玉米大斑病菌StPKS基因与黑色素合成及致病性的关系研究
|
摘要
本试验前期克隆获得了1个玉米大斑病菌聚酮体合成酶基因(StPKS),为了明确该基因在玉米大斑病菌DHN黑色素合成以及病菌致病性中的作用,本研究利用RNA干扰(RNA interference,RNAi)和基因敲除(Gene knock-out)两种方法分别创制StPKS基因沉默转化子和基因缺失突变体,通过对突变体进行分析,明确了该基因的功能,主要研究结果如下:
以pSilent-1质粒和pBU质粒为载体,分别构建了StPKS基因的RNAi和同源重组载体,采用聚乙二醇(PEG)介导的原生质体转化方法,经潮霉素筛选、PCR及RT-PCR鉴定得到了5株StPKS基因表达量下降的RNAi转化子和1株基因缺失突变体。
与野生型相比,5株RNAi转化子的菌丝形态很不规则,出现膨大、分枝、变形等表型变化,菌落颜色均趋向于白色,黑色素含量明显低于野生型;基因缺失突变菌株ΔStPKS不产生分生孢子,菌丝细胞壁透明,分隔不明显,细胞内部可见许多液泡状结构,菌落颜色为白色,黑色素含量明显降低,与RNAi转化子的表型相似,表明StPKS基因的编码产物与玉米大斑病菌DHN黑色素的生物合成、菌丝发育及分生孢子形成密切相关。
对基因缺失突变菌株ΔStPKS的致病相关因子进行分析发现,野生型菌株及StPKS基因缺失突变体的附着胞膨压分别为5.4 MPa、4.6 MPa,ΔStPKS的附着胞形成时间推迟,产生及穿透玻璃纸的附着胞数量明显减少,该结果表明黑色素缺失突变体的附着胞形成受抑制,膨压有所下降,并且影响了病原真菌的穿透能力;另外,研究结果还发现,StPKS基因缺失并不影响玉米大斑病菌毒素活性,但可降低纤维素酶活性,即黑色素缺失突变菌株的侵染能力可能会大大下降。
A polyketide synthase gene of Setosphaeria turcica was cloned in our laboratory at the earlier research. In order to confirm the function of StPKS gene in DHN melanin biosynthesis and pathogenicity, RNA interference and gene knock-out techniques were used to construct StPKS gene silent transformants and deleted mutants. The founction of the StPKS gene was identified by analyzing the mutant, the main results were as follows: The transformation vectors pSilent-1 and pBU, were used to construct StPKS RNAi and homologous recombination plasmids. Two vectors were transformed into S. turcica protoplasts through PEG-mediated transformation. Five StPKS down-regulated transformants and one disrupted mutant were obtained by screening of hygromycin B, PCR and RT-PCR.
Comparing with the wild-type isolate, the hypae morphology of five RNAi transformants was anomalistic, including intumescent, ramose and anamorphic. The colony colour was tend to be white and melanin production was significantly declined. The deletion mutantΔStPKS didn’t produce conidia. Cell wall was transparent and the hypha had no distinct septum but had lots of vacuole-like structures in cells.ΔStPKS showed an albino phenotype, the melanin content was reduced remarkably, these consistented with the results of RNAi transformants. These results indicated that the encoding products of StPKS gene was involved in DHN melanin biosynthesis, mycelial development and conidia formation.
The research of pathogenic factors ofΔStPKS revealed that, wild type isolate and StPKS mutant had 5.4 MPa、4.6 MPa turgor pressure respectively. Appressorium formation ofΔStPKS was put off, the numbers of appressorium and penetration were decreased. The results showed that appressorium formation of the melanin-deficient mutants was inhibited, the low turgor pressure would affect the ability of penetration. Targeted disruption of the StPKS gene showed no affection about toxin activity, but reduced the cellulose activity, this would influenced the pathogenicity of melanin-deficient mutant.
以pSilent-1质粒和pBU质粒为载体,分别构建了StPKS基因的RNAi和同源重组载体,采用聚乙二醇(PEG)介导的原生质体转化方法,经潮霉素筛选、PCR及RT-PCR鉴定得到了5株StPKS基因表达量下降的RNAi转化子和1株基因缺失突变体。
与野生型相比,5株RNAi转化子的菌丝形态很不规则,出现膨大、分枝、变形等表型变化,菌落颜色均趋向于白色,黑色素含量明显低于野生型;基因缺失突变菌株ΔStPKS不产生分生孢子,菌丝细胞壁透明,分隔不明显,细胞内部可见许多液泡状结构,菌落颜色为白色,黑色素含量明显降低,与RNAi转化子的表型相似,表明StPKS基因的编码产物与玉米大斑病菌DHN黑色素的生物合成、菌丝发育及分生孢子形成密切相关。
对基因缺失突变菌株ΔStPKS的致病相关因子进行分析发现,野生型菌株及StPKS基因缺失突变体的附着胞膨压分别为5.4 MPa、4.6 MPa,ΔStPKS的附着胞形成时间推迟,产生及穿透玻璃纸的附着胞数量明显减少,该结果表明黑色素缺失突变体的附着胞形成受抑制,膨压有所下降,并且影响了病原真菌的穿透能力;另外,研究结果还发现,StPKS基因缺失并不影响玉米大斑病菌毒素活性,但可降低纤维素酶活性,即黑色素缺失突变菌株的侵染能力可能会大大下降。
A polyketide synthase gene of Setosphaeria turcica was cloned in our laboratory at the earlier research. In order to confirm the function of StPKS gene in DHN melanin biosynthesis and pathogenicity, RNA interference and gene knock-out techniques were used to construct StPKS gene silent transformants and deleted mutants. The founction of the StPKS gene was identified by analyzing the mutant, the main results were as follows: The transformation vectors pSilent-1 and pBU, were used to construct StPKS RNAi and homologous recombination plasmids. Two vectors were transformed into S. turcica protoplasts through PEG-mediated transformation. Five StPKS down-regulated transformants and one disrupted mutant were obtained by screening of hygromycin B, PCR and RT-PCR.
Comparing with the wild-type isolate, the hypae morphology of five RNAi transformants was anomalistic, including intumescent, ramose and anamorphic. The colony colour was tend to be white and melanin production was significantly declined. The deletion mutantΔStPKS didn’t produce conidia. Cell wall was transparent and the hypha had no distinct septum but had lots of vacuole-like structures in cells.ΔStPKS showed an albino phenotype, the melanin content was reduced remarkably, these consistented with the results of RNAi transformants. These results indicated that the encoding products of StPKS gene was involved in DHN melanin biosynthesis, mycelial development and conidia formation.
The research of pathogenic factors ofΔStPKS revealed that, wild type isolate and StPKS mutant had 5.4 MPa、4.6 MPa turgor pressure respectively. Appressorium formation ofΔStPKS was put off, the numbers of appressorium and penetration were decreased. The results showed that appressorium formation of the melanin-deficient mutants was inhibited, the low turgor pressure would affect the ability of penetration. Targeted disruption of the StPKS gene showed no affection about toxin activity, but reduced the cellulose activity, this would influenced the pathogenicity of melanin-deficient mutant.
引文
[1]王会伟,李洪杰,朱振东,等. Ht2背景下玉米对大斑病菌1号小种抗性基因的表达差异研究[J].植物病理学报, 2010, 2: 135-143.
[2]王晓鸣,晋齐鸣,石洁,等.玉米病害发生现状与推广品种抗性对未来病害发展的影响[J].植物病理学报, 2006, 1: 1-11.
[3]范永山,谷守芹,董金皋,等. MAPK途径对玉米大斑病菌HT-毒素产生和生物学活性的调控作用[J].中国农业科学, 2008, 1: 86-92.
[4]曹志艳,范永山,董金皋,等.玉米大斑病菌黑色素缺失突变体的获得及其生物学特性[J].植物保护学报, 2007, 3: 268-272.
[5]浦子钢.玉米抗大斑病育种研究现状[J].黑龙江农业科学, 2010, 11: 129-132.
[6] Zheng W, Zhao Z, Chen J, et al. A Cdc42 ortholog is required for penetration and virulence of Magnaporthe grisea[J]. Fungal Genetics and Biology, 2009, 46(6-7): 450-460.
[7] Walker C A, Gomez B L, Mora-Montes H M, et al. Melanin externalization in Candida albicans depends on cell wall chitin structures[J]. Eukaryotic Cell, 2010, 9(9): 1329-1342.
[8] da Silva M B, Marques A F, Nosanchuk J D, et al. Melanin in the dimorphic fungal pathogen Paracoccidioides brasiliensis: effects on phagocytosis, intracellular resistance and drug susceptibility[J]. Microbes and Infection, 2006, 8(1): 197-205.
[9] Gomez B L, Nosanchuk J D, Diez S, et al. Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection[J]. Infection and Immunity, 2001, 69(9): 5760-5767.
[10] Nosanchuk J D, Gomez B L, Youngchim S, et al. Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection[J]. Infection and Immunity, 2002, 70(9): 5124-5131.
[11] Nosanchuk J D, van Duin D, Mandal P, et al. Blastomyces dermatitidis produces melanin in vitro and during infection[J]. FEMS Microbiology Letters, 2004, 239(1): 187-193.
[12] Nosanchuk J D, Yu J J, Hung C Y, et al. Coccidioides posadasii produces melanin in vitro and during infection[J]. Fungal Genetics and Biology, 2007, 44(6): 517-520.
[13] Nurudeen T A, Ahearn D G. Regulation of melanin production by Cryptococcus neoformans[J]. Journal of Clinical Microbiology, 1979, 10(5): 724-729.
[14] Taborda C P, 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.
[15] Kihara J, Moriwaki A, Tanaka N, et al. Characterization of the BMR1 gene encoding a transcription factor for melanin biosynthesis genes in the phytopathogenic fungus Bipolaris oryzae[J]. FEMS Microbiology Letters, 2008, 281(2): 221-227.
[16] Langfelder K, Jahn B, Gehringer H, et al. Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence[J]. Medical Microbiology and Immunology, 1998, 187(2): 79-89.
[17] Sawada H, Sugihara M, Takagaki M, et al. Monitoring and characterization of Magnaporthe grisea isolates with decreased sensitivity to scytalone dehydratase inhibitors[J]. Pest Management Science, 2004, 60(8): 777-785.
[18] 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.
[19] 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.
[20] 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.
[21] Jackson J C, Higgins L A, Lin X Conidiation color mutants of Aspergillus fumigatus are highly pathogenic to the heterologous insect host Galleria mellonella[J]. PLoS One, 2009, 4(1): e4224.
[22] Ikehata K, Buchanan I D, Pickard M A, et al. Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment[J]. Bioresource Technology, 2005, 96(16): 1758-1770.
[23] 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.
[24] Nosanchuk J D, Casadevall A. The contribution of melanin to microbial pathogenesis[J]. Cellular Microbiology, 2003, 5(4): 203-223.
[25] Jacobson E S. Pathogenic Roles for Fungal Melanins[J]. Clinical Microbiology Reviews, 2000, 13(4): 708-717.
[26] 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.
[27]王洪凯,林福呈,王政逸.植物病原真菌附着胞的机械穿透力[J].菌物学报, 2004, 1: 151-157.
[28] Lagunas-Munoz V H, Cabrera-Valladares N, Bolivar F, et al. Optimum melanin production using recombinant Escherichia coli[J]. Journal of Applied Microbiology, 2006, 101(5): 1002-1008.
[29] Gachomo E W, Seufferheld M J, Kotchoni S O. Melanization of appressoria is critical for the pathogenicity of Diplocarpon rosae[J]. Molecular Biology Reports, 2010, 37(7): 3583-3591.
[30] Zhang C Q, Zhu G N, Ma Z H, et al. Isolation, characterization and preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast fungus, Magnaporthe grisea[J]. Journal of Phytopathology, 2006, 154(7-8): 392-397.
[31] Parisot D, Dufresne M, Veneault C, et al. clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum[J]. Molecular Genetics and Genomics, 2002, 268(2): 139-151.
[32] 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.
[33] Singaravelan N, Grishkan I, Beharav A, et al. Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at "Evolution Canyon", Mount Carmel, Israel[J]. PLoS One, 2008, 3(8): e2993.
[34]陈赟娟,韦建福,等.丝状真菌遗传转化的研究进展[J].云南农业大学学报, 2009, 3: 448-454.
[35] Turgeon B G, Condon B, Liu J, et al. Protoplast transformation of filamentous fungi[J]. Methods in Molecular Biology, 2010, 638: 3-19.
[36] Lee M J, Duong C T, Han K, et al. Combination strategy to increase cyclosporin A productivity byTolypocladium niveum using random mutagenesis and protoplast transformation[J]. Journal of Microbiology and Biotechnology, 2009, 19(9): 869-872.
[37]张学炜,王笑梅,李明春,等.以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化[J].生物工程学报, 2007, 23(3): 462-466.
[38]周永力, Chihiro T, Satoshi M,等.玉米大斑病菌(Exserohilum turcicum)原生质体的分离及转化[J].生物工程学报, 2003, 19(3): 364-367.
[39] de Vries R P, Burgers K, van de Vondervoort P J, et al. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production[J]. Applied and Environmental Microbiology, 2004, 70(7): 3954-3959.
[40] Dlugonski J, Sedlaczek L, Jaworski A. Protoplast release from fungi capable of steroid transformation[J]. Canadian Journal of Microbiology, 1984, 30(1): 57-62.
[41] Liu X H, Lu J P, Wang J Y, et al. Promoter trapping in Magnaporthe grisea[J]. Zhejiang University SCIENCE B, 2006, 7(1): 28-33.
[42] Koukaki M, Giannoutsou E, Karagouni A, et al. A novel improved method for Aspergillus nidulans transformation[J]. Journal of Microbiological Methods, 2003, 55(3): 687-695.
[43]田媛,苑社强,袁耀武,等.康氏木霉原生质体制备及再生条件研究[J].中国酿造, 2010, 7: 75-79.
[44]刘玲,叶博,刘长江,等.白腐真菌原生质体制备和再生条件的研究[J].生物技术, 2006, 5: 41-44.
[45]宋庆涛,张国珍,董金皋,等.玉米大斑病菌原生质体的制备与再生[J].微生物学通报, 2003, 2: 41-44.
[46] Graziani S, Vasnier C, Daboussi M J. Novel Polyketide Synthase from Nectria haematococca[J]. Applied and Environmental Microbiology, 2004, 70(5): 2984-2988.
[47] Zhang A, Lu P, Dahl-Roshak A M, et al. Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis[J]. Molecular Genetics and Genomics, 2003, 268(5): 645-655.
[48] Askenazi M, Driggers E M, Holtzman D A, et al. Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains[J]. Nature Biotechnology, 2003, 21(2): 150-156.
[49] Campbell C D, Vederas J C. Biosynthesis of lovastatin and related metabolites formed by fungal iterative PKS enzymes[J]. Biopolymers, 2010, 93(9): 755-763.
[50] Yang G, Rose M S, Turgeon B G, et al. A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin[J]. Plant Cell, 1996, 8(11): 2139-2150.
[51] Shimizu T, Kinoshita H, Ishihara S, et al. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus[J]. Applied and Environmental Microbiology, 2005, 71(7): 3453-3457.
[52] Yu J H, Leonard T J. Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase[J]. Journal of Bacteriology, 1995, 177(16): 4792-4800.
[53] Gerber R, Lou L, Du L. A PLP-dependent polyketide chain releasing mechanism in the biosynthesis of mycotoxin fumonisins in Fusarium verticillioides[J]. Journal of the American Chemical Society, 2009, 131(9): 3148-3149.
[54] Moriwaki A, Kihara J, Kobayashi T, et al. Insertional mutagenesis and characterization of a polyketide synthase gene (PKS1) required for melanin biosynthesis in Bipolaris oryzae[J]. FEMS Microbiology Letters, 2004, 238(1): 1-8.
[55] Akamatsu H O, Chilvers M I, Stewart J E, et al. Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei[J]. Current Genetics, 2010, 56(4): 349-360.
[56] Paolo W F, Jr., Dadachova E, Mandal P, et al. Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature[J]. BMC Microbiology, 2006, 6: 55.
[57] Perpetua N S, Kubo Y, Yasuda N, et al. Cloning and characterization of a melanin biosynthetic THR1 reductase gene essential for appressorial penetration of Colletotrichum lagenarium[J]. Molecular Plant-Microbe Interactions, 1996, 9(5): 323-329.
[58] Tsai H F, Chang Y C, Washburn R G, et al. The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence[J]. Journal of Bacteriology, 1998, 180(12): 3031-3038.
[59] Tanguay P, Loppnau P, Morin C, et al. A spontaneous albino mutant of Ceratocystis resinifera results from a point mutation in the polyketide synthase gene, PKS1[J]. Canadian Journal of Microbiology, 2006, 52(6): 501-507.
[60]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, 4: 410-417.
[61]安鑫龙,董金皋,韩建民,等.玉米大斑病菌的RAPD分析Ⅰ.应用CTAB法提取玉米大斑病菌DNA[J].河北农业大学学报, 2001, 1: 38-41.
[62] Kihara J, Moriwaki A, Ueno M, et al. Cloning, functional analysis and expression of a scytalone dehydratase gene ( SCD1) involved in melanin biosynthesis of the phytopathogenic fungus Bipolaris oryzae[J]. Current Genetics, 2004, 45(4): 197-204.
[63] Bashyal B M, 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, 2009, 26(2): 309-316.
[64]曹志艳,贾慧,董金皋,等. DHN黑色素与玉米大斑病菌附着胞膨压形成的关系[J].中国农业科学, 2011, 44(5): 925-932.
[65]翟晖.玉米鞘腐病病原鉴定与致病机制研究[D].保定:河北农业大学, 2010.
[66] Tokuoka M, Seshime Y, Fujii I, et al. Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae[J]. Fungal Genetics and Biology, 2008, 45(12): 1608-1615.
[67] Royer M, Costet L, Vivien E, et al. Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes[J]. Molecular Plant-Microbe Interactions, 2004, 17(4): 414-427.
[68] de Jong J C, McCormack B J, Smirnoff N, et al. Glycerol generates turgor in rice blast[J]. Nature, 1997, 389(6648): 244-244.
[69] 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.
[70] Howard R J, Valent B. Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea[J]. Annual Review of Microbiology, 1996, 50: 491-512.
[71] Kawamura C, Moriwaki J, Kimura N, et al. The melanin biosynthesis genes of Alternariaalternata can restore pathogenicity of the melanin-deficient mutants of Magnaporthe grisea[J]. Molecular Plant-Microbe Interactions, 1997, 10(4): 446-453.
[72] Money N P, Howard R J. Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement[J]. Fungal Genetics and Biology, 1996, 20(3): 217-227.
[73] Chen Z, Nunes M A, Silva M C, et al. Appressorium turgor pressure of Colletotrichum kahawae might have a role in coffee cuticle penetration[J]. Mycologia, 2004, 96(6): 1199-1208.
[74] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391(6669): 806-811.
[75] Novina C D, Sharp P A. The RNAi revolution[J]. Nature, 2004, 430(6996): 161-164.
[76] Stevenson M. Therapeutic potential of RNA interference[J]. The New England Journal of Medicine, 2004, 351(17): 1772-1777.
[2]王晓鸣,晋齐鸣,石洁,等.玉米病害发生现状与推广品种抗性对未来病害发展的影响[J].植物病理学报, 2006, 1: 1-11.
[3]范永山,谷守芹,董金皋,等. MAPK途径对玉米大斑病菌HT-毒素产生和生物学活性的调控作用[J].中国农业科学, 2008, 1: 86-92.
[4]曹志艳,范永山,董金皋,等.玉米大斑病菌黑色素缺失突变体的获得及其生物学特性[J].植物保护学报, 2007, 3: 268-272.
[5]浦子钢.玉米抗大斑病育种研究现状[J].黑龙江农业科学, 2010, 11: 129-132.
[6] Zheng W, Zhao Z, Chen J, et al. A Cdc42 ortholog is required for penetration and virulence of Magnaporthe grisea[J]. Fungal Genetics and Biology, 2009, 46(6-7): 450-460.
[7] Walker C A, Gomez B L, Mora-Montes H M, et al. Melanin externalization in Candida albicans depends on cell wall chitin structures[J]. Eukaryotic Cell, 2010, 9(9): 1329-1342.
[8] da Silva M B, Marques A F, Nosanchuk J D, et al. Melanin in the dimorphic fungal pathogen Paracoccidioides brasiliensis: effects on phagocytosis, intracellular resistance and drug susceptibility[J]. Microbes and Infection, 2006, 8(1): 197-205.
[9] Gomez B L, Nosanchuk J D, Diez S, et al. Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection[J]. Infection and Immunity, 2001, 69(9): 5760-5767.
[10] Nosanchuk J D, Gomez B L, Youngchim S, et al. Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection[J]. Infection and Immunity, 2002, 70(9): 5124-5131.
[11] Nosanchuk J D, van Duin D, Mandal P, et al. Blastomyces dermatitidis produces melanin in vitro and during infection[J]. FEMS Microbiology Letters, 2004, 239(1): 187-193.
[12] Nosanchuk J D, Yu J J, Hung C Y, et al. Coccidioides posadasii produces melanin in vitro and during infection[J]. Fungal Genetics and Biology, 2007, 44(6): 517-520.
[13] Nurudeen T A, Ahearn D G. Regulation of melanin production by Cryptococcus neoformans[J]. Journal of Clinical Microbiology, 1979, 10(5): 724-729.
[14] Taborda C P, 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.
[15] Kihara J, Moriwaki A, Tanaka N, et al. Characterization of the BMR1 gene encoding a transcription factor for melanin biosynthesis genes in the phytopathogenic fungus Bipolaris oryzae[J]. FEMS Microbiology Letters, 2008, 281(2): 221-227.
[16] Langfelder K, Jahn B, Gehringer H, et al. Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence[J]. Medical Microbiology and Immunology, 1998, 187(2): 79-89.
[17] Sawada H, Sugihara M, Takagaki M, et al. Monitoring and characterization of Magnaporthe grisea isolates with decreased sensitivity to scytalone dehydratase inhibitors[J]. Pest Management Science, 2004, 60(8): 777-785.
[18] 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.
[19] 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.
[20] 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.
[21] Jackson J C, Higgins L A, Lin X Conidiation color mutants of Aspergillus fumigatus are highly pathogenic to the heterologous insect host Galleria mellonella[J]. PLoS One, 2009, 4(1): e4224.
[22] Ikehata K, Buchanan I D, Pickard M A, et al. Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment[J]. Bioresource Technology, 2005, 96(16): 1758-1770.
[23] 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.
[24] Nosanchuk J D, Casadevall A. The contribution of melanin to microbial pathogenesis[J]. Cellular Microbiology, 2003, 5(4): 203-223.
[25] Jacobson E S. Pathogenic Roles for Fungal Melanins[J]. Clinical Microbiology Reviews, 2000, 13(4): 708-717.
[26] 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.
[27]王洪凯,林福呈,王政逸.植物病原真菌附着胞的机械穿透力[J].菌物学报, 2004, 1: 151-157.
[28] Lagunas-Munoz V H, Cabrera-Valladares N, Bolivar F, et al. Optimum melanin production using recombinant Escherichia coli[J]. Journal of Applied Microbiology, 2006, 101(5): 1002-1008.
[29] Gachomo E W, Seufferheld M J, Kotchoni S O. Melanization of appressoria is critical for the pathogenicity of Diplocarpon rosae[J]. Molecular Biology Reports, 2010, 37(7): 3583-3591.
[30] Zhang C Q, Zhu G N, Ma Z H, et al. Isolation, characterization and preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast fungus, Magnaporthe grisea[J]. Journal of Phytopathology, 2006, 154(7-8): 392-397.
[31] Parisot D, Dufresne M, Veneault C, et al. clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum[J]. Molecular Genetics and Genomics, 2002, 268(2): 139-151.
[32] 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.
[33] Singaravelan N, Grishkan I, Beharav A, et al. Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at "Evolution Canyon", Mount Carmel, Israel[J]. PLoS One, 2008, 3(8): e2993.
[34]陈赟娟,韦建福,等.丝状真菌遗传转化的研究进展[J].云南农业大学学报, 2009, 3: 448-454.
[35] Turgeon B G, Condon B, Liu J, et al. Protoplast transformation of filamentous fungi[J]. Methods in Molecular Biology, 2010, 638: 3-19.
[36] Lee M J, Duong C T, Han K, et al. Combination strategy to increase cyclosporin A productivity byTolypocladium niveum using random mutagenesis and protoplast transformation[J]. Journal of Microbiology and Biotechnology, 2009, 19(9): 869-872.
[37]张学炜,王笑梅,李明春,等.以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化[J].生物工程学报, 2007, 23(3): 462-466.
[38]周永力, Chihiro T, Satoshi M,等.玉米大斑病菌(Exserohilum turcicum)原生质体的分离及转化[J].生物工程学报, 2003, 19(3): 364-367.
[39] de Vries R P, Burgers K, van de Vondervoort P J, et al. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production[J]. Applied and Environmental Microbiology, 2004, 70(7): 3954-3959.
[40] Dlugonski J, Sedlaczek L, Jaworski A. Protoplast release from fungi capable of steroid transformation[J]. Canadian Journal of Microbiology, 1984, 30(1): 57-62.
[41] Liu X H, Lu J P, Wang J Y, et al. Promoter trapping in Magnaporthe grisea[J]. Zhejiang University SCIENCE B, 2006, 7(1): 28-33.
[42] Koukaki M, Giannoutsou E, Karagouni A, et al. A novel improved method for Aspergillus nidulans transformation[J]. Journal of Microbiological Methods, 2003, 55(3): 687-695.
[43]田媛,苑社强,袁耀武,等.康氏木霉原生质体制备及再生条件研究[J].中国酿造, 2010, 7: 75-79.
[44]刘玲,叶博,刘长江,等.白腐真菌原生质体制备和再生条件的研究[J].生物技术, 2006, 5: 41-44.
[45]宋庆涛,张国珍,董金皋,等.玉米大斑病菌原生质体的制备与再生[J].微生物学通报, 2003, 2: 41-44.
[46] Graziani S, Vasnier C, Daboussi M J. Novel Polyketide Synthase from Nectria haematococca[J]. Applied and Environmental Microbiology, 2004, 70(5): 2984-2988.
[47] Zhang A, Lu P, Dahl-Roshak A M, et al. Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis[J]. Molecular Genetics and Genomics, 2003, 268(5): 645-655.
[48] Askenazi M, Driggers E M, Holtzman D A, et al. Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains[J]. Nature Biotechnology, 2003, 21(2): 150-156.
[49] Campbell C D, Vederas J C. Biosynthesis of lovastatin and related metabolites formed by fungal iterative PKS enzymes[J]. Biopolymers, 2010, 93(9): 755-763.
[50] Yang G, Rose M S, Turgeon B G, et al. A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin[J]. Plant Cell, 1996, 8(11): 2139-2150.
[51] Shimizu T, Kinoshita H, Ishihara S, et al. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus[J]. Applied and Environmental Microbiology, 2005, 71(7): 3453-3457.
[52] Yu J H, Leonard T J. Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase[J]. Journal of Bacteriology, 1995, 177(16): 4792-4800.
[53] Gerber R, Lou L, Du L. A PLP-dependent polyketide chain releasing mechanism in the biosynthesis of mycotoxin fumonisins in Fusarium verticillioides[J]. Journal of the American Chemical Society, 2009, 131(9): 3148-3149.
[54] Moriwaki A, Kihara J, Kobayashi T, et al. Insertional mutagenesis and characterization of a polyketide synthase gene (PKS1) required for melanin biosynthesis in Bipolaris oryzae[J]. FEMS Microbiology Letters, 2004, 238(1): 1-8.
[55] Akamatsu H O, Chilvers M I, Stewart J E, et al. Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei[J]. Current Genetics, 2010, 56(4): 349-360.
[56] Paolo W F, Jr., Dadachova E, Mandal P, et al. Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature[J]. BMC Microbiology, 2006, 6: 55.
[57] Perpetua N S, Kubo Y, Yasuda N, et al. Cloning and characterization of a melanin biosynthetic THR1 reductase gene essential for appressorial penetration of Colletotrichum lagenarium[J]. Molecular Plant-Microbe Interactions, 1996, 9(5): 323-329.
[58] Tsai H F, Chang Y C, Washburn R G, et al. The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence[J]. Journal of Bacteriology, 1998, 180(12): 3031-3038.
[59] Tanguay P, Loppnau P, Morin C, et al. A spontaneous albino mutant of Ceratocystis resinifera results from a point mutation in the polyketide synthase gene, PKS1[J]. Canadian Journal of Microbiology, 2006, 52(6): 501-507.
[60]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, 4: 410-417.
[61]安鑫龙,董金皋,韩建民,等.玉米大斑病菌的RAPD分析Ⅰ.应用CTAB法提取玉米大斑病菌DNA[J].河北农业大学学报, 2001, 1: 38-41.
[62] Kihara J, Moriwaki A, Ueno M, et al. Cloning, functional analysis and expression of a scytalone dehydratase gene ( SCD1) involved in melanin biosynthesis of the phytopathogenic fungus Bipolaris oryzae[J]. Current Genetics, 2004, 45(4): 197-204.
[63] Bashyal B M, 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, 2009, 26(2): 309-316.
[64]曹志艳,贾慧,董金皋,等. DHN黑色素与玉米大斑病菌附着胞膨压形成的关系[J].中国农业科学, 2011, 44(5): 925-932.
[65]翟晖.玉米鞘腐病病原鉴定与致病机制研究[D].保定:河北农业大学, 2010.
[66] Tokuoka M, Seshime Y, Fujii I, et al. Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae[J]. Fungal Genetics and Biology, 2008, 45(12): 1608-1615.
[67] Royer M, Costet L, Vivien E, et al. Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes[J]. Molecular Plant-Microbe Interactions, 2004, 17(4): 414-427.
[68] de Jong J C, McCormack B J, Smirnoff N, et al. Glycerol generates turgor in rice blast[J]. Nature, 1997, 389(6648): 244-244.
[69] 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.
[70] Howard R J, Valent B. Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea[J]. Annual Review of Microbiology, 1996, 50: 491-512.
[71] Kawamura C, Moriwaki J, Kimura N, et al. The melanin biosynthesis genes of Alternariaalternata can restore pathogenicity of the melanin-deficient mutants of Magnaporthe grisea[J]. Molecular Plant-Microbe Interactions, 1997, 10(4): 446-453.
[72] Money N P, Howard R J. Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement[J]. Fungal Genetics and Biology, 1996, 20(3): 217-227.
[73] Chen Z, Nunes M A, Silva M C, et al. Appressorium turgor pressure of Colletotrichum kahawae might have a role in coffee cuticle penetration[J]. Mycologia, 2004, 96(6): 1199-1208.
[74] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391(6669): 806-811.
[75] Novina C D, Sharp P A. The RNAi revolution[J]. Nature, 2004, 430(6996): 161-164.
[76] Stevenson M. Therapeutic potential of RNA interference[J]. The New England Journal of Medicine, 2004, 351(17): 1772-1777.