Table of Content

25 December 2017, Volume 33 Issue 12

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Role of MicroRNA in Plant Resistance to Salt Stress

LIU Xiao-wei, YANG Xiu-yan, LIU Zheng-xiang, WU Hai-wen, ZHANG Hua-xin, ZHU Jian-feng

2017, 33(12):  12-21.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0538

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Salt stress is a major limiting factor in the process of plant growth and development，which can affect the process of plant organ development，morphogenesis，signal transduction，and so on. MicroRNA（miRNA）is a class of non-coding single stranded RNA about 19-25 nt long，and more and more studies have found that plant miRNAs play an important roles in salt resistance by participating in the regulation of plant seed germination，organ development，morphogenesis and active oxygen scavenging. In this paper，plant miRNAs，which respond to salt stress，are reviewed in order to provide references for the study of salt tolerance mechanism and molecular breeding of plant salt tolerance.

Research Progress on Phenolic Compounds of Plant Secondary Metabolites

HUA Xiao-yu, TAO Shuang, SUN Sheng-nan, GUO Na, YAN Xiu-feng, LIN Ji-xiang

2017, 33(12):  22-29.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0546

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With the rapid development of biological theories and technologies in recent years，plant secondary metabolites are growingly concerned. Among them，the phenolic compounds are the most widely distributed and have critical physiological functions. They not only have natural antioxidant activity，but also contain a variety of medicinal values，and play an important regulating role in plant development and signal transduction under stress conditions，thus have become a hot spot of research. Based on this，we summarized the classification，synthesis pathways，physiological functions and medicinal values of the phenolic compounds from domestic and foreign researches，and put forward the future research direction and the problems that should be paid attentions to，aimed at providing some references for the in-depth study of secondary metabolites，especially phenolic compounds.

Roles of Key Genes and Relevant Plant Hormones in the Early and Late Stages of Plant Embryogenesis

ZHAO Fang-dong, LI Lin-kun, HE Xu-sheng, ZENG Hui-ming

2017, 33(12):  30-36.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0502

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Both LEAFY COTYLEDON 1-LIKE and LEAFY COTYLEDON1 are AHAP3 subunit of the LEAFY COTYLEDON1 type，but LEAFY COTYLEDON 1-LIKE is capable of converting the cells of vegetable stage into the cells of embryo periods in the early stages of somatic embryogenesis，differing from the LEAFY COTYLEDON1 that plays an important role in the early stages of embryonic development. LEAFY COTYLEDON2，which has the same specific B3 domain as the LEAFY COTYLEDON gene，can participate in creating the environment of embryogenesis in somatic tissue by IPA-YUC auxin synthesis pathway. Besides，LEAFY COTYLEDON2 promotes the occurrence and development of embryos by regulating the ratio of ABA/GA. The expression of WUSCHE and SHOOTMERISTEMLESS is independent but coordinated to maintain the function of shoot apical meristem（SAM）. The KNOX pathway that is independent from the WUSCHE expression but interacts with plant hormone can maintain the environment of high-concentration CK and low-concentration GA for embryonic SAM，which is beneficial to the pathway of WUSCHE -CLAVATA responding to CK and continuously produce stem cells. In summary，it is feasible to improve the understanding of the molecular regulation network of embryogenesis，and to lay the molecular basis for further studying embryogenesis.

Research Progress on Mechanism of ARF and Aux/IAA Regulating Fruit Development and Ripening

HU Xiao, HOU Xu, YUAN Xue, GUAN Dan, LIU Yue-ping

2017, 33(12):  37-44.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0506

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The auxin is one of key essential plant hormones regulating fruit development and ripening. Aux/IAAs and auxin response factors（ARFs）play central roles in auxin-mediated signal transduction mechanism. ARF proteins combine with the auxin-responsive elements in the promoters of auxin-responding genes to promote or inhibit gene expression. Aux/IAA proteins，through specific binding of their common domains III and IV with ARF，regulate the transcriptional activity of early response genes of auxin. The researches indicate that ARF participates in the regulation of fruit morphology development，hardness，sugar accumulation，etc.；Aux/IAA plays a significant role in pollination and fruit morphology etc. In addition，the interactions of Aux/IAA and ARF or themselves regulate downstream gene expressions，which is the main mechanism of plant response to auxin regulation. In this review，we summarized the structures of ARF and Aux/IAA，the distribution characteristics in varied plants，and the regulating function in fruit ripening development and signal transduction. The research status on the interaction of ARFs and Aux/IAA was also discussed, which will provide knowledge for reveal the mechanism of auxin control of fruit development and ripening.

Research Progress on BYL Cell-free Systems in Plant RNA Virus Translation and Replication Mechanisms

AN Meng-nan, WU Yuan-hua

2017, 33(12):  45-50.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0514

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Cell-free translation system is an in vitro molecular study system for efficient expression of recombinant proteins. The BYL is tobacco BY-2 lysate cell-free system from which vacuoles are removed by density gradient centrifugation. The BYL system supports recombinant protein expression and many plant viral protein expressions. Because the intracellular membrane fraction required for viral RNA replication is retained，the BYL system also supports negative-strand，positive-strand and subgenomic RNA synthesis of plant positive-strand RNA viruses. This paper reviewed the applications of BYL system in the molecular insight of translation and replication mechanism of plant RNA viruses as well as RNA interference mechanism，and prospected their future research trend. This review systematically illustrated the BYL system，which is expected to be applied in the study of of other plant RNA viruses and provides intriguing insights.

Research Advances on Metabolism of Higher Fatty Acids in Nicotiana tabacum and Its Affecting Factors

YANG Li-yun, YANG Shuang-long, LI Jun-ying, PANG Tao, HE Bin, GONG Ming

2017, 33(12):  51-60.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0391

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As one kind of important compounds in tobacco plants，fatty acids play remarkable roles in their growth and development，as well as tobacco quality and flavor. Recently，it also has been verified that fatty acids are involved in the formation of stress tolerance in tobacco plants. Tobacco seeds are rich in lipids and various kinds of fatty acids，and the contents of linoleic acid，oleic acid and palmitic acid increase gradually with the developing of tobacco seeds；and they can be considered to be utilized as a potential biomass source. During the process of growth and development of tobacco leaves，fatty acid contents increase gradually，reach the maximum when tobacco plants are blooming，then decrease during maturing and senescing of tobacco leaves；at the same time，the saturated fatty acids are transformed into the unsaturated ones. Moreover，the contents of fatty acids are higher in lower and middle leaves than those in upper ones of tobacco plants，and also higher in palisade tissue than in spongy tissue in tobacco leaves. In addition，genotype and environmental factors such as light irradiation，temperature，geographical factors，fertilization，flue-curing methods，mechanical injury and so on apply significant effects on composition and content of fatty acids，as well as key enzyme activities and their gene expressions in the biosynthesis and metabolism of fatty acids in tobacco leaves. Chill-hardening may enhance chilling resistance of tobacco plants by increasing the contents of the polyunsaturated fatty acids and reducing the contents of saturated fatty acids. It is also an effective method to increase the contents of unsaturated fatty acids in tobacco plants with over-expressing the genes of the acyl-ACP desaturase，ACP，fatty acids desaturase and fatty acids dehydrogenase by genetic engineering，which in turn improves the stress tolerance of tobacco plants to abiotic stresses，such as drought，high temperature and highlight stress and so on. Based on the above-mentioned works，the paper presents the future research direction，aiming at providing the theoretical and practical guidance for the study of this field.

Research Advances on Antimicrobial Peptides of Fish and Amphibians

XUE Lin-gui, MA Ping, SHANG Hai, HE Xiao-yan, CHEN Xi-ming, LIU Guang-xiu, CHEN Tuo, ZHANG Wei

2017, 33(12):  61-66.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0599

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At present，750 antimicrobial peptides have been successfully isolated from the biological community，of which with antitumor activity account for about 13.1%. The antibacterial peptides derived from amphibians and fish are about 13.3% and 9.3%，respectively. Fish and amphibians are the most primitive vertebrate，and most of them rely on the gills，skin and intestinal breathing. Due to their extremely fragile acquired immune system，they have a powerful innate immune system to avoid the infection of the pathogen，thus the antimicrobial peptides distributed in different parts have become the important component of first defense line of broad-spectrum in these lower organisms. This paper reviewed the broad-spectrum antibacterial activity and immune regulation ability of antibacterial peptides such as moronecidin，epinecidin-1，paradaxin，and misgurin distributed in hybrid striped bass，Epinephelus coioides，Pardachirus marmoratus，and Misgurnus anguillicaudatusis，as well as antibacterial spectrum of smelly frog antimicrobial peptide. For instance，the moronecidin can kill all gram positive bacteria and some gram negative bacteria. Epinecidin-1 can effectively prevent the infection of vulnificus（Vibrio vulnificus）in grouper and kill the nervous necrosis virus. Paradaxin reveals the antitumor activities. Misgurin has strong bactericidal ability. Antibacterial peptides from the smelly frog can kill Candida albicans. Then，the paper summarized the antibacterial peptides successfully expressed in Pichia pastoris and those derived from fish and amphibian of having antiviral and antitumor activities. Finally，the paper discussed the problems and solutions in the application of these antibacterial peptides，providing a theoretical basis for green and effective antibacterial peptide drugs in various fields to play a great role.

High-throughput Genotyping Techniques and Their Applications in Rice

ZHANG Xian-wen, HE Zhi-zhou, JIANG Nan, DENG Hua-feng, Li Ji-ming

2017, 33(12):  67-73.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0467

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Deep understanding and efficient utilization of genetic basis of crop germplasm resources is of great importance for crop cultivar improvement and food safety. The traditional methods based on pedigree played a vital role in breeding practice. With the rapid development of molecular biology，genotyping methods based on genomics and high-throughput SNP markers have been developed and may efficiently identify the germplasm resources. This article reviews the main genotyping methods，focusing on the methods based on single nucleotide polymorphism（SNP）marker such as the next-generation sequencing（NGS），competitive allele-specific PCR（KASP），and SNP chip as well as the popular SNP analysis tools and databases. Furthermore，this article introduces the progresses of high-throughput SNP genotyping techniques in studying rice，and prospects the application and developmental trends of genotyping techniques in the fields such as crop breeding and so on.

Genetic Engineering for Production of Phloroglucinol

CUI Xin, YAN Zhen-xin, SONG Wei-guo, LIN Wen-han, Peter PROKSCH

2017, 33(12):  74-80.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0542

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This work is to further improve the efficiency of phloroglucinol biosynthesis，and to investigate the molecular mechanism of phloroglucinol biosynthesis and more efficient fermentation conditions and media components. We cloned the phlD gene from Pseudomonas fluorescens and reconstructed the intracellular synthesis pathway of phloroglucinol in the BL21（DE3）. At the same time the multi-drug resistant regulator（marA）and acetyl-coA carboxylase（ACCase）were also super-expressed in the BL21（DE3），and the effects of different carbon sources and medium ion environment on the phloroglucinol accumulation were analyzed. The results showed that intracellular synthesis pathway of phloroglucinol was successfully established，and the yield reached 450 mg/L. The marA gene increased the resistance of engineering strain to phloroglucinol and thus phloroglucinol yield increased to 1060 mg/L. The ACCase improved the content of the intracellular malonyl-CoA，and increased the phloroglucinol yield up to 2120 mg/L. Compared to glycerin and sodium pyruvate，glucose was the most suitable for the phloroglucinol biosynthesis，and the phloroglucinol yield with glucose was 1.8 times and 2.4 times of that with glycerol and pyruvate，respectively. Calcium and magnesium ions in medium had no impact on the phloroglucinol synthesis. Conclusively，the superexpression of the marA and ACCase genes can greatly increase the efficiency of phloroglucinol biosynthesis，and the medium using glucose as carbon source and without no calcium and magnesium is the optimal for phloroglucinol biosynthesis.

A Nano Biosensor for Rapid Detecting Staphylococcus aureus Containing tst Gene

LI Ming-yang, HE Qi-zhi, MA Chun-xia, YU Jian-kun

2017, 33(12):  81-86.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0291

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This work is to construct a nano biosensor of simple operation，low cost，high sensitivity and specificity to rapid detect Staphylococcus aureus containing tst gene with single-walled carbon nanotubes（SWCNTs）based on the principle of fluorescence energy resonance transfer（FRET）. SWCNTs combined with carboxyfluorescein（FAM）-labeled DNA molecule probes（FAM-P）were used to construct FAM-P/SWCNTs composite detection system，and the probe was complementary molecular sequence of tst gene. The sensitivity and specificity of the system to the target sequence and the target strain were investigated. As results，the lower detection limit of target sequence was 10 nmol/L and presented a fine linear relationship（R2 was 0.994）in the range of 10-100 nmol/L. The detection limit of the target strain was as low as 102 and in the range of 102-107 CFU/mL（R2 was 0.999）. In addition，the sensor exhibited a high specificity in mismatch experiments and for non-target bacteria detection. In conclusion，the composite detection system by FAM-P/SWCNTs has the advantages of rapid detection，simple operation，high sensitivity and strong specificity，which opens up a new approach for food detection and clinical diagnosis.

Preparation of a Novel Aβ42 Oligomer Mimotope Vaccine

LIU Xiang-meng, YU Ya-dong, WANG Shao-wei, YU Xiao-lin, WANG Rui-ming

2017, 33(12):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0488

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The objective of this study is to obtain a mimotope vaccine specifically targeting to pathogenic Aβ42 oligomer. In previous studies，we purified an antibody IVIG-AOB，which specifically recognizes Aβ42 oligomers，from intravenous immunoglobulin（IVIG）by affinity chromatography. Using phage display technique，a series of ring-shape mimotope polypeptides were screened for specific binding to IVIG-AOB. These oligomer epitope peptide genes were then cloned to HBc-VLP vector and the recombinant proteins were expressed in Escherichia coli. After that，the recombinant Aβ42 oligomer conformational epitope HBc-VLP vaccine were applied to Balb/c mice. The antibody responses induced by the Aβ42 oligomer-targeted VLP vaccine were determined. Five mimotope polypeptides specifically-bound with IVIG-AOB were successfully screened from phage cyclic peptide library and cloned into HBc-VLP vector. The recombinant proteins were successfully expressed in E. coli. HBc-C4 elicited the highest antibody titer after vaccination to Balb/c mice. Conclusively，Aβ oligomer is the major factor in the pathological processes of Alzheimer’s disease（AD）. The mimotope HPc-VLP vaccine based on the unique structure of Aβ42 oligomer induced body to generate the antibody targeting and neutralizing the toxic Aβ42 oligomers，while not affecting its normal physiological function，thus it plays a fundamental role in the treatment of AD.

Preliminary Study on Physiological Function of Gene Bph14 in Rice

LI San-he, ZHA Wen-jun, CHEN Zhi-jun, ZHOU Lei, LIU Kai, YOU Ai-qing

2017, 33(12):  93-98.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0754

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Gene Bph14 is the first cloned rice insect-resistant gene，however，there were few reports about the effects of Bph14 gene on rice physiology. The study of the physiological effects of the gene on rice would contributes to a comprehensive understanding of the resistance mechanism of gene Bph14 to brown planthopper. The gene Bph14 was transferred into rice cultivar m5274，and genes related to ethylene biosynthesis and abiotic stress response were detected by Q-PCR in transgenic lines carrying gene Bph14. In addition，transgenic lines and the accepter line were treated with 5 μmol/L ABA and 250 mmol/L NaCl respectively. In these lines，OsACO2，OsACS2，OsACS6，O1gP5CS，and O5gP5CS expressed differently from those in m5274. Genes such as CatA，CatB，CatC，RAB16A，LEA3，LIP9，SalT，and AdhI related to abiotic stress of drought and salt-resistance also differently expressed from those in m5274. At the same time，the introduction of Bph14 gene improved the sensitivity of rice to ABA and the resistance to salt stress. These results indicate that gene Bph14 in rice probably participates in the regulation of ethylene biosynthesis and expression of functional genes in abiotic stress response.

Screening Interacted Factors of ZmSCL1 in Yeast Two-hybrid cDNA Libraries of Developing Maize Seeds

ZHAO Qian-qian, ZHOU Xiao-jin, CHEN Ru-mei

2017, 33(12):  99-107.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0408

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ZmSCL1 is a member of GRAS gene family. Although highly expressed in maize seeds in the key period for the filling period of maize，especially in the endosperm，the molecular function of ZmSCL1 for maize seeds development remains unclear. Therefore，to screen interaction factors for ZmSCL1，yeast two-hybrid cDNA libraries of maize inbred line（B73）developing seeds（12 and 20 days after pollination）were constructed in yeast strain AH109 using homologous recombination-mediated SMART technology. The qualities of the cDNA libraries were evaluated as the followings：the titers were 1.10×105 CFU/mL，the capacity of cDNA library was 1.32×106，the recombinant rate was 81.90%，and the protein length encoded by the inserted cDNA ranged from 133 aa to 500 aa. Finally，109 clones were obtained for sequencing. GO enrichment analysis showed that，ZmSCL1 may be involved in the regulation of starch and nutrition accumulation.

Tempo-spatial Distribution of Cry1Ab/c Protein in the Main Stem Leaves of Transgenic Bt Cotton

XIAO Hai-bing, WANG Peng-jun, LI Xian-feng, DONG Hong-qiang, YANG Ming-lu

2017, 33(12):  108-111.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0462

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The objective of this study is to reveal the tempo-spatial distribution of Cry1Ab/c protein in main stem leaves of transgenic cotton variety M-49 and X-37. The Cry1Ab/c protein content and the water-soluble protein in the main stem leaves from transgenic Bt cotton were detected by enzyme linked immunoassay（ELISA）and Coomassie brilliant blue method，respectively. The results showed that the content of Cry1Ab/c protein in the main stem leaves of the cotton variety X-37 were greatly higher than that in M-49. The contents of Cry1Ab/c protein in both varieties increased gradually from the top leaves to the bottom leaves. The Cry1Ab/c protein content of the bottom leaves were greatly higher than that of the top leaves at squaring period（3 June 2016）and initial flowering stage（27 June 2016）（P < 0.05），but there were no significant differences on the content of Cry1Ab/c protein between the upper and middle parts of the main stems at blossoming and boll forming stage（6 August 2016）and boll period（26 August 26 2016）. Besides，the proportion of the Cry1Ab/c protein content to the water-soluble protein content in the upper leaves were greatly higher than that in the middle and lower leaves in main stem of transgenic Bt cotton（27 June 2016）. In summary，the content of Cry1Ab/c protein in the main stem leaves of both varieties decreased gradually from the bottom leaves to the top leaves，and the trend of the content of Cry1Ab/c protein lowered after increased with its growth period going on，and the ratio of the Cry1Ab/c protein to the water-soluble protein content from the upper leaves changed more significantly at initial flowering stage than other developmental stages.

Allelopathy Effects of Aquatic Extracts from Mirabilis himalaica Roots on Morphology and Cell Structure of Wheat and Mung Bean Root

LIU Sheng-li, MA Shi-yao, WU Xiao-fei, SHI Jun-na, ZHOU Xiao-yang, LAN Xiao-zhong, LU Cun-fu

2017, 33(12):  112-118.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0389

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To figure out the allelopathy effects of aquatic extracts from Mirabilis himalaica roots，we used wheat and mung bean as target plants for this study. Results showed that the cell and tissue morphologies of the two target plants were in significantly allelopathy and inhibitory effects after the treatment by the aquatic extracts from M. himalaica roots for 8 d. We observed and analyzed the root morphology and cell structure of the two crops treated with the aquatic extracts using transmission electron microscopy and scanning electron microscopy. The root tip structure in wheat and mung bean were incomplete，part of the epidermis cell felled off，the space between the cells turned larger，interstitial faulting and cavity appeared，and tightly structure between cells were disrupted. In the cell of wheat and mung bean root，membrane shrank，plasmolysis appeared，vacuoles in the cytoplasm turned bigger，nucleus ablated，and the structure of main organelles such as endoplasmic reticulum，mitochondria and Golgi apparatus varied or collapsed and depleted in numbers；i.e.，plant life severely restrained. These results imply that the aquatic extracts from M. himalaica roots presented the strong allelopathic inhibition on two common crops.

Development of Microbial Inoculum of Streptomyces silaceus and Its Effect on Wheat Seedling Growth

HE Wen, LIU Jin-long, KOU Juan-ni, TANG Zheng-shuai, LIU Li-ying, SUN Zhong-tao

2017, 33(12):  119-124.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0402

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In order to improve the stability of microbial agents with Streptomyces silaceus SN16，the amounts of carriers，protectants and dispersants were optimized and the optimal formula of them was determined，and the influence of which on wheat seedling growth was studied by pot experiment. Results showed that the optimal formula of the microbial agent was Streptomyces SN16 20%，sodium alginate solution（1 mg/mL）24%，sodium carboxymethyl cellulose（CMC）solution（10 mg/mL）24%，and diatomaceous earth 32%. By this formula，the biomass of microbial inoculum was 2.45×108 CFU/g while conserved at 4℃ and 0.94×108 CFU/g at room temperature after 60 d. Pot experiment demonstrated that the plant height，fresh weight and dry weight of wheat seedlings applied with microbial agent of 1% Streptomyces silaceus SN16 increased by 9.85%，57.90% and 66.67% compared to the CK，respectively，which reached significant level（P<0.05）. However，its promoting effect on root was insignificant（P>0.05）. This research provides a new type of Streptomyces silaceus agent for agricultural production.

Screening the Strains Degrading Glycyrrhiz uralensis Residues and Its Enzyme Production

ZENG Fei, ZHANG Sen, QIAN Da-wei, ZHU Zheng-hua, ZHOU Jia-lin, DUAN Jin-ao

2017, 33(12):  125-131.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0419

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This work aims to screen the strains that can degrade Glycyrrhiza uralensis residues（GUR）and to optimize the production process of cellulase by the strains. First，the strains were screened from decayed Glycyrrhiza uralensis and soil，and then were identified through morphological observation and 18S rDNA. Second，the effects of different factors on the fermentation of the isolated strains were studied，and the orthogonal test L9（34）was used to determine the optimal condition of producing enzyme. The produced cellulase complex was used to investigate how the enzymatic hydrolysis of GUR was proceeding. According to the results，the isolated strain was identified as Penicillium oxalicum，designated as G2. The effects of concentrations of herb residues and fermentation time on cellulase production of G2 were significant（P<0.05 and P<0.05）. Under the optimal conditions，the FPase activity reached 3.43 U/mL and EG activity reached 16.89 U/mL. Compared with commercial cellulase，the cellulase by G2 was more efficient in degrading GUR. In addition，the extraction rate of total flavonoids from Glycyrrhiza uralensis Fisch was significantly higher after enzymatic hydrolysis. Here screening the GUR-degrading bacteria and studying the process of producing the enzyme from the GUR laid the foundation for the rational utilization of GUR and the production of cellulase with low cost and fine performance.

Screening of High Selenium-enriched Yeast and Enhancement of Its Selenium-enriched Capacity

LOU Xing-dan, ZHANG Gen-lin, YE Bang-ce

2017, 33(12):  132-137.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0604

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Selenium yeast is important for health and pharmaceutical industry，and high selenium-enriched yeast is the basis for the development of selenium-enriched yeast products. In this study，Rhodotorula glutinis X-20 with relatively high amount of enriched selenium was screened from selenium-enriched soil of Xinjiang via preliminary screening with Martin medium and re-screening of selenium-enriching capacity. The optimal culture conditions were determined through single experimental factors. The selenium content enriched by the yeast was significantly improved by using 2% glycerol as carbon source. Supplying sodium selenite at 12 h of cultivation ensured the higher selenium content and biomass of the yeast. Addition of 0.4 mM phosphate efficiently increased the transport and accumulation of selenium，and ultimately the enriched selenium content by strain X-20 reached 5009 μg/g.

Isolation and Identification of a Melanin-producing Marine Fungi and Characterization of the Pigment

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

2017, 33(12):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0437

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Our aims are to isolate melanin-producing fungi from Haizhou Bay，to clarify the taxonomic status，and to study the properties of the melanin. The pigments-producing fungi were isolated from the collected samples of sea water，sea anemone，and sea mud from Haizhou Bay with the method of enrichment culture. The melanin-producing fungi were re-screened by shake flask fermentation. Then the melanin-producing fungi were identified and the properties of the crudly extracted melanin were measured. The strain CXPF01 was obtained and identified as Cladosporium cladosporioides by its morphological and ITS sequence homology. The pigment had maximum absorption value at 220 nm. There was non-significant effect of light on the pigment while high temperature and alkalinity or acidity had a remarkable impact on it. The pigment presented inhibitory effect on Staphylococcus aureus and Escherichia coli. Conclusively，the melanin-producing fungus CXPF01 was obtained，from which the melanin was stable in light and it had antibacterial activity.

Prokaryotic Expression，Purification and Bioinformatics Analysis of Pediocin pedA Gene

HUANG Yu-liang, WANG Li-ping, LU Ke-wen, SHAO Hui-juan, WANG Zheng-quan

2017, 33(12):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0373

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The aim of this study is to construct a prokaryotic expression vector for pedA gene of pediocin PA-1 to realize the exogenous expression of PA-1，and to analyze the physicochemical properties and molecular structure of recombinant protein by bioinformatics. The pedA gene was amplified by DNA of Pediococcus pentosaceus C-2-1 as template，and the PCR product was cloned into prokaryotic expression vector pET28a（+）. The recombinant plasmid pET28a-pedA was thus constructed and transformed into Escherichia coli BL21（DE3）competent cells. Next，the transformant was induced to express protein by IPTG（1 mmol/L）at 30℃ for 5 h. Then，the recombinant protein was purified by Ni-NTA resin affinity chromatography，and the antibacterial activity of the recombinant protein was detected. Finally，the physicochemical properties and molecular structure of recombinant protein was explored by bioinformatics method. The results showed that the recombinant plasmid pET28a-pedA was successfully constructed，and recombinant pediocin of PA-1 with His-tag was expressed in E. coli. The molecular size of the purified recombinant protein by Ni-NTA affinity chromatography was determined to be about 7.8 kD via Tricine-SDS-PAGE，which was consistent with the theoretical value. Agar diffusion method demonstrated that antibacterial colony was remarkable，indicating that the purified recombinant pediocin had antibacterial activity. Comparative analysis of the protein structure and characteristics by bioinformatics method showed that the recombinant protein contained the signal peptide and consisted of 23.53% alpha helix，meaning that its hydrophobicity increased，thus which might enhance the soluble expression of the recombinant protein. Conclusively，in this experiment，the prokaryotic expression vector of pediocin PA-1 was successfully constructed，and the recombinant protein was at soluble expression in E. coli. The soluble expression may be related to signal peptide and the hydrophobicity of the signal peptide，and the antibacterial activity of the purified recombinant protein was significant.

Effects of Metal Ions on Activities of Mixed Moderately Thermophilic Bacteria

ZHAO Xue-song, WANG Dong-xu, LIU Xin, SHI Qian-qian, SHI Shuai

2017, 33(12):  151-155.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0335

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The effects of metal ions Mo6+，Cu2+，and Zn2+ on the activities of mixed moderately thermophilic bacteria such as Leptospirillum ferriphilum and Acidithiobacillus caldus，were tested by the metal tolerance experiment. As results，the activities of mix moderately thermophilic bacteria decreased with increasing concentration of metal ions. The tolerated concentration of bacteria tested for metal ions Mo6+，Cu2+ and Zn2+ were 0.6 g/L，10 g/L and 30 g/L respectively. The toxicity order of metal ions was in Mo6+ >Cu2+>Zn2+. A. caldus presented a higher degree of resistance to metal ions than L. ferriphilum. Results in this study demonstrated that L. ferriphilum and A. caldus in co-culture had strong resistance to metal ions，especially to Mo6+，thus it can be applied to the bioleaching for molybdenum sulphide ore，molybdenum waste and molybdenum sludge.

Role of Long Non-coding RNA AK043773 in Adipocyte Differentiation

ZHANG Juan-juan, QIAO Ling, ZHU En-dong

2017, 33(12):  156-161.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0556

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This work is to investigate the expression changes and regulating role of long non-coding RNA（lncRNA）AK043773 in adipocyte differentiation. First，pre-adipocyte cells in mice were induced for adipogenesis. Then，the expressions of adipogenic differentiation regulating gene（KLF7）and AK043773 were measured by real-time quantitative PCR（RT-qPCR）. Next，the overexpression vector for AK043773 was constructed for detecting its effects on adipogenic differentiation. According to search in UCSC database，the locus of AK043773 was in the 3'-end downstream of KLF7’s exon 4. The expressions of KLF7 and AK043773 were down-regulated coordinately in adipogenesis-induced and differentiated bone marrow stromal cell line ST2 and pre-adipocyte cells 3T3-L1. Furthermore，the overexpression vector pCDNA3.1-AK043773 was constructed and transfected into ST2 cells，which significantly enhanced the expression of AK043773，but inhibited the adipogenesis in ST2 cells by oil red O staining and OD520nm absorbance test after the induction of adipocyte differentiation. All of results indicate that AK043773 and KLF7 coordinately down-regulate in adipocyte differentiation，and increasing the expression of AK043773 remarkably suppresses the differentiation of adipocyte.

Anti-diabetic Activity of Alcohol Extracts from Lessonia nigrescens and Its Effects on Intestinal Microflora in Mice

CHEN Yu-qing, YAN Xin, CHEN Ming-jun, LIN Luan, YANG Cheng-feng , LI Qiu-zhe , LIU Bin , ZHAO Chao

2017, 33(12):  162-169.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0464

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The aim of this article is to screen the seaweed active material and study the effect on intestinal flora in mice with type 2 diabetes for providing functional nutrition food with hypoglycemic effect. First，the 35 different extracts from 13 kinds of seaweeds prepared by step-by-step extraction method were screened by evaluating in vitro α-glucosidase activity and using HepG2 insulin resistance cell model. Then，the impacts of extracts with solid hypoglycemic effects on intestinal flora in type 2 diabetic mice were evaluated through 16s rRNA high-throughput sequencing. Among the 35 seaweed extracts，55%-ethanol extracts from Lessonia nigrescens and Ascophyllum nodosum showed the most significant inhibitory effects on α-glucosidase activity；moreover，the 55%-ethanol extracts from L. nigrescens significantly increased the glucose consumption of insulin-resistant HepG2 cells，also remarkably increased the abundance of Bacteroidetes in the intestine of mice and decreased the number of Firmicutes bacteria. The 55%-ethanol extract of Lessonia nigrescens showed the most significant hypoglycemic activity and the ability to maintain the balance of intestinal microflora，which may have potential medicinal value.

The Promoting Role of Gene Wnt10A in Proliferation and Migration of Gastric Cancer Cells and the Underlying Mechanism

GUO Hui-can

2017, 33(12):  170-175.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0434

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This work aims to elucidate the function of gene Wnt10A during the proliferation and migration of gastric cancer cells and the underlying molecular mechanism，for providing new target molecule in the diagnosis and treatment of gastric cancer. Real-time quantitative PCR and Western bolt analysis were used to detect gene expression；RNAi technology was applied to knockdown and decrease the expression of Wnt10A，and MTT assay，wound healing assay and transwell assay were employed to detect the biological behavior of gastric cancer cells. Results showed that gene Wnt10A expressed more highly in tumor tissues than that in adjacent normal tissues，approximately 3 times. The level of Wnt10A in gastric cancer cell lines was also higher than the normal cells GES. The proliferation rate of AGS cells reduced by 40%，the migration rate decreased by 40%，and the invasive ability was also downregulated about 70%，when the expression level of gene Wnt10A in AGS cells decreased about 60%. Furthermore，Western blot analysis demonstrated that the expressions of β-catenin，Cyclin D，TCF and gene Myc reduced after Wnt10A knockdown in AGS cells，while the expression levels of DKK1 and GSK3β increased，which was in consistent with the results by the treatment of LGK-974，a specific inhibitor against Wnt/β-catenin signaling. Conclusively，gene Wnt10A promotes the proliferation and migration of gastric cancer cells through simulating activation of Wnt/β-catenin/Myc signaling pathway，i.e.，functions as cancer-promoting gene.

Overexpression of Glypican-3 and Its Effects on the Biological Behaviors in Highly Metastatic Hepatocarcinoma Cells HCCLM3

ZHANG Jing-jing, JIN Xiao-bao, LI Xiao-bo, WANG Jie, MA Yan

2017, 33(12):  176-184.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0616

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This work aims to construct the recombinant eukaryotic expression vector for sGPC3/GPC3（soluble glypican-3/glypican-3）gene，and to study the effects of overexpressing sGPC3/GPC3 on the biological behaviors of human highly metastatic hepatocarcinoma cells HCCLM3. The gene fragment encoding sGPC3/GPC3 was obtained by PCR and cloned into eukaryotic expression vector pcDNA3.0-GFP，then with which HCCLM3 was transfected by liposome-mediated method. The expression of GPC3 was detected by RT-PCR and Western blot. The effects of sGPC3/GPC3 on the biological behavior of high metastatic hepatocarcinoma cells were detected by CCK-8，clonal plate formation experiment，cell scratch test and flow cytometry. Results showed that the recombinant eukaryotic expression vector of sGPC3/GPC3 gene was successfully constructed by restriction enzyme digestion and sequencing. The results by RT-PCR and Western blot demonstrated that sGPC3/GPC3 was successfully overexpressed in HCCLM3. Compared with normal group and transfected control vector，the proliferation ability and clonogenic ability of HCCLM3 were significantly inhibited by the overexpression of sGPC3/GPC3（P< 0.05），the mobility significantly decreased and the cell cycle was arrested in G1 phase，thus，the rate of apoptosis increased. Moreover，the effect by overexpressing sGPC3 was significantly better than by GPC3（P < 0.05）. Conclusively，the up-regulation of sGPC3/GPC3 inhibits the growth and migration of highly metastatic hepatocarcinoma cells HCCLM3，induces the cell apoptosis and affects the redistribution of cell cycle，which plays an important role in the development of highly metastatic liver cancer.

Construction and Expression of Mutant A-Raf-S214C and Studies on Its Role in MAPK Signaling Pathway

LI An-yi, YANG Yang, LIU Ying, GUO Xiao-xi, HAO Qian, XU Tian-rui, AN Shu

2017, 33(12):  185-190.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0445

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This study is to observe the influence of A-Raf mutant A-Raf-S214C on MAPK（mitogen-activated protein kinase）signal pathway and to explore the molecular mechanism of lung cancer and Langerhans cell histiocytosis caused by A-Raf-S214C. The pcDNA5-FRT-TO-A-Raf-S214C plasmid was constructed through point mutation，and then it was used to establish HEK293 cell lines stably expressing A-Raf-S214C. Further，the influence of A-Raf-S214C on MAPK signaling pathway was determined by detecting the level of ERK phosphorylation. Results showed that HEK293 cell line with stable expression of A-Raf-S214C was successfully constructed，and A-Raf-S214C expression levels increased under induction with increasing concentrations of doxycycline. In comparison with wild type A-Raf，mutant A-Raf-S214C significantly enhanced the phosphorylation level of ERK . Conclusively，A-Raf-S214C mutant is an activating mutant of A-Raf kinase，resulting in significantly increased phosphorylation level of ERK，a downstream signaling molecule in MAPK pathway.