玉米大斑病菌漆酶基因StLAC在黑色素合成中的功能分析
摘要
为研究玉米大斑病菌漆酶基因在黑色素合成及致病过程中的作用,本研究首次发现漆酶可以促进玉米大斑病菌在寄主玉米组织中的扩展;利用同源重组的方法获得了玉米大斑病菌漆酶基因StLAC1的敲除突变体;通过对突变体进行分析,明确了StLAC1基因在黑色素合成中起着关键作用;利用PCR和RACE技术获得了玉米大斑病菌漆酶基因StLAC2的全长序列,同源性分析确定了StLAC2与StLAC1基因编码氨基酸序列间的相似性为29.22%;主要研究结果如下:
1.对玉米大斑病菌及9种常见植物病原真菌进行漆酶活性比较,发现供试菌株均具有漆酶活性和降解木质素的能力;漆酶活性测定结果表明,玉米大斑病菌的胞内漆酶活性较高,为18.984 U·mL~(-1);致病力测定发现,在玉米大斑病菌的致病过程中,漆酶可以促进病原菌在寄主组织中的扩展。
2.利用基因敲除技术,通过PEG介导转化法将构建好的玉米大斑病菌漆酶基因StLAC1的敲除载体转化玉米大斑病菌野生型菌株原生质体,经潮霉素筛选获得了潮霉素抗性转化子,利用PCR、Southern blot及RT-PCR技术进行验证,获得了StLAC1基因的功能缺失突变体△StLAC1。
3.对突变体△StLAC1进行表型分析发现,△StLAC1菌落呈灰白色,黑色素合成受阻,菌丝透明且呈不规则状,不产生分生孢子,生长速率减慢,渗透调节能力增强,附着胞膨压、侵染能力显著降低,漆酶活性有所降低。说明StLAC1基因在玉米大斑病菌黑色素合成、附着胞穿透力和分生孢子形成等方面具有重要作用。
4.利用PCR和RACE技术,获得了玉米大斑病菌漆酶基因StLAC2的全长序列。通过对该基因的结构进行分析,确定了其DNA全长1 763 bp,含有3个外显子,2个内含子;cDNA为1 665 bp,编码554个氨基酸。同源性分析发现,StLAC2基因编码的氨基酸序列与许多其他真菌漆酶具有45%~76%的相似性,与已知StLAC1基因编码的氨基酸序列间相似性仅为29.22%。聚类分析发现,Stlac1和Stlac2属于进化树中不同的分支,二者亲缘关系较远。保守结构域分析发现,Stlac1和Stlac2都含有铜离子的结合位点。Southern blot分析,确定了StLAC2基因在玉米大斑病菌基因组中是以单拷贝形式存在的。
This research intend to determine the laccases’function of melanin synthesis and pathogenicity in Setosphaeria turcica. It had been found that laccase could accelerate the extention of S. turcica against the host tissue. A null StLAC1 mutant was obtained using homologous recombination and it was found that the gene played a critical role in the melanin synthesis. Laccase gene StLAC2 of S. turcica was cloned by using PCR and RACE technologies, The amino acid sequence of StLAC2 gene shared 29.22% homology with Stlac1 at the protein level. Main results in this paper were as follows:
1. Laccase activity was compared between S. turcica and nine other plant pathogenic fungi which played critical roles in lignin degradation. S. turcica contained the higher intracellular laccase activity, which was 18.984 U·mL~(-1). The analysis of histopathology in S. turcica showed that corn leaves were oxidated by laccase which could promote the expansion of pathogen against the host.
2. By using homologous recombination technology, StLAC1 gene-knockout vector was transformed into S. turcica by PEG-mediated transformation method. A StLAC1 gene-deletion transformants were screened by hygromycin B. One StLAC1 gene-disruption mutant named as△StLAC1 was determined by PCR, Southern blot and semiquantitative RT-PCR analysis.
3. The mutant△StLAC1 displayed a gray-white colony which had less melanization compare with the wide type. Unlike the wide type, the null StLAC1 mutant could not produce conidia, it was transparent and had irregular-shaped mycelium, had slower vegetative growth rate, enhanced osmotic ability, weaked turgor pressure and penetration of appressorium, decreased laccase activity. StLAC1 gene involved in melanin biosynthesis and related to penetration of appressorium, form of conidiophore, respectively.
4. StLAC2 was cloned using PCR and RACE technologies. StLAC2 possess 1 763 bp DNA sequence which contained 1 665 bp coding region, encoded 554 amino acids and consisted of 3 exons and 2 introns. The amino acid sequence of StLAC1 gene shared 45%~76% indentity with other fungal laccases, shared 29.22% with the amino acid sequence of StLAC1 gene at the protein level. Analyzed the cluster tree and conserved domains, we found that Stlac1 and Stlac2 had the conserved copper binding domains, but belonged to different branches of phylogenetic trees and related distantly. Southern blot results showed that StLAC2 gene had single copy in the genome of S. turcica.
1.对玉米大斑病菌及9种常见植物病原真菌进行漆酶活性比较,发现供试菌株均具有漆酶活性和降解木质素的能力;漆酶活性测定结果表明,玉米大斑病菌的胞内漆酶活性较高,为18.984 U·mL~(-1);致病力测定发现,在玉米大斑病菌的致病过程中,漆酶可以促进病原菌在寄主组织中的扩展。
2.利用基因敲除技术,通过PEG介导转化法将构建好的玉米大斑病菌漆酶基因StLAC1的敲除载体转化玉米大斑病菌野生型菌株原生质体,经潮霉素筛选获得了潮霉素抗性转化子,利用PCR、Southern blot及RT-PCR技术进行验证,获得了StLAC1基因的功能缺失突变体△StLAC1。
3.对突变体△StLAC1进行表型分析发现,△StLAC1菌落呈灰白色,黑色素合成受阻,菌丝透明且呈不规则状,不产生分生孢子,生长速率减慢,渗透调节能力增强,附着胞膨压、侵染能力显著降低,漆酶活性有所降低。说明StLAC1基因在玉米大斑病菌黑色素合成、附着胞穿透力和分生孢子形成等方面具有重要作用。
4.利用PCR和RACE技术,获得了玉米大斑病菌漆酶基因StLAC2的全长序列。通过对该基因的结构进行分析,确定了其DNA全长1 763 bp,含有3个外显子,2个内含子;cDNA为1 665 bp,编码554个氨基酸。同源性分析发现,StLAC2基因编码的氨基酸序列与许多其他真菌漆酶具有45%~76%的相似性,与已知StLAC1基因编码的氨基酸序列间相似性仅为29.22%。聚类分析发现,Stlac1和Stlac2属于进化树中不同的分支,二者亲缘关系较远。保守结构域分析发现,Stlac1和Stlac2都含有铜离子的结合位点。Southern blot分析,确定了StLAC2基因在玉米大斑病菌基因组中是以单拷贝形式存在的。
This research intend to determine the laccases’function of melanin synthesis and pathogenicity in Setosphaeria turcica. It had been found that laccase could accelerate the extention of S. turcica against the host tissue. A null StLAC1 mutant was obtained using homologous recombination and it was found that the gene played a critical role in the melanin synthesis. Laccase gene StLAC2 of S. turcica was cloned by using PCR and RACE technologies, The amino acid sequence of StLAC2 gene shared 29.22% homology with Stlac1 at the protein level. Main results in this paper were as follows:
1. Laccase activity was compared between S. turcica and nine other plant pathogenic fungi which played critical roles in lignin degradation. S. turcica contained the higher intracellular laccase activity, which was 18.984 U·mL~(-1). The analysis of histopathology in S. turcica showed that corn leaves were oxidated by laccase which could promote the expansion of pathogen against the host.
2. By using homologous recombination technology, StLAC1 gene-knockout vector was transformed into S. turcica by PEG-mediated transformation method. A StLAC1 gene-deletion transformants were screened by hygromycin B. One StLAC1 gene-disruption mutant named as△StLAC1 was determined by PCR, Southern blot and semiquantitative RT-PCR analysis.
3. The mutant△StLAC1 displayed a gray-white colony which had less melanization compare with the wide type. Unlike the wide type, the null StLAC1 mutant could not produce conidia, it was transparent and had irregular-shaped mycelium, had slower vegetative growth rate, enhanced osmotic ability, weaked turgor pressure and penetration of appressorium, decreased laccase activity. StLAC1 gene involved in melanin biosynthesis and related to penetration of appressorium, form of conidiophore, respectively.
4. StLAC2 was cloned using PCR and RACE technologies. StLAC2 possess 1 763 bp DNA sequence which contained 1 665 bp coding region, encoded 554 amino acids and consisted of 3 exons and 2 introns. The amino acid sequence of StLAC1 gene shared 45%~76% indentity with other fungal laccases, shared 29.22% with the amino acid sequence of StLAC1 gene at the protein level. Analyzed the cluster tree and conserved domains, we found that Stlac1 and Stlac2 had the conserved copper binding domains, but belonged to different branches of phylogenetic trees and related distantly. Southern blot results showed that StLAC2 gene had single copy in the genome of S. turcica.
引文
[1]董金皋.农业植物病理学(北方本) [M].北京:中国农业出版社, 2001.
[2] Polak A. Melanin as a virulence factor in pathogenic fungi[J]. Mycoses, 1990, 33(5): 215-224.
[3] Morris-Jones R, Gomez B L, Diez S, et al. Synthesis of melanin pigment by Candida albicans in vitro and during infection[J]. Infection and Immunity, 2005, 73(9): 6147-6150.
[4] Saparrat M C, Fermoselle G E, Stenglein S A, et al. Pseudocercospora griseola causing angular leaf spot on Phaseolus vulgaris produces 1,8-dihydroxynaphthalene-melanin[J]. Mycopathologia, 2009, 168(1): 41-47.
[5] Butler M J, Gardiner R B, Day A W. Melanin synthesis by Sclerotinia sclerotiorum[J]. Mycologia, 2009, 101(3): 296-304.
[6] Coppin E, Silar P. Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina[J]. Mycological Research, 2007, 111(8): 901-908.
[7] Thompson J E, Fahnestock S, Farrall L, et al. The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase[J]. The Journal of Biological Chemistry, 2000, 275(45): 34867-34872.
[8] Langfelder K, Streibel M, Jahn B, et al. Biosynthesis of fungal melanins and their importance for human pathogenic fungi[J]. Fungal Genetics and Biology, 2003, 38(2): 143-158.
[9] Eliahu N, Igbaria A, Rose M S, et al. Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, Chk1 and Mps1, and the transcription factor Cmr1[J]. Eukaryotic Cell, 2007, 6(3): 421-429.
[10] Kogej T, Wheeler M H, Lanisnik Rizner T, et al. Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions[J]. FEMS Microbiology Letters, 2004, 232(2): 203-209.
[11] Baldrian P. Fungal laccases-occurrence and properties[J]. FEMS Microbiology Reviews, 2006, 30(2): 215-242.
[12] Yoshi H. Chemistry of lacquer[J]. Journal of the Chemical Society, 1883, 43: 472-486.
[13] Durante D, Casadio R, Martelli L, et al. Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera[J]. Journal of Molecular Catalysis B: Enzymatic, 2004, 27(4-6): 191-206.
[14] Camarero S, García O, Vidal T, et al. Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system[J]. Enzyme and Microbial Technology,2004, 35(2-3): 113-120.
[15] Michizoe J, Ichinose H, Kamiya N, et al. Biodegradation of phenolic environmental pollutants by a surfactant-laccase complex in organic media[J]. Journal of Bioscience and Bioengineering, 2005, 99(6): 642-647.
[16] Quaratino D, Federici F, Petruccioli M, et al. Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79[J]. Antonie Van Leeuwenhoek, 2007, 91(1): 57-69.
[17] Arica M Y, Altintas B, Bayramoglu G. Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation[J]. Bioresource Technology, 2009, 100(2): 665-669.
[18] Uhnakova B, Petrickova A, Biedermann D, et al. Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor[J]. Chemosphere, 2009, 76(6): 826-832.
[19] Saha B, Taylor K E, Bewtra J K, et al. Laccase-catalyzed removal of diphenylamine from synthetic wastewater[J]. Water Environment Research, 2008, 80(11): 2118-2124.
[20] Widsten P, Kandelbauer A. Laccase applications in the forest products industry: A review[J]. Enzyme and Microbial Technology, 2008, 42(4): 293-307.
[21] Johannes C, Majcherczyk A. Laccase activity tests and laccase inhibitors[J]. Journal of Biotechnology, 2000, 78(2): 193-199.
[22] Niladevi K N, Prema P. Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization[J]. Bioresource Technology, 2008, 99(11): 4583-4589.
[23]黄乾明.漆酶高产菌株的诱变选育及其酶的分离纯化、性质和基因克隆研究[D].雅安:四川农业大学; 2006.
[24]张敏,肖亚中,蒲春雷,等.白腐真菌AH28-2菌株发酵合成漆酶初步研究[J].微生物学通报, 2002, 29(3): 37-42.
[25] Sole M, Kellner H, Brock S, et al. Extracellular laccase activity and transcript levels of putative laccase genes during removal of the xenoestrogen technical nonylphenol by the aquatic hyphomycete Clavariopsis aquatica[J]. FEMS Microbiology Letters, 2008, 288(1): 47-54.
[26] Cusano A M, Mekmouche Y, Meglecz E, et al. Plasticity of laccase generated by homeologous recombination in yeast[J]. FEBS Journal, 2009, 276(19): 5471-5480.
[27] De Vries O M H, Kooistra W H C F, Wessels J G H. Formation of an Extracellular Laccase by a Schizophyllum commune Dikaryon[J]. Journal of General Microbiology, 1986, 132(10): 2817-2826.
[28] Coll P M, Fernandez-Abalos J M, Villanueva J R, et al. Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971)[J]. Applied and Environmental Microbiology, 1993, 59(8):2607-2613.
[29] Pihet M, Vandeputte P, Tronchin G, et al. Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia[J]. BMC Microbiology, 2009, 9: 177.
[30]詹旭,曹志艳,邢继红,等.植物病原真菌产漆酶菌株的筛选[J].中国农业科学, 2011, 44(4): 723-729.
[31] Choi G H, Larson T G, Nuss D L. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain[J]. Molecular Plant-Microbe Interactions, 1992, 5(2): 119-128.
[32] Castro-Sowinski S, Martinez-Drets G, Okon Y. Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J]. FEMS Microbiology Letters, 2002, 209(1): 119-125.
[33] Zhu X, Gibbons J, Zhang S, et al. Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans[J]. Molecular Microbiology, 2003, 47(4): 1007-1014.
[34] Bohlin C, Jonsson L J, Roth R, et al. Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger[J]. Applied Biochemistry and Biotechnology, 2006, 129-132: 195-214.
[35] Tetsch L, Bend J, Janssen M, et al. Evidence for functional laccases in the acidophilic ascomycete Hortaea acidophila and isolation of laccase-specific gene fragments[J]. FEMS Microbiology Letters, 2005, 245(1): 161-168.
[36] Nakade K, Watanabe H, Sakamoto Y, et al. Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence[J]. Microbiological Research. 2010.
[37] Pukkila-Worley R, Gerrald Q D, Kraus P R, et al. Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade[J]. Eukaryotic Cell, 2005, 4(1): 190-201.
[38] Tsuji G, Fujikawa J, Ishida H, et al. Laccase gene LAC1 of Colletotrichum lagenarium is not essential for melanin biosynthesis and pathogenicity[J]. Journal of General Plant Pathology, 2001, 67(3): 182-190.
[39]曹志艳.玉米大斑病菌黑色素合成途径相关基因的克隆及功能分析[D].保定:河北农业大学, 2009.
[40] Shin K S, Lee Y J. Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus[J]. Archives of Biochemistry and Biophysics, 2000, 384(1): 109-115.
[41]蔡磊,尹峻峰,杨丽萍,等.几种简便的木质素降解真菌定性筛选方法[J].微生物学通报, 2002, 29(1): 67-69.
[42] Edens W A, Goins T Q, Dooley D, et al. Purification and characterization of asecreted laccase of Gaeumannomyces graminis var. tritici[J]. Applied and Environmental Microbiology, 1999, 65(7): 3071-3074.
[43] Bashyal B, Chand R, Kushwaha C, et al. Association of melanin content with conidiogenesis in Bipolaris Sorokiniana of barley (Hordeum vulgare L.)[J]. World Journal of Microbiology and Biotechnology, 2010, 26(2): 309-316.
[44]曹志艳,董金皋,杨胜勇,等.玉米大斑病菌黑色素的一些理化性质和光谱吸收特征[J].植物病理学报, 2007, (04): 410-417.
[45] Money N P. Osmotic pressure of aqueous polyethylene glycols : Relationship between molecular weight and vapor pressure deficit[J]. Plant Physiolog, 1989, 91(2): 766-769.
[46] Money N P. Measurement of pore size in the hyphal cell wall of Achlya bisexualis[J]. Experimental Mycology, 1990, 14(3): 234-242.
[47] Lin J K, Ladisch M R, Patterson J A, et al. Determining pore size distribution in wet cellulose by measuring solute exclusion using a differential refractometer[J]. Biotechnology and Bioengineering, 1987, 29(8): 976-981.
[48] Howard R J, Ferrari M A, Roach D H, et al. Penetration of hard substrates by a fungus employing enormous turgor pressures[J]. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(24): 11281-11284.
[49]张利辉,刘云惠,董金皋,等.玉米大斑病菌特异性毒素组分的分离与纯化[J].植物病理学报, 2003, 1: 67-71.
[50] Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.
[51] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4): 406-425.
[52] Asakura M, Okuno T, Takano Y. Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium[J]. Applied and Environmental Microbiology, 2006, 72(9): 6345-6354.
[53] Takagaki M, Kaku K, Watanabe S, et al. Mechanism of resistance to carpropamid in Magnaporthe grisea[J]. Pest Management Science, 2004, 60(9): 921-926.
[54] Mayer A M, Staples R C. Laccase: new functions for an old enzyme[J]. Phytochemistry, 2002, 60(6): 551-565.
[55] Skalova T, Dohnalek J, Ostergaard L H, et al. The structure of the small laccase from Streptomyces coelicolor reveals a link between laccases and nitrite reductases[J]. Journal of Molecular Biology, 2009, 385(4): 1165-1178.
[56] Komori H, Miyazaki K, Higuchi Y. X-ray structure of a two-domain type laccase: a missing link in the evolution of multi-copper proteins[J]. FEBS Letters, 2009, 583(7): 1189-1195.
[57] Hajdok S, Conrad J, Leutbecher H, et al. The laccase-catalyzed domino reaction between catechols and heterocyclic 1,3-dicarbonyls and the unambiguous structure elucidation of the products by NMR spectroscopy and X-ray crystal structure analysis[J]. The Journal of Organic Chemistry, 2009, 74(19): 7230-7237.
[58] Jeon J R, Murugesan K, Kim Y M, et al. Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase[J]. Applied Microbiology and Biotechnology, 2008, 81(4): 783-790.
[59] Orzaez D, Granell A. Reverse genetics and transient gene expression in fleshy fruits: overcoming plant stable transformation[J]. Plant Signaling and Behavior, 2009, 4(9): 864-867.
[60] Bhadauria V, Banniza S, Wei Y, et al. Reverse genetics for functional genomics of phytopathogenic fungi and oomycetes[J]. Comparative and Functional Genomics, 2009, 380719.
[61] Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science, 1992, 257(5072): 967-971.
[62] Hubank M, Schatz D G. Identifying differences in mRNA expression by representational difference analysis of cDNA[J]. Nucleic Acids Research, 1994, 22(25): 5640-5648.
[63] Schena M, Shalon D, Davis R W, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray[J]. Science, 1995, 270(5235): 467-470.
[64] Ramsay G. DNA chips: state-of-the art[J]. Nature Biotechnology, 1998, 16(1):40-44.
[65] Velculescu V E, Zhang L, Vogelstein B, et al. Serial analysis of gene expression[J]. Science, 1995, 270(5235): 484-487.
[66] Bussey T J, Saksida L M, Rothblat L A. Discrimination of computer-graphic stimuli by mice: a method for the behavioral characterization of transgenic and gene-knockout models[J]. Behavioral Neuroscience, 2001, 115(4): 957-960.
[67] Ding C, Butler G. Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation[J]. Eukaryotic Cell, 2007, 6(8): 1310-1319.
[68]曹志艳.玉米大斑病菌黑色素性质与功能研究[D].保定:河北农业大学, 2006.
[69] Yan R, Hou J, Ding D, et al. In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi[J]. Journal of Basic Microbiology, 2008, 48(4): 293-301.
[70] Taborda C P, da Silva M B, Nosanchuk J D, et al. Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview[J]. Mycopathologia, 2008, 165(4-5): 331-339.
[71] Choi G H, Larson T G, Nuss D L. Molecular analysis of the laccase gene from thechestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain[J]. Molecular Plant-Microbe Interactions, 1992, 5(2): 119-128.
[72] Valeru S P, Rompikuntal P K, Ishikawa T, et al. Role of melanin pigment in expression of Vibrio cholerae virulence factors[J]. Infection and Immunity, 2009, 77(3): 935-942.
[73] Castro-Sowinski S, Martinez-Drets G, Okon Y. Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J]. FEMS Microbiology Letters, 2002, 209(1): 119-125.
[74] Pedras M S, Yu Y. Salt stress induces production of melanin related metabolites in the phytopathogenic fungus Leptosphaeria maculans[J]. Natural Product Communications, 2009, 4(1): 53-58.
[75] Giardina P, Cannio R, Martirani L, et al. Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus[J]. Applied and Environmental Microbiology, 1995, 61(6): 2408-2413.
[76] Otterbein L, Record E, Longhi S, et al. Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris[J]. Eur J Biochem, 2000, 267(6): 1619-1625.
[77] Guo M, Lu F, Pu J, et al. Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica[J]. Applied Microbiology and Biotechnology, 2005, 69(2): 178-183.
[78] Giardina P, Aurilia V, Cannio R, et al. The gene, protein and glycan structures of laccase from Pleurotus ostreatus[J]. European Journal of Biochemistry, 1996, 235(3): 508-515.
[79] Kumar S V, Phale P S, Durani S, et al. Combined sequence and structure analysis of the fungal laccase family[J]. Biotechnol Bioeng, 2003, 83(4): 386-394.
[80] Hilden K, Hakala T K, Maijala P, et al. Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus[J]. Applied Microbiology and Biotechnology, 2007, 77(2): 301-309.
[81] Hoegger P J, Navarro-Gonzalez M, Kilaru S, et al. The laccase gene family in Coprinopsis cinerea (Coprinus cinereus)[J]. Current Genetics, 2004, 45(1): 9-18.
[82] Shleev S V, Morozova O V, Nikitina O V, et al. Comparison of physico-chemical characteristics of four laccases from different basidiomycetes[J]. Biochimie, 2004, 86(9-10): 693-703.
[2] Polak A. Melanin as a virulence factor in pathogenic fungi[J]. Mycoses, 1990, 33(5): 215-224.
[3] Morris-Jones R, Gomez B L, Diez S, et al. Synthesis of melanin pigment by Candida albicans in vitro and during infection[J]. Infection and Immunity, 2005, 73(9): 6147-6150.
[4] Saparrat M C, Fermoselle G E, Stenglein S A, et al. Pseudocercospora griseola causing angular leaf spot on Phaseolus vulgaris produces 1,8-dihydroxynaphthalene-melanin[J]. Mycopathologia, 2009, 168(1): 41-47.
[5] Butler M J, Gardiner R B, Day A W. Melanin synthesis by Sclerotinia sclerotiorum[J]. Mycologia, 2009, 101(3): 296-304.
[6] Coppin E, Silar P. Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina[J]. Mycological Research, 2007, 111(8): 901-908.
[7] Thompson J E, Fahnestock S, Farrall L, et al. The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase[J]. The Journal of Biological Chemistry, 2000, 275(45): 34867-34872.
[8] Langfelder K, Streibel M, Jahn B, et al. Biosynthesis of fungal melanins and their importance for human pathogenic fungi[J]. Fungal Genetics and Biology, 2003, 38(2): 143-158.
[9] Eliahu N, Igbaria A, Rose M S, et al. Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, Chk1 and Mps1, and the transcription factor Cmr1[J]. Eukaryotic Cell, 2007, 6(3): 421-429.
[10] Kogej T, Wheeler M H, Lanisnik Rizner T, et al. Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions[J]. FEMS Microbiology Letters, 2004, 232(2): 203-209.
[11] Baldrian P. Fungal laccases-occurrence and properties[J]. FEMS Microbiology Reviews, 2006, 30(2): 215-242.
[12] Yoshi H. Chemistry of lacquer[J]. Journal of the Chemical Society, 1883, 43: 472-486.
[13] Durante D, Casadio R, Martelli L, et al. Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera[J]. Journal of Molecular Catalysis B: Enzymatic, 2004, 27(4-6): 191-206.
[14] Camarero S, García O, Vidal T, et al. Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system[J]. Enzyme and Microbial Technology,2004, 35(2-3): 113-120.
[15] Michizoe J, Ichinose H, Kamiya N, et al. Biodegradation of phenolic environmental pollutants by a surfactant-laccase complex in organic media[J]. Journal of Bioscience and Bioengineering, 2005, 99(6): 642-647.
[16] Quaratino D, Federici F, Petruccioli M, et al. Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79[J]. Antonie Van Leeuwenhoek, 2007, 91(1): 57-69.
[17] Arica M Y, Altintas B, Bayramoglu G. Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation[J]. Bioresource Technology, 2009, 100(2): 665-669.
[18] Uhnakova B, Petrickova A, Biedermann D, et al. Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor[J]. Chemosphere, 2009, 76(6): 826-832.
[19] Saha B, Taylor K E, Bewtra J K, et al. Laccase-catalyzed removal of diphenylamine from synthetic wastewater[J]. Water Environment Research, 2008, 80(11): 2118-2124.
[20] Widsten P, Kandelbauer A. Laccase applications in the forest products industry: A review[J]. Enzyme and Microbial Technology, 2008, 42(4): 293-307.
[21] Johannes C, Majcherczyk A. Laccase activity tests and laccase inhibitors[J]. Journal of Biotechnology, 2000, 78(2): 193-199.
[22] Niladevi K N, Prema P. Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization[J]. Bioresource Technology, 2008, 99(11): 4583-4589.
[23]黄乾明.漆酶高产菌株的诱变选育及其酶的分离纯化、性质和基因克隆研究[D].雅安:四川农业大学; 2006.
[24]张敏,肖亚中,蒲春雷,等.白腐真菌AH28-2菌株发酵合成漆酶初步研究[J].微生物学通报, 2002, 29(3): 37-42.
[25] Sole M, Kellner H, Brock S, et al. Extracellular laccase activity and transcript levels of putative laccase genes during removal of the xenoestrogen technical nonylphenol by the aquatic hyphomycete Clavariopsis aquatica[J]. FEMS Microbiology Letters, 2008, 288(1): 47-54.
[26] Cusano A M, Mekmouche Y, Meglecz E, et al. Plasticity of laccase generated by homeologous recombination in yeast[J]. FEBS Journal, 2009, 276(19): 5471-5480.
[27] De Vries O M H, Kooistra W H C F, Wessels J G H. Formation of an Extracellular Laccase by a Schizophyllum commune Dikaryon[J]. Journal of General Microbiology, 1986, 132(10): 2817-2826.
[28] Coll P M, Fernandez-Abalos J M, Villanueva J R, et al. Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971)[J]. Applied and Environmental Microbiology, 1993, 59(8):2607-2613.
[29] Pihet M, Vandeputte P, Tronchin G, et al. Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia[J]. BMC Microbiology, 2009, 9: 177.
[30]詹旭,曹志艳,邢继红,等.植物病原真菌产漆酶菌株的筛选[J].中国农业科学, 2011, 44(4): 723-729.
[31] Choi G H, Larson T G, Nuss D L. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain[J]. Molecular Plant-Microbe Interactions, 1992, 5(2): 119-128.
[32] Castro-Sowinski S, Martinez-Drets G, Okon Y. Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J]. FEMS Microbiology Letters, 2002, 209(1): 119-125.
[33] Zhu X, Gibbons J, Zhang S, et al. Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans[J]. Molecular Microbiology, 2003, 47(4): 1007-1014.
[34] Bohlin C, Jonsson L J, Roth R, et al. Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger[J]. Applied Biochemistry and Biotechnology, 2006, 129-132: 195-214.
[35] Tetsch L, Bend J, Janssen M, et al. Evidence for functional laccases in the acidophilic ascomycete Hortaea acidophila and isolation of laccase-specific gene fragments[J]. FEMS Microbiology Letters, 2005, 245(1): 161-168.
[36] Nakade K, Watanabe H, Sakamoto Y, et al. Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence[J]. Microbiological Research. 2010.
[37] Pukkila-Worley R, Gerrald Q D, Kraus P R, et al. Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade[J]. Eukaryotic Cell, 2005, 4(1): 190-201.
[38] Tsuji G, Fujikawa J, Ishida H, et al. Laccase gene LAC1 of Colletotrichum lagenarium is not essential for melanin biosynthesis and pathogenicity[J]. Journal of General Plant Pathology, 2001, 67(3): 182-190.
[39]曹志艳.玉米大斑病菌黑色素合成途径相关基因的克隆及功能分析[D].保定:河北农业大学, 2009.
[40] Shin K S, Lee Y J. Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus[J]. Archives of Biochemistry and Biophysics, 2000, 384(1): 109-115.
[41]蔡磊,尹峻峰,杨丽萍,等.几种简便的木质素降解真菌定性筛选方法[J].微生物学通报, 2002, 29(1): 67-69.
[42] Edens W A, Goins T Q, Dooley D, et al. Purification and characterization of asecreted laccase of Gaeumannomyces graminis var. tritici[J]. Applied and Environmental Microbiology, 1999, 65(7): 3071-3074.
[43] Bashyal B, Chand R, Kushwaha C, et al. Association of melanin content with conidiogenesis in Bipolaris Sorokiniana of barley (Hordeum vulgare L.)[J]. World Journal of Microbiology and Biotechnology, 2010, 26(2): 309-316.
[44]曹志艳,董金皋,杨胜勇,等.玉米大斑病菌黑色素的一些理化性质和光谱吸收特征[J].植物病理学报, 2007, (04): 410-417.
[45] Money N P. Osmotic pressure of aqueous polyethylene glycols : Relationship between molecular weight and vapor pressure deficit[J]. Plant Physiolog, 1989, 91(2): 766-769.
[46] Money N P. Measurement of pore size in the hyphal cell wall of Achlya bisexualis[J]. Experimental Mycology, 1990, 14(3): 234-242.
[47] Lin J K, Ladisch M R, Patterson J A, et al. Determining pore size distribution in wet cellulose by measuring solute exclusion using a differential refractometer[J]. Biotechnology and Bioengineering, 1987, 29(8): 976-981.
[48] Howard R J, Ferrari M A, Roach D H, et al. Penetration of hard substrates by a fungus employing enormous turgor pressures[J]. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(24): 11281-11284.
[49]张利辉,刘云惠,董金皋,等.玉米大斑病菌特异性毒素组分的分离与纯化[J].植物病理学报, 2003, 1: 67-71.
[50] Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.
[51] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4): 406-425.
[52] Asakura M, Okuno T, Takano Y. Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium[J]. Applied and Environmental Microbiology, 2006, 72(9): 6345-6354.
[53] Takagaki M, Kaku K, Watanabe S, et al. Mechanism of resistance to carpropamid in Magnaporthe grisea[J]. Pest Management Science, 2004, 60(9): 921-926.
[54] Mayer A M, Staples R C. Laccase: new functions for an old enzyme[J]. Phytochemistry, 2002, 60(6): 551-565.
[55] Skalova T, Dohnalek J, Ostergaard L H, et al. The structure of the small laccase from Streptomyces coelicolor reveals a link between laccases and nitrite reductases[J]. Journal of Molecular Biology, 2009, 385(4): 1165-1178.
[56] Komori H, Miyazaki K, Higuchi Y. X-ray structure of a two-domain type laccase: a missing link in the evolution of multi-copper proteins[J]. FEBS Letters, 2009, 583(7): 1189-1195.
[57] Hajdok S, Conrad J, Leutbecher H, et al. The laccase-catalyzed domino reaction between catechols and heterocyclic 1,3-dicarbonyls and the unambiguous structure elucidation of the products by NMR spectroscopy and X-ray crystal structure analysis[J]. The Journal of Organic Chemistry, 2009, 74(19): 7230-7237.
[58] Jeon J R, Murugesan K, Kim Y M, et al. Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase[J]. Applied Microbiology and Biotechnology, 2008, 81(4): 783-790.
[59] Orzaez D, Granell A. Reverse genetics and transient gene expression in fleshy fruits: overcoming plant stable transformation[J]. Plant Signaling and Behavior, 2009, 4(9): 864-867.
[60] Bhadauria V, Banniza S, Wei Y, et al. Reverse genetics for functional genomics of phytopathogenic fungi and oomycetes[J]. Comparative and Functional Genomics, 2009, 380719.
[61] Liang P, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science, 1992, 257(5072): 967-971.
[62] Hubank M, Schatz D G. Identifying differences in mRNA expression by representational difference analysis of cDNA[J]. Nucleic Acids Research, 1994, 22(25): 5640-5648.
[63] Schena M, Shalon D, Davis R W, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray[J]. Science, 1995, 270(5235): 467-470.
[64] Ramsay G. DNA chips: state-of-the art[J]. Nature Biotechnology, 1998, 16(1):40-44.
[65] Velculescu V E, Zhang L, Vogelstein B, et al. Serial analysis of gene expression[J]. Science, 1995, 270(5235): 484-487.
[66] Bussey T J, Saksida L M, Rothblat L A. Discrimination of computer-graphic stimuli by mice: a method for the behavioral characterization of transgenic and gene-knockout models[J]. Behavioral Neuroscience, 2001, 115(4): 957-960.
[67] Ding C, Butler G. Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation[J]. Eukaryotic Cell, 2007, 6(8): 1310-1319.
[68]曹志艳.玉米大斑病菌黑色素性质与功能研究[D].保定:河北农业大学, 2006.
[69] Yan R, Hou J, Ding D, et al. In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi[J]. Journal of Basic Microbiology, 2008, 48(4): 293-301.
[70] Taborda C P, da Silva M B, Nosanchuk J D, et al. Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview[J]. Mycopathologia, 2008, 165(4-5): 331-339.
[71] Choi G H, Larson T G, Nuss D L. Molecular analysis of the laccase gene from thechestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain[J]. Molecular Plant-Microbe Interactions, 1992, 5(2): 119-128.
[72] Valeru S P, Rompikuntal P K, Ishikawa T, et al. Role of melanin pigment in expression of Vibrio cholerae virulence factors[J]. Infection and Immunity, 2009, 77(3): 935-942.
[73] Castro-Sowinski S, Martinez-Drets G, Okon Y. Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J]. FEMS Microbiology Letters, 2002, 209(1): 119-125.
[74] Pedras M S, Yu Y. Salt stress induces production of melanin related metabolites in the phytopathogenic fungus Leptosphaeria maculans[J]. Natural Product Communications, 2009, 4(1): 53-58.
[75] Giardina P, Cannio R, Martirani L, et al. Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus[J]. Applied and Environmental Microbiology, 1995, 61(6): 2408-2413.
[76] Otterbein L, Record E, Longhi S, et al. Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris[J]. Eur J Biochem, 2000, 267(6): 1619-1625.
[77] Guo M, Lu F, Pu J, et al. Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica[J]. Applied Microbiology and Biotechnology, 2005, 69(2): 178-183.
[78] Giardina P, Aurilia V, Cannio R, et al. The gene, protein and glycan structures of laccase from Pleurotus ostreatus[J]. European Journal of Biochemistry, 1996, 235(3): 508-515.
[79] Kumar S V, Phale P S, Durani S, et al. Combined sequence and structure analysis of the fungal laccase family[J]. Biotechnol Bioeng, 2003, 83(4): 386-394.
[80] Hilden K, Hakala T K, Maijala P, et al. Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus[J]. Applied Microbiology and Biotechnology, 2007, 77(2): 301-309.
[81] Hoegger P J, Navarro-Gonzalez M, Kilaru S, et al. The laccase gene family in Coprinopsis cinerea (Coprinus cinereus)[J]. Current Genetics, 2004, 45(1): 9-18.
[82] Shleev S V, Morozova O V, Nikitina O V, et al. Comparison of physico-chemical characteristics of four laccases from different basidiomycetes[J]. Biochimie, 2004, 86(9-10): 693-703.