16%,84.98% and 55.76% respectively at the concentration of 50 mg/L. Conclusively,the strain YC-WZ5 has highly-efficient degradation of cyfluthrin and highly environmental adaptability.

Biological Characteristics of Three Pseudomonas Cold-active Bacteriophages

LI Ming-yuan^{1, 2}, WANG Ji-lian¹, REN Yu¹, JI Xiu-ling³, Gulbahar·Sawut¹

2016, 32(9):  123-130.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.017

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Three cold-active bacteriophages infected Pseudomonas named MuztagBP1,MuztagBP2,and MYBP2A-15 were isolated from Karakul Lake of East Pamir and Mingyong glacier,and their biological characteristics were studied. All the phages cultured by double-layer agar method were characterized by observing the shape of plaque,and the morphologies of plaque particles were observed by the electron microscopic after negative staining of phosphotungstic acid. The physiological characteristics of 3 phages such as the proliferation temperature,the stability to heat and pH,the sensitivity to chemical reagent,and the one-step growth curves were compared and analyzed. The results indicated that the morphologies and biological characteristics differed significantly though the host of 3 phages belonged to the same genus of Pseudomonas. Three phages were in the same type with the structure of head to tail. Among them,both MuztagBP1 and MYBP2A-15 had an isometric head（about 68 nm and 74 nm in diameter）with a contractile tail（about 130 nm and 160 nm in length respectively）;and they were identified as the family of Myoviridae. While MuztagBP2 belonged to the family of Siphoviridae that had a long tail（about 640 nm in length）. All the phages presented thermolability and UV resistance. MuztagBP1 was extremely sensitive to chloroform,isopropyl alcohol and ether,which indicated that MuztagBP1 was a cold-active phage with a lipid envelope,while MuztagBP2 and MYBP2A-15 were not so. The one-step growth curves demonstrated that the latent period,burst period,and burst size of 3 phages were different.

Analysis on Diversity of Archaea in the Soil of Bole River’s Entrance in Ebinur Lake Wetland,Xinjiang

MO Chao, HU Wen-ge, GUO Yang, WANG Cui-hua, WU Fei

2016, 32(9):  131-139.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.018

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This study is to investigate the diversity and community composition of archaea in the soil of Ebinur Lake Wetland. The clone library was constructed for the first time using the specific 16S rDNA primers of archaea with the samples of soil from the entrance of Bole River in Ebinur Lake Wetland. The targeted fragments were typed by amplified rDNA restriction analysis（ARDRA）using restriction endonuclease Afa I and Msp I. Positive clones randomly chosen from every spectral type were sequenced,and the phylogenetic tree of archaea 16S rDNA was constructed by sequence alignment. The results showed that 117 sequences gained from sequencing were classified into 61 operational taxonomic units（OTUs）. By aligning the sequences and phylogenetic analysis,they were classified into 2 divisions of Euryarchaeota and Thaumarchaeota. Halobacteriaceae of Euryarchaeota was the most dominant family including 10 genera such as Halomicrobium,Natronococcus,Halalkalicoccus etc.,which contained 48 OTUs（78.7%）and 77 positive clones（63.6%）. Conclusively,the diversity of archaea is abundant in the soil from the entrance of Bole River in Ebinur Lake Wetland and there should be a plentiful new unknown taxon in this environment.

Antioxidant Activity of Inonotus weigelae During the Liquid Cultivation

MENG Ge¹, ZHENG Fei¹, LIU Hong-xia², SI Jing¹, CUI Bao-kai¹

2016, 32(9):  140-148.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.019

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In order to better study and develop the pharmacological effect of the medicinal fungus Inonotus weigelae in the future,based on the tested indicators of mycelial biomass,pH,reducing sugar content,laccase activity,polyphenol content,MDA（Malonaldehyde）content,SOD（Superoxide dismutase）activity,T-AOC（Total antioxidant capacity）,and DPPH（1,1-diphenyl-2-picrylhydrazyl）radicals scavenging activity,the antioxidant activity of I. weigelae was evaluated during the liquid cultivation. The results demonstrated that the strain’s antioxidant activity was closely correlated with its mycelial growth,extracellular enzyme activity,and secondary metabolite secretion. The variations of mycelial biomass presented S-like curve,which increased rapidly from 6 to 12 d,reaching its peak at the 12th day. During the liquid cultivation,the increased peaks on reducing sugar content,laccase activity,and polyphenol content were also observed. Variation trends among the mycelial biomass,laccase activity,and polyphenol content were approximately consistent,indicating that there existed a mutual dependence between the nutrition utilization of this fungal strain and its physiological metabolism. The SOD activity and T-AOC during the culture tended to increase,indicating that the strain activated its antioxidant system to resist the damage from external environment under the oxidative stress. Additionally,I. weigelae exhibited remarkable scavenging capacity against DPPH radicals,showing the scavenging efficiency was up to 78.56% on the 2nd day and remaining over 50% on the 14th day. The above results confirmed that the medicinal fungus I. weigelae possesses the superior antioxidant activity.

Expression and Function of Embryonic Skeletal Muscle Development of Leizhou Black Duck

XU Chong, CHEN Qi, SU Ying, HUANG Han-guang, CUI Hong-yan, HUANG Jun-teng, CHANG Yu

2016, 32(9):  149-155.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.020

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To protect the excellent meat performance of Leizhou black duck,develop and utilize this precious germplasm,we selected Leizhou black duck as the experimental material and observed the tissue section of breast thigh meat in its fetal period,and used qRT-PCR and Western blot to detect the developmental expression patterns of gene Pax3,DCN and MSTN in breast thigh meat at 8th,13th,18th,23th,and 28th fetal period. Concurrently,qRT-PCR was used to test the mRNA difference expressions of genePax3,DCN and MSTN in all tissues of 28th fetal period. The observation of tissue section of breast thigh meat showed that in fetal period of Leizhou black duck,the development of thigh muscle was obviously earlier than the chest muscle. Fluorescence quantitative PCR showed that,in each stage of breast thigh meat,the relative expressions of gene Pax3,DCN and MSTN were the highest at 23th and 28th fetal period,and significantly higher than other fetal periods（P<0.05）. In all tissues of Leizhou black duck,the expressions of Pax3,DCN and MSTN in thigh muscle and chest muscle was higher than other tissues in 28th fetal period（P<0.05）;The results of Western blot showed that the expressions of Pax3,DCN and MSTN protein were the lowest in 8th fetal period and its expression in 23th fetal period was significantly higher than that in other fetal period（P< 0.05）,and reached a peak. Combined the profiles of sections,comparing them with the expression pattern analysis showed that the gene Pax3,DCN and MSTN play a determining role for the physiological characteristics of skeletal muscles in the formation and growth of muscle fibers,and regulate the differentiation,proliferation and the formation of muscle cells of Leizhou black duck early embryo at different development stages of skeletal muscle in embryonic stage.

Cloning and Bioinformatic Analysis of γ-Actin Gene（Bbgγ-Actin） from Bufo bufo gargarizans

LIU Yan-fang^{1, 2}, WANG Qiu-shuang^{1, 2}, LING Hong^{1, 2}, LIU Dong-chun^{1, 2}, DAI Ying-hui¹, WANG Dong^{1, 2}

2016, 32(9):  156-164.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.021

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In order to obtain the full-length of actin’s sequence in Bufo bufo gargarizans,the total RNA was extracted from the parotid gland of B. bufo gargarizans,and high-throughput sequencing of transcriptome was used to obtain the full-length cDNA of actin （BbgActin）in B. gargarizansas 2 055 bp. The sequence of ORF（open reading frame）was verified by RT-PCR and sequencing. Sequence analysis showed that the ORF was 1 131 bp encoding for a polypeptide of 376 amino acid residues. Homologous alignment by software BLAST P revealed that it shared over 89% of nucleotide identities and over 90% of amino acid identities with actins from other animals in GenBank. Phylogenetic analysis showed that BbgActin clustered in a class with γ-Actin and highly conserved during evolution. In conclusion,it is the first time to acquire the full-length sequence of γ-Actin cDNA in B. bufo gargarizans,which lays a foundation for further studies on the role of gene Bbgγ-Actin on the life activities of B. bufo gargarizans.

Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene

ZHANG Yan-fen¹, LIU Li-peng², CHEN Yao², CUI Zhe², BI Ke-wei², WANG Nan-nan², LIU Zhong-cheng²

2016, 32(9):  165-171.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.022

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FcγRIIb is an important inhibitory receptor with immune negative regulation function. The purpose of this study is to clone the rat FcγRIIb gene,construct the prokaryotic expression system of sFcγRIIb,and prepare the recombinant rat sFcγRIIb protein. The extracellular domain gene of FcγRIIb was cloned from RBL-2H3 cells using RT-PCR and the recombinant expression plasmid containing gene sFcγRIIb was constructed and transferred to Escherichia coli. The recombinant protein was purified by nickel column affinity chromatography and identified by Western blotting and ELISA after refolding. As results,the gene sFcγRIIb was successfully cloned,and the prokaryotic expression vector sFcγRIIb-pET17b was constructed and transferred into E. coli BL21（DE3）. By optimizing the expression system,the protein expression efficiency was the highest when the IPTG concentration was 1.0 mmol/L,and the induction time was 4 h. The recombinant proteins were mainly the inclusion bodies and dissolved by 8 mol/L urea. Then the purified recombinant protein was obtained by Ni-NTA column affinity chromatography. The results of Western blotting,ELISA and competitive ELISA showed that the recombinant protein sFcγRIIb after refolding was recognized by specific antibodies and combined with IgG. Conclusively,the prokaryotic expression system of gene sFcγRIIb was successfully established,and the recombinant protein with biological function was prepared.

Cloning,Expression and Anticancer Mechanism of Scolopin 2,an Antimicrobial Peptide from Centipede Venoms（Scolopendra subspinipes mutilans）

HOU Huan-huan, LU Jia, ZHANG Wei-jing, HUANG Fang, REN Wen-hua

2016, 32(9):  172-178.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.023

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This work is to explore the antimicrobial activity and anticancer mechanism of AMP-scolopin 2. The small ubiquitin-related modifier（SUMO）fusion technology was applied in the expression of AMP-scolopin 2 in vitro. The technologies of inhibition zone assay,MTT,cytotoxicity assay,flow cytometry and Western blotting were utilized to analyze its antimicrobial and hemolytic activity as well as apoptotic effects on cancer cells. As results,AMP-scolopin 2 was successfully expressed and purified. The AMP-scolopin 2 inhibited the proliferation of K562 leukemia cells and HepG2 hepatocellular carcinoma,while it had only a minimal effect on erythrocytes and normal HEK293 cells. AMP-scolopin 2（40 μmol/L）induced the apoptosis of 21.4% K562 leukemia cells and 18.5% HepG2 hepatocellular carcinoma respectively. In addition,AMP-scolopin 2 down-regulated the expression level of apoptosis proteins（pro-caspase-3,-9 and pro-PARP）that were related to mitochondria in a dose-dependent mode. In summary,the present study indicates that AMP-scolopin 2 promotes the apoptosis of tumor cells in vitro through mitochondrial caspase-dependent pathway.

Cloning of Gene cyt b₅ and cyt b₅r and Their Co-expression with cyp51A in Penicillium digitatum

QIN Ting-ting, GENG Hui, WANG Sheng-qiang, NIU Yu-hui, WU Zhi, LIU De-li

2016, 32(9):  179-188.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.024

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In order to investigate the role of Cytochrome b₅（Cyt b₅）and Cytochrome b₅ reductase（Cyt b₅r）in the electron transport of cytochrome P450 CYP51A in Penicillium digitatum,the co-expression mechanism of CYP51A and Cyt b₅-Cyt b₅r in P. digitatum was studied,and its effects on the expression of gene cyp51A was detected. By analyzing and screening transcriptome as well as PCR cloning,gene cyt b5 and cyt b₅r were acquired and designated as HS-Pdcyt b₅ and HS-Pdcyt b₅r. Further,a co-expressed plasmid vector ppbrA（pPIC-Pdcyp51A-cyt b₅-cyt b₅r）was constructed successfully using multiple-gene series cloning vector pPICZαA. This recombinant plasmid ppbrA was transformed into Pichia pastoris X-33 by electroporation. Analysis by qRT-PCR revealed that after CYP51A was co-expressed with Cyt b₅-Cyt b₅r,the expression level of cyp51A increased 54%-97% and it remained in a long period（48-72 h）. This indicated that the Cyt b₅-Cyt b₅r complex was capable of transferring electrons to CYP51A,which thus enhanced the transcript expression level of cyp51A. This is the first report regarding cloning and expressing HS-PdCyt b₅ and HS-PdCyt b₅r proteins from P. digitatum,and explore the function of P. digitatum’s gene cyp51A by co-expressing system.

Separation,Purification,and Enzymatic Properties of Thermo-tolerant β-glucosidase from Tridchoderma viride

PENG Li-sha^{1, 2}, ZHANG Yong-xiang^{1, 2}, YAN Qing^{1, 2}, WANG Xiang^{1, 2}, LI Jun^{1, 2}

2016, 32(9):  189-196.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.025

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This study aims to separate and purify the β-glucosidase of Tridchoderma viride and to investigate its enzyme characterization. T. viride GIM3.139 was fermented in shaking flask,and the enzymatic properties of the purified β-glucosidase by centrifugal ultrafiltration and Sephadex G-200 gel chromatography were studied. Further,the separated and purified constituents were tested by PAGE（polyacrylamide gel electrophoresis）,and it reached the electrophoretic-purity. The results showed that the optimal reaction temperature was 80℃ and it was still in a high enzymatic activity for a long time in the range of 75-95℃;also the β-glucosidase presented a strong stability under acidic and alkaline conditions and its optimal pH value was 6.5. Some metal ions including Fe³⁺,Mg²⁺,and K⁺ inhibited the activity of β-glucosidase,among which Fe³⁺ did it the most significantly. However,Fe²⁺ and Mn²⁺ activated the enzyme,and Mn²⁺ did it the most significantly. Organic solvents such as methanol and acetone increased the enzymatic activity,and methanol did it the most significantly. However,the ethyl acetate showed obvious inhibition effect. In conclusion,electrophoretic-purity β-glucosidase was separated and purified from T. viride and the enzymatic properties were characterized as well.

Green Fluorescent Protein Marker of Biocontrol Streptomyces SSD49 and Its Colonization on the Populus tomentosa Somaclone

LIU Xiao-yu, MA Yu-chao

2016, 32(9):  197-202.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.026

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Poplar canker is one of the major biohazard of poplar plantation in China,and the sustained and effective method to control poplar canker is biological control. This study aims to obtain a strain which has solid bio-control effect on Botryosphaeria dothidea and can colonize in poplar efficiently. This study imported a strong promoter（ermEp）and constructed highly-expressed green fluorescent protein（GFP）recombinant plasmid,then led the plasmid into bio-control strain Streptomyces SSD49 through joint experiment,and constructed the GFP-tagged strain SSD49-pIJ8660Ep. Fluorescence microscope was used to study the colonization of this biocontrol strain in Populus tomentosa somaclone. Results showed that the GFP-tagged SSD49 didn’t affect its bacteriostatic activity against poplar canker pathogen and colonized in stems and leaves of P. tomentosa somaclone. This study successfully constructed the GFP-tagged strain SSD49,and the strain presented certain colonization in P. tomentosa somaclone.

Study of Plasma Differential Proteins in fat-1 Transgenic Cow

LIU Xin-feng^{1, 2}, DING Xiang-bin², WANG Yi-min², LI Xin², GUO Hong², LI Guang-peng¹

2016, 32(9):  203-209.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.027

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Using the plasma proteins in fat-1 transgenic cow as the research material,the potential mechanism of fat-1 regulating lipid metabolism were studied using two-dimensional electrophoresis,bioinformatics and plasma lipid biochemical detection technology. The result showed that 8 differential proteins（AHSG,KNG1,Serpin A3-1,ENSBTAG00000021729,Serpin A3-5,FAM208A,CTAGE5,and LOC511240）were identified. Bioinformatics predicted that there were 63 proteins interacting with these 8 differential proteins,and it was discovered that differential proteins and interacting proteins enriched in 11 biological pathways related to lipid metabolism,and the APOA1 of which enriched at the highest frequency in the 11 pathways. Accordingly,the significantly higher plasma level of APOA1 was detected in fat-1 transgenic cow than wild-type cow. Then,the plasma level of APOA1 was negatively related to the plasma level of low density lipoproten-cholesterol（LDL-C,r=-0.90）,whereas positively related to the ratio of high density lipoproten-cholesterol/total cholesterol（HDL-C/TC,r=0.69）. These results indicated the fat-1 gene regulated the lipid metabolism by mediating the expression of APOA1 in transgenic cow.

Analysis of the TXT Motifs’Effect on the Antifreeze Activity of Antifreeze Protein ApAFP914 from Anatolica polita borealis Using Differential Scanning Calorimetry

DU Rong-feng, LIU Zhong-yuan, MAO Xin-fang

2016, 32(9):  210-217.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.028

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This work is to study the prokaryotic expression and activities of antifreeze protein ApAFP914 and its mutants,and deduce the effects of mutations of TXT motifs on the insect’s antifreeze protein’s activities. By site directed mutagenesis in regular site number of TXT motifs of gene apafp914 in Anatolica polita borealis,then the mutant gene was cloned into pET32a vector,and expressed in Escherichia coli BL21（DE3）. The fusion protein TrxA-ApAFP914 and the other three mutant proteins were purified using Ni-NTA. The three-dimensional structure of the protein ApAFP914 was predicted and analyzed using SwisS-Model server. The thermal hysteresis activities of TrxA-ApAFP914’s and its mutants were detected by differential scanning calorimetry（DSC）. The results showed that the molecular weight of the 4 fusion proteins was about 30 kD,and the mutant protein TrxA-A19T had the highest thermal hysteresis activity,while the thermal hysteresis activities of mutant TrxA-T33&45F and TrxA-T33F were significantly lower than un-mutated TrxA-914. The results indicate that the more regular the TXT motif of insect antifreeze protein is,the stronger the thermal hysteresis activity of it is.

The Role of Myrosinase TGG1 in Drought Resistance in Arabidosis thaliana

MAO Ming-yu¹, ZHANG Wen-yu¹, XIA Hong-yu¹, WANG Yang², LI Jing¹

2016, 32(9):  218-224.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.029

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In order to illuminate the function of myrosinase TGG1 in drought resistance,an over-expressing vector with TGG1 gene driven by 35S promoter was constructed and transferred into Arabidopsis thaliana. Drought stress were tested using the wild and TGG1-overexpressed transgenic plants. Our results showed that in a simulated drought stress by the treatment of mannitol,the seed germination rate of transgenic seedlings was significantly higher than those of wild type,while relative conductivity rate of leaves in transgenic plants was significantly lower than that in wild type. In the condition of natural drought stress followed by re-watering,35S∶TGG1 showed higher survival rate and was more sensitive to ABA in the process of stomatal closure,and consequently presented the decreased water loss rate and permeability of cell membrane. In general,our study suggested that overexpressed TGG1 gene improved the drought tolerance of A. thaliana,and the mechanism is likely related to degree of stomata closing in stress.

Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase

LIN Yan-mei, LI Rui-jie, ZHANG Hui-jie, QIN Xiu-lin, FENG Jia-xun

2016, 32(9):  225-231.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.030

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The purpose of this work is to optimize the conditions of formatting and transforming the protoplast of Trichoderma atroviride HP35-3,aiming at genetically manipulating the strain for increasing the yield of cellulase. The individual condition for formatting the protoplast of Trichoderma atroviride,i.e.,mycelium age,hydrolysis time of enzyme,constituents and ratios of enzymes,as well as transformation conditions were optimized respectively. Results showed that the combination of 3 mg/mL snailase,3 mg/mL lysing enzyme,and 3 mg/mL lysozyme enzyme was effective in releasing protoplasts from T. atroviride HP35-3 mycelium（at the age of 10 h）after digestion for 2 h,the concentration was over 3.5×10⁷ protoplasts/mL,and the regeneration rate was 61%. The protoplasts were mediated and transformed by PEG,and the transformation rate was 35 transformants/μg DNA when 1×10⁸ protoplasts were employed with 5 μg DNA. In conclusion,establishing efficient formation and transformation of protoplasts may be applied in strain improvement and gene transformation of T. atroviride in biotech industries and laboratories.

Expression and Antibacterial Activity of CHAP Catalytic Domain of Staphylococcus aureus Phage Lysin Ply187

WU Meng¹, LU Hai-rong^{1, 2}, HUANG Qing-shan^{1, 2}

2016, 32(9):  232-238.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.031

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Bacteriophage lysins are efficiently antibacterial proteins,thus systematically analysing the activity of catalytic domain of it is conducive to the design of efficient heterozygous lysins. The coding sequence of the CHAP catalytic domain of Ply187（CHAP_Ply187）was synthesized,and a recombinant expression plasmid pET28a-CHAP_Ply187 was constructed,which then was transformed into Escherichia coli BL21（DE3）. The highly pure recombinant CHAP_Ply187 protein was produced by IPTG induction,further purified by a two-step method,reaching > 95% purity,and finally compared with the catalytic domain of lysostaphin（CAT_Lysn）. The results from turbidimetry showed that both CHAP_Ply187 and CAT_Lysn possessed the solid antibacterial activities to Staphylococcus aureus.CHAP_Ply187 showed a much broader antibacterial spectrum and optimal activity in a wider range of the pH,tolerated higher ionic strength,and more easily affected by EDTA. Ca²⁺,Mg²⁺,and Mn²⁺ in low concentration significantly promoted the catalytic domains of both proteins,while the Zn²⁺,Cu²⁺,and Fe²⁺ in low concentration strongly inhibited the catalytic domains.

Identification of Aeromonas hydrophila and Histopathological Observation of Artificial Infected Zebrafish

LIN Jin-xing¹, YANG Chi^{1, 2}, FENG Li-ping¹, HU Jian-hua¹

2016, 32(9):  239-245.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.032

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The aims of this paper are to isolate and identify pathogenic strain from diseased zebrafish,and to observe the histopathological changes of artificial infected zebrafish. The isolated strain ZF4 was identified by comprehensive analysis of its morphology,physiological and biochemical characteristics,conserved gene and phylogenetic tree. The results showed that the ZF4 was gram negative bacillus,positive in indole test,fermented glucose,sucrose sugars,etc. The gene AHA_0438,ASP,gyrB and 16S rRNA of ZF4 were all positive. Based on gyrB and 16S rRNA gene sequence,clustering analysis result showed that ZF4 was Aeromonas hydrophila. The infected zebrafish presented obvious clinical symptoms,and histopathological changes were observed,such as the liver tissue degeneration,intestinal lining shedding,hyperemia of the spleen,pancreas fibrous tissue hyperplasia.,etc. In conclusion,the ZF4 isolated from the diseased zebrafish was identified as pathogenicity A. hydrophila. Meanwhile,due to its susceptibility,zebrafish could be used as the animal model to study the A. hydrophila.

Expression and Activity Analysis of DSPAα1 Deletion Mutants in Pichia pastoris

CHEN Yun¹, NIU Chun-qing¹, SONG Xiao-shuang¹, SU Chang¹, HUA Zi-chun², LIU Yan¹

2016, 32(9):  246-252.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.033

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There are 4 types of desmodus salivary plasminogen activators（DSPAs）,i.e.,DSPAα1,DSPAα2,DSPAβ and DSPAγ. DSPAα1 and DSPAα2 contain a finger domain（F）,an epidermal growth factor domain（E）,a kringle domain（K）and a serine proteinase domain（P）;DSPAβ contains E,K and P domains;and DSPAγ contains K and P. The effect of DSPAα1’s structure on fibrinolytic activity was also investigated. A deletion mutant of DSPAα1（mDSPAα1）lacking the E domain was synthesized by splicing overlap extension PCR（SOE-PCR）method,and the recombinant plasmids of mDSPAα1/pPIC9K,DSPAβ/pPIC9K and DSPAγ/pPIC9K were constructed and transformed into Pichia pastoris GS115. Fibrinolytic activity was determined by fibrin plate assay. Results showed that the expression of DSPAγ was not detected,the activities of DSPAα1,DSPAβ,and mDSPAα1 were 2.64 × 10⁵ U/mg,1.32 × 10⁴ U/mg and 151.52 U/mg respectively. The total activity hardly changed when using DSPAα1,mDSPAα1 and DSPAβ together,but there led a 2-3folds of reduction by using either two of them. The mDSPAα1 exhibited almost no fibrinolytic activity,whilst DSPAβ retained comparable catalytic activity to the wild-type DSPAα1. Deletion mutant studies illustrated that N-terminal region of DSPAα1 greatly affected the PA activity,and it without E domain nearly lost its activity,indicating that the E domain of DSPAα1 plays a key role during fibrinolytic process.

Establishment and Application of a High-throughput Drug Screening Assay Targeting Macrophage Migration Inhibitory Factor

ZHANG Jing^{1, 2}, SUN Rui-qiu², TANG Yan-ting², LIU Xiang²

2016, 32(9):  253-259.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.034

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The aim is to establish a high-throughput drug screening assay targeting macrophage migration inhibitory factor（MIF）by UV-spectrophotometry. The target gene was molecularly cloned,prokaryotic expression system of Escherichia coli was used to purify,and then the highly-purified target protein was acquired. UV-spectrophotometry was utilized to establish the enzymatic system,in which then the conditions were optimized,and the proper high-throughput drug screening assay was set up and screened new MIF inhibitors from 384 small molecules. As results,the screening assay was established successfully,and 2 small molecules with high inhibitory rate were identified,while median inhibitory concentration（IC₅₀）was 59.07 μmol/L and 44.12 μmol/L, respectively. In conclusion,targeting MIF protein,the ideal high-throughput drug screening assay was established,which is appropriate for screening MIF inhibitors and conducive to the future drug development.

Construction and Characterization of the Pathways of Synthesizing Lactate in Vitro Related to NADP（H）

MAO Jia-ling, XU Lin, YAN Ming

2016, 32(9):  260-266.  doi:10.13560/j.cnki.biotech.bull.1985.2016.09.035

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The regulating circles of the metabolic pathways in cells in vivo are complicated,and the intuitive researches may be carried out if the target metabolic pathways could be moved to the cells in vitro. Selecting 10 thermophilic enzymes,synthesizing pathways in vitro were constructed,which achieved the cycle balance of energy ATP and redox cofactors NAHP（H）,as well as the produced lactate from glucose metabolism. The preliminary study revealed this system’s optimal reaction condition was pH 7 and 50℃. Under this condition,adding small amount of NADP⁺,12.4 g/L glucose was converted to 9.5 g/L lactate within 8 hours.