Table of Content

20 April 2018, Volume 34 Issue 4

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Research Progress of CRISPR/Cas9 System in Filamentous Fungi

WANG Pei， LIU Wen， SHEN Xiang-ling

2018, 34(4):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1128

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Filamentous fungi,which are closely related to human survival,have been widely used in industrial development and application in addition to its great edible and medicinal values. In recent years,CRISPR/Cas9 has been rapidly developed in various fields due to its simple design,convenient operation,precise positioning and high efficiency,and has been received great attentions in the world. However,because of the relatively low level of homologous recombination in some fungi,such as Aspergillus fumigatus,genetic manipulation of them is rather difficult. Therefore,this technique is rarely used in filamentous fungi,especially in large fungi. Based on the latest application and research progress of CRISPR/Cas9 technology in filamentous fungi,this paper introduced various editing mechanisms in filamentous fungi,and analyzed the reported its application in model fungi,aiming at providing a reference to conduct gene editing for more fungi.

Research Advances in Pathogenesis of Sclerotinia sclerotiorum

YANG Guo-gen, CHENG Jia-sen

2018, 34(4):  9-15.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0108

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Sclerotinia sclerotiorum（Lib.） de Bary is an important phytopathogenic fungus in the worldwide. S. sclerotiorum has a wide host range and can infect many kinds of crops. Sclerotinia diseases causes significant yield losses and economic damage of many crops. S. sclerotiorum is an necrotrophic phytopathogenic fungus,which obtain nutrient from plant by killing host cells and destroying host tissue. Previous studies focused on the role of hydrolytic enzymes（cutinase,cell wall degrading enzymes and proteases） and oxalic acid during infection. The pathogenicity mechanism of S. sclerotiorum is extremely complicated,more and more recent studies have shown that secreted proteins also play an important role in the interaction of host and S. sclerotiorum. Secreted proteins facilitate the invasion and colonization of S. sclerotiorum by inducing plant cell death or suppressing host cell immune responses. This review summarizes the latest research progress on the pathogenesis of S. sclerotiorum including hydrolases,oxalic acid,secreted proteins and other pathogenic proteins. We also prospect the research of pathogenicity mechanism of S. sclerotiorum,in order to provide new ideas and theoretical basis for the safety and control of Sclerotinia disease.

Research Progress on Genetic Diversity of Shiraia bambusicola,Biosynthesis and Anticancer Activity of Hypocrellin

ZHAO Ning, CHEN Shuang-lin

2018, 34(4):  16-23.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0116

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Shiraia bambusicola is an important species of medicinal fungi in China. Its major active metabolites are perylenequinone pigment,such as hypocrellin. The former researches on S. bambusicola were mainly focused on the analysis of active products,clinical application,and biological fermentation,etc. The genetic diversity of S. bambusicola results in the differences of hypocrellin anabolism among different populations and strains. With the antitumor mechanism of hypocrellin and derivatives continuously discovered,studies on the genetic diversity of S. bambusicola,the synthesis and metabolism and anticancer mechanism of hypocrellin are proceeding more deeply. Applying different molecular markers reveal that the genetic diversity of S. bambusicola might be mainly from a population or among populations. Moreover,hypocrellin presents a promising prospect in the photodynamic therapy（PDT）of tumor by chemical modification or physical modification. Meanwhile,new results of research on the polyketide synthase genes and other genes,which affect the production of hypocrellin,will eventually help to reveal the biosynthesis pathway of hypocrellin in S. bambusicola. This article reviews the research progress on the genetic diversity of S. bambusicola and the role of hypocrellin and derivatives on cancer cells,the biosynthesis of hypocrellin,aiming at providing valuable references for the conservation of S. bambusicola,the further research and development of hypocrellin.

Applications of Fungal Laccase in Green Chemistry

GONG Rui, SUN Kai, XIE Dao-yue

2018, 34(4):  24-34.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0824

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Laccase is a kind of extracellular multi-copper oxidase secreted by fungi. It is widely used in green environmental chemistry because of its multifunctional property. It is proved that the copper-cluster of laccase can catalyze one-electron oxidative of phenolic and non-phenolic substrates via yielding radical intermediates. Then these radical intermediates involved in oxidative coupling or bond rupture,resulting in substrate oxidation,decomposition or polymerization. At present,the methods for immobilization of laccase are primarily divided into four categories including encapsulation in a carrier,adsorption on a carrier,covalent binding,and cross-linking. Compared with the free laccase,the underlying reasons for immobilization of laccase are the need to enhance the activity,stability,and recyclability of fungal laccase. In this paper,we review the molecular composition,structural characteristics,and catalytic oxidation mechanism of fungal laccase. Additionally,the main methods,advantages and disadvantages for immobilization of laccase are comparably evaluated,mainly focusing on the latest advances in the transformation and/or degradation of organic contaminants,the decolorization of synthetic dye,the treatment of paper wastewater,food processing,bio-sensors,and bio-indicators of environmental quality. It is aimed at providing new theoretical evidences and guidance for the multi-functional application of fungal laccase in green chemistry.

Research Process on the Endophyte Improving the Grass’s Salt and Alkali Resistance

CHEN Shui-hong, CAO Ying, CHEN Tai-xiang, LI Chun-jie

2018, 34(4):  35-42.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0429

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Soil salinization results in the slow growth of plants,and even inhibits their normal development. How to eliminate the adverse influences of high salinity and high pH on the growth and productivity of plants in saline-alkali land has become a key issue in agricultural sustainable development. Recently,some great benefits of endophytes and symbiotic fungi in salt and alkali resistance of host plants have been arousing great interest among scientific community. In this paper we summarized the advances on the effects of endophytic fungi on salt and alkali tolerance of host grasses. Firstly,we reviewed the impacts of endophytic fungi on the seed germination and growth of host grasses under salt-alkali stress,i.e.,increasing the seed germination rate,accelerating the growth of germ and radicle,and improving the tiller number,leaf number,plant height,biomass,root-shoot ratio,mature inflorescence length,seed number and seed production of host grasses. While as there were no promoting effects among some grasses infected by endophyte under salt stress. Secondly,we reviewed the roles of endophytic fungi in terms of reducing the original salt injury of host grasses in salt and alkali stresses,such as affecting the membrane permeability,improving photosynthesis,increasing water use efficiency,changing the nutritional metabolism and ion poisoning,and impacting the metabolism of plant hormones. Finally,we summarized the roles of endophytic fungi in reducing secondary salt damages to the host grass;endophytic fungi promoted the secretion of antioxidants in grasses,increased the activity of antioxidant enzymes,and adjusted the contents of the organic osmotic substances. This review systematically summarized the relevant research achievements on the endophytic fungi and the salt and alkali resistance of host grasses,brought up possible future research questions and approaches,and may provide references for later research.

Research Progress on Blue-light Photoreceptors in Zygomyceta

GE Xin, CUI Tian-qi, LI Xing-wang, XIE Lu-han, XIN Qi

2018, 34(4):  43-50.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0035

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Light is a key environmental signal. Light,most notably blue light,may regulate many aspects of the biology of fungi,including vegetative growth,development of fruiting body,the circadian rhythm,pigment formation and varied metabolic pathways. Zygomycete fungi are generally sensitive to environmental signals,especially light stimulation and have obvious tendency,and have served as model organisms to studying light signal transduction in fungi. Zygomycete fungi are at early stage of fungal evolution,i.e.,in low evolution. Their multi-copy blue photo-receptor proteins usually form perfect photoreceptor system during long-term evolution,which may sense blue and near ultraviolet lights at different wavelengths,intensities and directions,thus play a critical role in phototaxis and carotenoid synthesis of zygomyceta,as well as in the photo-response of asexual and sexual reproduction. In this paper,we took the representative strains of Zygomyceta as an example,summarized the blue-light photoreceptor proteins reported by several researchers,and made a comparative analysis from the aspects of gene discovery,function identification,photochemical characteristics,regulatory pathways,etc. Moreover,based on the above three（four）aspects,we described special physiological process mediated and regulated by blue-photoreceptors thatwere entirely different from it in higher plant. In the end,we suggested the future focuses and the confronting difficulties on the studies of blue-photoreceptors in zygomycetes,expecting for providing direction and reference for the researchers in this field.

Research Advances on Verticillium dahliae Genes Resulting in Pathogenicity and Microsclerotia Formation

XIE Cheng-jian

2018, 34(4):  51-59.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1025

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Verticillium dahliae causes serious damages to agricultural production because of its strong pathogenicity and wide host area,as well as it may live many years as microsclerotia in soil,and germinate as long as there is a appropriate host,thus it is particularly difficult to control it. Currently,the many genes affecting the pathogenicity of V. dahliae have been discovered,and the most critical result is that penetration pegs are generated to infect plant,and some effectors were secreted to be V. dahliae at the hyphal neck of penetration pegs,which regulates plant immunity. Furthermore,the studies have suggested that melanin was vital for the formation of mature microsclerotia,and some genes involved in microsclerotia formation also were related to V. dahliae pathogenicity. However,current researches did not completely elucidate the molecular mechanisms of V. dahliae causing plant wilting and necrosis and microsclerotia formation. This review summarizes research advances on V. dahliae genes involved in pathogenicity and microsclerotia formation,aiming at establishing the theoretical basis for further research on the mechanisms of V. dahliae pathogenicity and microsclerotia formation.

Research Progresses on Monoterpene Synthesis in Saccharomyces cerevisiae

FU Bei-bei, ZHAO Jian-zhi, LI Chen, LIU Xin-li, BAO Xiao-ming, HOU Jin

2018, 34(4):  60-69.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0880

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Terpenoids are a large class of natural compounds which are synthesized from the precursor of 5-carbon isoprene unit,and they exist widely in plants,microorganisms and insects. Among them,monoterpene-like compounds are mainly used in production of flavors,cosmetics,food additives,pesticides,herbicides and advanced biofuels,i.e.,it presents wide application potential. In recent years,many terpenoid-producing recombinant Saccharomyces cerevisiae strains have been constructed,and the terpenoid yield is efficiently increased through approaches of metabolic engineering and synthetic biology. However,the synthesis of monoterpene by microorganism is relatively lagging behind because the lack of precursors,its toxicity to microorganisms,etc. limit the efficient synthesis of monoterpene. Here we discuss the recent advances of monoterpene synthesis in S. cerevisiae including the expression of monoterpene synthase,the enhancement of precursor geranyl pyrophosphate through dynamic regulation and the protein engineering,blocking the endogenous conversion of monoterpene,and improving the tolerance of the strains to monoterpene. There fore,we propose the feasible strategies toward resolving the bottleneck in microbial synthesis of monoterpene,aiming at providing potential references for optimizing the monoterpene-producing microbial cell factories.

Optimization of Preparation Conditions of Pleurotus geesterani Monocaryon Mycelia Protoplast

SUN Xiao-rui, CHEN Bo-wen, ZHANG Xiao-lin, MENG Jun-long

2018, 34(4):  70-76.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0797

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The optimal conditions for preparing protoplast from lywallzyme by cultured monocaryon mycelia of Pleurotus geesterani were investigated via single factor and orthogonal array design. The results showed：the yield of protoplast and regeneration rate of protoplast reached 3.5×107 CFU/mL and 1.4% respectively,under the conditions of 8 days mycelium in the enzyme solution of 1.6% lywallzyme digested for 160 min at temperature of 29℃ with 0.6 mol/L mannitol +10 mmol/L Tris-HCl as osmotic pressure stabilizer.

Preparation and Regeneration Method of Phytophthora infestans Protoplast in Potato

WANG Wei-wei, XIAO Yan, ZHANG Yi-xi, LIU Jing, TANG Wei, LI Can-hui

2018, 34(4):  77-82.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0151

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For better understanding the pathogenicity of Phytophthora infestans,a highly efficient system of preparing and regenerating protoplast should be established. In this study,two lyases,Driselase and Cellulase R-10,were used to prepare protoplasts. The effects of different hyphal age,lyase,lyase concentration,and enzymolysis time on the protoplast preparation were discussed. The optimized method was established with the 18-day-old mycelia,the yield of protoplast reached 9.5 × 104 cells/g after 60 min treatment with 10 mg/mL Driselase. The protoplast regeneration rate was up to 3.1% in rye V8 culture medium supplemented with 0.1 mol/L calcium chloride and 0.4 mol/L mannitol as the osmotic stabilizer.

Response Surface Analysis for Optimizing Antimicrobial Protein from Mushroom Coprinus comatus Fermentation Liquid and Its Antioxidant Activity

QIN Nan, LIU Yu, WANG Li-yu

2018, 34(4):  83-90.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0850

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This study is to extract the antimicrobial protein from Coprinus comatus fermentation liquid. The inhibitive circle diameter was used as evaluating indicator,and the fermentation condition was optimized by single factor experiments and response surface methodology（RSM）. The optimized extraction condition was 5 d fermentation time,30℃,pH7,inoculation volume of 5%,and the shaker rotation speed 100 r/min,under which the inhibitive circle diameter was up to 21.14 mm. The antimicrobial protein was proved to have weak scavenging effect on DDPH and ABTS,however,have strong scavenging effect on ·OH,and its antioxidant capacity enhanced with the increase of antibacterial protein concentration.

Optimization of Liquid Medium Composition for the Production of Cellulase from Brown Rot Fungus Antrodia bambusicola by Response Surface Methodology

MA Hong-fei, CUI Bao-kai, YUAN Yuan, CHEN Yuan-yuan, DAI Yu-cheng, SI Jing

2018, 34(4):  91-101.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0067

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Antrodia bambusicola,a brown rot fungus,produces cellulase with important practical values. To improve its cellulase activity,Plackett-Burman design,the steepest ascent design,and Box-Behnken design in response surface methodology were employed to establish mathematical model while the cellulase activity was taken as the response. The optimal composition for the production of cellulase from A. bambusicola was obtained as follows：glucose 7.44 g/L,peptone 5.0 g/L,wheat bran 35.31 g/L,Tween 80 9.57 mL/L,KH2PO4 1.0 g/L,MgSO4·7H2O 0.5 g/L,CoCl2·6H2O 0.06 g/L,FeSO4·7H2O 0.03 g/L,CaCl2 0.5 g/L,ZnSO4·7H2O 0.05 g/L,and vitamin B1 0.01 g/L. Under the optimized conditions,the cellulase activity was 53.17 U/mL,which agreed closely with the theoretical result of 52.65 U/mL predicted by the developed model,and this yield was 2.84-fold higher than that produced in basal medium alone. Above results indicate that the response surface methodology is viable for optimizing medium composition in improving cellulase activity.

Comparing the Methods of Isolating Genomic DNA and Construction of Its Small Genomic Library of Ustilaginoidea virens

FU Rong-tao, WANG Jian, CHEN Cheng, GONG Xue-shu, LU Dai-hua, LUO Xi, ZHENG Ai-ping

2018, 34(4):  102-106.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0686

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Rice false smut caused by Ustilaginoidea virens is one of important fungal diseases for rice. This experiment aimed to select an optimal method of isolating genomic DNA,and to construct small genomic library of U. virens. The genomic DNA of U. virens was extracted by 3 methods of CTAB,SDS,and fungal DNA isolation kit. The DNA sample was sequenced using Illumina system,and small genomic library was constructed. The results showed that the high-quality genomic DNA was obtained by CTAB. Meanwhile,the small genomic library of U. virens was contained 29 350 268 bases and 17 904 Scaffold,and the GC content was 49.79% of total bases. We found that CTAB was the best method of isolating genomic DNA of U. virens. The successful construction of the small genomic library provided data resource for filling the genome gap and identifying pathogenetic gene.

Sequence Characterization and Differential Expression Analysis of a Aryl Alcohol Oxidase Gene vvaao1 from Volvariella volvacea

YAN Jun-jie, TONG Zong-jun, LIU Yuan-yuan, ZHANG Lei, ZHANG Yun, XIE Bao-gui

2018, 34(4):  107-114.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0166

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A gene encoding aryl alcohol oxidase named vvaao1 was obtained from straw mushroom Volvariella volvacea. To reveal the role of vvaao1 during substrate degradation and fruiting-body development in V. volvacea,the structure,sequence characters and expression profile of vvaao1 were analyzed. ZOOM software was used to confirm the accuracy of gene sequence and analyze the gene structure. The protein sequence was submitted to Expasy ProtParam for physicochemical properties prediction,SignalP server for signal peptide prediction,TargetP server for sub-cellular localization prediction,and the Phyre 2 server for three dimensional（3D）structure analysis. The MEGA 5.1 was used for multiple sequence alignment and phylogenetic tree analysis. Digital gene expression profiling（DGE）,real time fluorescent quantitative PCR（RT-qPCR）and iTRAQ methods were used to determine the expression pattern of vvaao1. Results showed that the full sequence of vvaao1 covered 3 091 bp,containing 9 exons and 8 introns,and the open reading frame length was 1803bp,encoded a protein with 600 amino acids. Bioinformatic analysis showed that the VvAAO1 contained a secretory signal peptide,and located in secretory pathway. The 3D structures predicted showed that VvAAO1 consisted with 20 α-helices and 19 β-strands,and had 48% identity with model protein 3FIM. The phylogenetic tree showed that VvAAO1 of V. volvacea belonged to the AAO class of GMC superfamily,and had the closest relationship with AAO in Pleurotus eryngii and P. pulmonarius. The analysis of DGE,RT-qPCR and iTRAQ results showed that the expression value of vvaao1 in H1521 were 51.0,49.8 and 2.13 times as much as the sum of two homokaryons,respectively. In addition,the gene expression of vvaao1 in primordia showed significant higher than other fruiting-body samples. VvAAO1 is a secretory protein,and its high level expression may be beneficial for fruiting-body formation.

Identification of Candidate Genes Related to the Development of Pleurotus tuoliensis Fruiting Bodies

RONG Cheng-bo, ZHAO Shuang, SONG Shuang, WANG Jing, LIU Yu

2018, 34(4):  115-120.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0139

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Pleurotus tuoliensis contains rich nutrition and has many functions such as anti-tumor and enhancing immunity,etc.,thus it is precious edible white mushroom with independent intellectual property right in China. Identifying the candidate genes related to the formation of P. tuoliensis fruiting bodies would provide effective and rapid markers for its breeding. Two strains were obtained from the previous studies,one strain JZB2106010 formed fruiting bodies,while another dikaryotic hybrid strain JZB2106103 from protoplast monokaryons of JZB2106010 couldn’t form fruiting body. The transcriptome sequencing of JZB2106010 and JZB2106103 were performed to identify the differentially-expressed genes and functional enrichment analysis was carried out. GO enrichment displayed that there were the most differentially-expressed genes in catalytic activity and metabolic process. qPCR verification of partially the differentially-expressed genes showed that phenylalanine ammonium lyase（CL4509）and aryl-alcohol oxidase gene（CL2173）had significant high gene expressions in JZB2106010,revealing that CL4509 and CL2173 were related to the development of P. tuoliensis fruiting bodies. In addition,ATP dependent DNA helicase（Unigene7793）displayed 1 046-fold higher expression in JZB2106103 than in JZB2106010,inferring that Unigene7793 was negatively related to the development of P. tuoliensis fruiting bodies.

Evaluation of the Genetic Diversity of Pleurotus citrinopileatus by Combined ISSR and SRAP Analyses

WU Hai-yue, ZHAO Shuang, LIU Yu, LI Hua-min, SONG Shuang, NIU Yu-rong, RONG Cheng-bo

2018, 34(4):  121-126.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1067

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The Pleurotus citrinopileatus have been one of the most important cultivated edible mushrooms in recent years,because they taste delicious,are full of nutriments,and contain high essential amino acids. Genetic diversity of 19 P. citrinopileatus cultivars,collected from all over the China,was analyzed using combined ISSR and SRAP molecular markers in the experiment,which provides theoretical guidance for the parental selection of cross breeding. Ten ISSR primers and 10 SRAP primer pairs were selected for the producing reproductive bands using genomic DNA of P. citrinopileatus as templates. In total,194 amplification patterns were obtained,and in which 150 bands were polymorphic specific,accounting for 77.3% of all bands. The obtained results by cluster analysis indicated that the tested strains fell into four clades at a coefficient of 0.70,revealing that there was significant genetic diversity among P. citrinopileatus cultivars. The diagram of cluster analysis laid the foundation for the parental selection in the cross breeding of P. citrinopileatus.

Purification and Enzymatic Characterization of Laccase from Stropharia rugosoannulata

QIAN Lei, ZHANG Ye-ni, CHEN Xue, LIU Jin-fu, ZHANG Zhi-jun, LIU Jian-hua

2018, 34(4):  127-132.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0629

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The laccase from the fermentation liquid of Stropharia rugosoannulata strain Sr-01 was isolated and the effects of temperature,pH and metal ions on laccase activity and stability were assayed. The laccase was purified by ammonium sulfate fractional precipitation,anionic exchange chromatography on Q-Sepharose,and gel filtration chromatography on Superdex 200. The laccase activity was measured by spectrophotometry with ABTS[2,2-azinobis-（3-ethylbenzothiazoline-6-sulfonate）]as the substrate. The results showed that the specific activity of the purified laccase was 152.79 U/mg and the recovery was 35.8%. SDS-PAGE analysis demonstrated that it was a monomeric protein with the molecular weight of 40 kD. The optimal reaction temperature and pH of the laccase were 35℃ and 4.0,respectively. The laccase activity was stimulated by Mg²⁺ and Cu²⁺,whereas it was significantly inhibited by Fe²⁺,Cd²⁺ and Hg²⁺. Under the optimal reaction conditions,the specific activity of the purified laccase achieved 222.93 U/mg.

Genetic Relationship Analysis of 22 Cordyceps militaris Based on Split Network and Preliminary Screening of High-yield Cordycepin Strains

LIU Jian-bing, QI Meng, DU Yuan-ru, FU Jun-sheng, HU Kai-hui

2018, 34(4):  133-138.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0852

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We used the split network based on ITS sequence for analyzing the genetic relationships of 22 Cordyceps militaris strains,and we compared the capacities of strains producing cordycepin and screened the strains highly yielding cordycepin. The results showed that length of ITS sequence（including ITS1,5.8S rDNA and ITS 2）of 22 strains was 518-539 bp,there were 39 variable sites and 12 parsimony informative sites in the ITS sequence after alignment,and all occurred in the ITS1 region. The split network showed that 22 C. militaris strains were roughly divided into 3 clades,and the strains of each clade were distinguished from others by its unique base substitutions. Cordycepin was mainly in extracellular liquid,cordycepin contents by the 22 strains varied significantly,and the content of cordycepin in MF 27 strain was the highest at 332.74 mg/L,significantly higher than that by other strains（P<0.05）. This study provides a basis for understanding the systematic relationship among strains of C. militaris and the management of germplasm resources.

Effects of PMT1 and PMT3 Double-gene Deficiency on the Lifespan of Saccharomyces cerevisiae

CUI Hong-jing, ZHOU Tao, QIU Kun-pei, SONG Hao-chang, LIU Xin-guang

2018, 34(4):  139-143.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0847

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We herein aimed to explore the effects of the PMT3 of protein O-mannosyltransferase（PMT）single-gene deficiency,and PMT1 and PMT3 double-gene deficiency on the replicative lifespan of Saccharomyces cerevisiae. PMT3 deficiency strain（pmt3Δ）was constructed through polymerase chain reaction（PCR）-mediated one-step gene disruption,and PMT1 and PMT3 double-gene deficiency strain（pmt1Δpmt3Δ）was prepared based on homologous recombination. The replicative lifespan of yeast cells were counted by separating daughter cells from mother cells using a manual micromanipulator equipped with a fiber-optic needle under the microscope. The replicative lifespan of the pmt3Δ yeast strain（24 generations）and pmt1Δpmt3Δ strain（25 generations,P > 0.05）exhibited no significant difference,compared with that of wild-type yeast strain（26 generations）（P > 0.05）,i.e.,there was no statistical significance among the differences. Conclusively,PMT3 single-gene deficiency and PMT1 and PMT3 double-gene deficiency exert no evident effects on the lifespan of yeast cells.

Cloning and Identification of Gene Promoter for Gliotoxin Biosynthesis from Deep-Sea-Derived Fungus Dichotomomyces cejpii

HUANG Zi-lei, ZHANG Wei-min, YE Wei, LI Sai-ni, LI Hao-hua, ZHU Mu-zi

2018, 34(4):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0941

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The promoters of gliotoxins biosynthesis genes including GliG,GliI and GliO from deep-sea-derived fungus Dichotomomyces cejpii were cloned by the method of genome walking,and the core regions of these promoters were inserted into the reporter gene vector of luciferase pGL-3-basic,and the transcriptional activities of GliG promoter was the highest according to its fluorescence intensity. Further,the core region of GliG promoter was further inserted into the pAN7-1 vector with hygromycin resistance marker to replace original pgpdA promoter,then the recombinant plasmid was transferred into Saccharomyces cerevisiae by electronic transformation. The positive clones were screened by plate with hygromycin resistance,and the results demonstrated that the GliG promoter initiated the expression of hygromycin resistance gene.

Isolation,Purification,Characterization and Structural Analysis of a Pectinase PGL1 Produced by Fusarium sp. Q7-31T

QIN Ri-tian, XIE Zhan-ling

2018, 34(4):  151-160.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0864

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To explore the role of pectinase degrading cell wall and further improve the information of cellulose-degrading enzymes in Fusarium sp. Q7-31T,the pectinase from Fusarium sp. Q7-31T was isolated,purified,and identified,followed by bioinformatics analyses. Q7-31T was cultured by induction and fermentation with 0.8% peptone as nitrogen source and 0.5% oat straw as carbon source. Then the pectinase PGL1 was purified from crude enzyme liquid using Sephacry S-100 chromatography and DEAE-sepharose ion-exchange column chromatography. Further,its enzymatic properties were analyzed and MADIL-TOF-TOF identification was conducted. Finally,its structure was analyzed and the function was predicted by bioinformatics. The molecular weight and isoelectric point（pI）of PGL1 was 36.21 kD and 8.13,respectively. PGL1 had optimal activity at 50℃ and p H 8.0,was highly stable at 20℃ to 50℃ and pH 8.0 to 10.0,and was inhibited by Na⁺,K⁺,Mg²⁺,Zn²⁺,Ca²⁺,and Fe²⁺. MADIL-TOF-TOF identification showed that the PGL1 was a new PL-1 family pectinase. The secondary structure prediction suggested that random coil and extended strand were major secondary components（37.96% and 31.17% respectively）. PGL1 had two hydrophilic ends and a hydrophobic middle part. Its phosphorylation sites were 13（Ser：9;Thr：3;Tyr：1）. There were 2 potential N-glycosylation sites in PGL1. The prediction of transmembrane domain revealed that PGL1 was a secretory enzyme. The functional domain prediction of PGL1 showed the role of its degrading plant cell wall. Conclusively,a new PL-1 family pectinase PGL1 was isolated and purified from Fusarium sp. Q7-31T and analyzed by bioinformatics.

Isolation,Purification and Identification of Metalloprotease FQME14 from Fusarium sp. Q7-31T

QIN Ri-tian1LU Yu-li1LIANG Gao-li1XIE Zhan-ling1, 2LEI Ya-nan3

2018, 34(4):  161-167.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0896

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The objectives of this work include：（1）to isolate the metalloproteinase from the fermented crude enzymatic solution of Fusarium sp. Q7-31T by oat straw,and then purify and identify them;（2）to study the enzymatic characteristics of protease for enriching and improving the basic data of metalloproteinases;and（3）to explore the role of cellulase in the degradation process of cellulose for understanding the mechanisms of cellulase degradation. Strain was cultured in liquid fermentation with oat straw as carbon source,and then crude enzyme solution was collected on the sixth day. The metalloprotease was purified using ammonium sulfate precipitation,Sephacry S-100 gel filtration chromatography and DEAE weak ion-exchange chromatography,its enzymatic properties were studied,and it was identified by MADIL-TOF-TOF. As results,the protease FQME14 isolated from the crude enzyme solution of Fusarium sp. Q7-31T was proved by SDS-PAGE to be a single band with a relative molecular mass of 30 kD. The MADIL-TOF-TOF results suggested that protease FQME14 belonged to M14 family. The characterization of purified protease showed that the specific activity of protease was 2.85 U/mg,its optimal pH and temperature for the enzyme activity were 7.0 and 40℃,respectively. The protease was very stable at pH 5.0-9.0 and temperature up to 50℃. The protease presented high degradation capacity to casein,ovalbumin and bovine serum albumin. The Co²⁺and Mn²⁺ were stimulators for protease,while Fe²⁺,Mg²⁺,K⁺,Ca²⁺,Zn²⁺and Na⁺ ions inhibited the activity of the protease at varied level. In conclusion,a protease from Fusarium sp. Q7-31T,named FQME14,was isolated,purified,identified and characterized. The enzymatic properties and MADIL-TOF-TOF results suggest that protease FQME14 belongs to M14 family,and is a new metalloprotease.

Effects of Oxidative Stress on Hyphal Growth and Chlamydospore Formation of Clonostachys rosea

WANG Qi, FENG Jing-hong, SUN Zhan-bin, JIANG Wei-zhi, LI Shi-dong, SUN Man-hong, MA Gui-zhen

2018, 34(4):  168-173.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0070

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The effects of oxidative stress on hyphal growth and chlamydospore production in Clonostachys rosea 67-1was investigated through adjusting ventilatory volume and adding various kinds of reactive oxygen（O2-,H2O2 and OH·）. The results showed that the effects of oxidative stress on fungal mycelia were different from that on chlamydospores. When aeration rate was low,the growth status of the fungus changed;with the increasing of loading volume,the branches of mycelia reduced,and the yield of chlamydospores decreased sharply. Reactive oxygen at certain amount affected the formation of chlamydospores during 67-1fermentation. When 5-6 mmol/L H₂O₂ was added before inoculation,the production of chlamydospores significantly increased,and the growth and sporulation of 67-1were completely inhibited while the concentration was 9 mmol/L. The oxygen-tolerance of the fungus was remarkably enhanced when H₂O₂ was supplied at 16 h after inoculation. Menadione inhibited chlamydospore formation when added into the fermentation liquor directly,but adding 350 µmol/L menadione significantly promoted fungal sporulation after incubated for 16 h. In Fe²⁺-H₂O₂ system,high oxygen stress in different time significantly increased chlamydospore production（P < 0.05）.

Identification and Biological Characteristics of One Antagonistic Fungal Strain Against Sclerotinia sclerotiorum

DING Rui, ZHANG Hao-qi, CHEN Xu-hui, LIN Hao, QU Bo

2018, 34(4):  174-178.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0073

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Our aims are to isolate and select antagonistic endophytic fungi against Sclerotinia sclerotionum from the roots of Liparis japonica（Orchidaceae）,to identify their taxonomic status and study the characteristics of in vitro culturing it. Conventional PDA culture technology was applied to isolate and culture endophytic fungi and plate confrontation method to screen those strains with strong antagonistic activities against S. sclerotionum and to identify their taxonomic status,as well as the biological characteristics were studied under different carbon sources,nitrogen sources,temperature and pH values. One strongly antagonistic endophytic fungus against S. sclerotionum were obtained and identified as Nodulisporium sp. by its morphological characters and ITS molecular sequences. The carbon sources that this fungus relied on were diverse and it grew well by any of maltose,fructose,glucose and sucrose as carbon resource. There existed significant differences among nitrogen sources for this fungus’ growth,and the optimal nitrogen source was beef extract. The optimal temperature was 25℃,and the hypha was inhibited significantly when the temperature fell below 10℃or rose above 30℃. This fungus also tolerated very wide range of pH,and grew well under pH 5 to 9.

Diversity and Functional Activity of Trichoderma in the Rhizosphere Soil from Facility Tomato Production

ZHANG Guang-zhi, WANG Jia-ning, WU Xiao-qing, ZHOU Fang-yuan, ZHANG Xin-jian, ZHAO Xiao-yan, XIE Xue-ying, ZHOU Hong-zi

2018, 34(4):  179-185.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0690

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This work aimed to explore the diversity of Trichoderma from the rhizosphere soil of vegetables under facility culture as well as their aniti-phytopathogenic fungi and organophosphorus-degrading activity. Trichoderma were isolated from the rhizosphere soil and the roots of the sampled tomato from facility culture fields for more than 20 years of cultivation,and were identified by tef1-α sequencing combined with phylogenetic analysis. Besides,the relative population density of each strain was estimated,and the biocontrol activity and the degradation activity were further studied. Results showed that 27 different strains were isolated,and then identified based on the morphological characteristics and tef1-α,they belonged to T. virens,T. atrobrunneum,T. simmonsii,T. atroviride,T. crassum and one suspected new species T. sp. nov. Conidiation of Trichoderma strains usually was distributed unevenly,more or less concentric,and bright green to dull green. All the strains showed varied levels of inhibiting activity to several plant pathogenic fungi such as Rhizoctoma solani,Fusarium oxysporum and Verticillium dahlia. In the degradation experiment of chlorpyrifos,except strains belonging to T. atrobrunneum,strains in other Trichoderma species effectively degraded chlorpyrifos. The relative population density of Trichoderma in the rhizosphere soil was 104 cfu/g,and there was significant difference among different strains. The results indicate that there is rich Trichoderma germplasm diversity in the soil under facility culture,and they show obvious strain difference in morphological characteristics and functional activities. Trichoderma strains have general inhibiting activity to three plant pathogenic fungi,and some of them can effectively degrade organophosphorus pesticide. Thus,they play a synergistic effect of biocontrol and biodegradation of pesticides and fertilizers. The results of this paper are of great significance for the study of the adaptability of Trichodema in the soil of facility agriculture and the exploitation and utilization of Trichoderma resources.

Identification of Filamentous Fungus Strain DWL-C010 and Thermal Stability Analysis of Its Soluble Pigment

MA Bo, ZHANG Ting-ting, WU Bao-xiang

2018, 34(4):  186-193.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0129

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Strain DWL-C010 capable of secreting diffusible red pigment,isolated from primary forest humus soil of Dawangling Nature Reserve in Gxuangxi,was identified by morphological characteristics and phylogenetic analysis of multiple gene loci,and then the thermal stability of its red pigment was analyzed as well. In addition,the enzyme activity of its cellulase,hemicellulase,and glucoamylase was also evaluated. The results showed that morphological characteristics of strain DWL-C010 are consistent with Talaromyces albobiverticillius on the whole,and the identity of ITS RBP2,RBP1 and BenA between strain DWL-C010 and T. albobiverticillius CBS133440T reach 99%,while the identity of CaM has only 96%;but they all resolve in a clade with T. albobiverticillius CBS133440T. Referring to the traditional morphological observation,strain DWL-C010 belongs to be T. albobiverticillius. At the same time,the thermal stability of red pigment from DWL-C010 is promising,the degradation rate of red pigment from strain DWL-C010 was in fine linear relation with time,and it could be well described by a first order model. The temperature-dependent degradation constants also followed the Arrhenius model,and the activation energy of the red pigment is 51.82 kJ/mol. Besides,the enzyme activity of strain DWL-C010 for xylanase,cellulase and glucoamylase was 33.098,1.880 and 0.976 U/mL,respectively.

Expression Analysis of Ribosomal Protein Genes in Aspergillus flavus

GENG Long-po, WANG Xin-wang, HUANG Lu-hua, DENG Ji-li, WANG Shi-hua, ZHANG Feng

2018, 34(4):  194-200.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1049

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Ribosome is a major organelle of protein synthesis in organism. Ribosomal protein is one of main components of ribosome. In order to explore the relationship between Aspergillus flavus ribosomal protein and its development,A. flavus were collected at mycelial stage,spore stage,and sclerotial stage. After total RNA extraction and the cDNA preparation,the expression levels of 74 genes of A. flavus ribosomal protein were detected by quantitative RT-PCR. The results showed that,none of the tested genes was expressed invariably at all three stages. Compared with mycelial stage,the expression levels of 54 ribosomal protein genes were up-regulated at the spore stage,while 2 genes were down-regulated. At sclerotial stage,the expression levels of 57 genes were up-regulated,while 12 genes were down-regulated. The GO functional analysis showed that the ribosomal protein genes were enriched into structural molecular activity,binding,cell,macromolecular complex,organelle,cell part,organelle part,cell process,and metabolic process. It can be concluded that ribosomal proteins are closely related to the growth and development of A. flavus.

Responses of Asperigillus niger to Oxidative Stress Under H₂O₂ Exposure

CHEN Hao-yu, XU Rui-tao, CHENG Zhi-xiang, GAO Qiang, ZHANG Jian

2018, 34(4):  201-207.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0026

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To investigate the response of Aspergillus niger to oxidative stress by adding H₂O₂ to the medium will provide the basis for controlling the mycelial growth and ochratoxin A（OTA）biosynthesis of A. niger. The effects of H₂O₂ on the growth,OTA biosynthesis,intracellular reactive oxygen species（ROS）formation,lipid peroxidation level,and antioxidase activities in A. niger were investigated. H₂O₂ inhibited the mycelial growth of A. niger,and the inhibition was dose dependent. Also H₂O₂ promoted OTA biosynthesis in A. niger,especially the most remarkably at the day 4 of growth. H₂O₂ caused intracellular ROS and malondialdehyde（MDA）content increasing in A. niger. H₂O₂ enhanced the activities of intracellular catalase（CAT）and glutathione peroxidase（GPX）,while significantly at the day 5 of growth for CAT and at the day 6 for GPX in A. niger. However,H₂O₂ presented no significant effect on the activity of superoxide dismutase（SOD）in A. niger. A. niger remediates oxidative stress by enhancing the activity of antioxidant enzymes and producing OTA,which may maintain the growth and metabolism of A. niger under oxygen stress.

Identification and Carbon Metabolic Analysis of a Mold Isolated from Uranium Acid Leaching Solution

LIU Huan, XU Ling-ling, LI Jiang

2018, 34(4):  208-213.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0592

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This study aims to identify a mold by morphological observation,rDNA ITS sequence analysis,and Biolog microbial identification system,to investigate the growth of the mold and its consumption of carbon source by calculating the average well color development（AWCD）,and to analyze carbon source utilization of the mold using data. Results revealed as such,combined rDNA ITS sequence with morphological observation and Biolog identification system,the strain was identified as Aspergillus niger. Compared the consumptions of 8 carbon sources,the types of amino acid,lipid and acid were the most widely used. According to the utilizations of carbon sources,15 first-used carbon sources were listed. The optimal carbon source of the strain was P-hydroxyphenylacetic acid,which could be used as a material for the research of acid-resistant Aspergillus metabolism.

Cloning,Expression and Enzyme Production of Laccase Gene lac1680 in Phanerochaete chrysosporium

CHEN Jian-jun, LIU Liang-tao, CAO Xiang-lin

2018, 34(4):  214-220.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0946

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In this study,previously screened highly laccase-yielding Phanerochaete chrysosporium as template,homologous cloning technology was employed to synthesize a full-length 1680 bp laccase gene. Alignment of nucleotide and amino acid sequences showed that the gene had high homology with fungal laccase gene,named as lac1680. Then the gene was connected to the pET24a vector,and transferred to the expression strain Escherichia coli BL21（DE3）. The whole cell lysates by the pre-expression of the recombinant strain was detected by SDS-PAGE,and 75 kD band was obtained,thus indicating that the gene expressed successfully. NI-NTA column was then used to purify the eluted lac1680 protein,and the purity was over 98%. Comparing the enzyme-producing activities between the gene engineering strain and wild P. chrysosporium strain in different culture time showed that the activity of engineering strain obviously improved by nearly 39% than the wild one.