大丽轮枝菌VdLac基因克隆与功能分析
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摘要
为探讨漆酶基因VdLac在大丽轮枝菌中的功能,克隆该基因并利用农杆菌介导的遗传转化方法和PEG介导的原生质体转化方法对VdLac进行敲除和功能回复,获得敲除和互补突变体。以野生型JY为对照,对敲除突变体和互补突变体进行生物学功能测定。结果显示,VdLac基因全长1 978bp,cDNA序列全长1 713bp,编码570个氨基酸组成的蛋白质;VdLac基因缺失突变体(△VdLac)对硝化压力更敏感,其在CM培养基上仍可产生漆酶,但产生漆酶的能力降低;表明大丽轮枝菌漆酶基因VdLac不影响菌株的营养生长、产孢、致病力以及黑色素合成。
In order to explore the laccase gene(VdLac)function of Verticillium dahliae,the VdLac was cloned from the wild type strain JY of Verticilliumdahliae.The deletion and its complementation were obtained through the gene knock-out and complementation methods by Agrobacterium tumifaciens-mediated transformation and PEG-mediated protoplasts transformation.Compared with the wild type strain JY of Verticillium dahliae,the deletion and complementation mutants were selected for VdLac biological function examination.The results showed that the complete DNA and cDNA sequences of the gene named VdLac were 1 978 bp and 1 713 bp in length,respectively,encoding aprotein of 570 amino acid.The deletion mutants(△VdLac)were more sensitive to nitrosative stress,and could produce laccase on CM plates,but the production was reduced.The results indicated that VdLac had no effect on fungus vegetative growth,conidia production,pathogenicity and melanin synthesis in Verticillium dahliae.
In order to explore the laccase gene(VdLac)function of Verticillium dahliae,the VdLac was cloned from the wild type strain JY of Verticilliumdahliae.The deletion and its complementation were obtained through the gene knock-out and complementation methods by Agrobacterium tumifaciens-mediated transformation and PEG-mediated protoplasts transformation.Compared with the wild type strain JY of Verticillium dahliae,the deletion and complementation mutants were selected for VdLac biological function examination.The results showed that the complete DNA and cDNA sequences of the gene named VdLac were 1 978 bp and 1 713 bp in length,respectively,encoding aprotein of 570 amino acid.The deletion mutants(△VdLac)were more sensitive to nitrosative stress,and could produce laccase on CM plates,but the production was reduced.The results indicated that VdLac had no effect on fungus vegetative growth,conidia production,pathogenicity and melanin synthesis in Verticillium dahliae.
引文
[1]BHAT R G,SUBBARAO K V.Host range specificity in Verticillium dahliae[J].Phytopathology,1999,89(12):1218-1225.
[2]KLOSTERMAN S,JATALLAH Z K,VALLAD G E.Diversity,pathogenicity,and management of Verticillium species[J].Annual Review of Phytopathology,2009,47(1):39-62.
[3]MENZIES J D,GRIEBEL G E.Survival and saprophytic growth of Verticillium dahliae in uncropped soil[J].Phytopathology,1967,57(2):703-709.
[4]POWELSON R L.Significance of Population Level of Verticilliumin Soil[M]∥Root Diseases and Soil-borne Pathogens.University of California Press,Berkeley,USA,1970:31-33.
[5]WILHELM S.Longevity of Verticillium wilt fungus in the laboratory and field[J].Phytopathology,1955,45(3):180-181.
[6]NOSANCHUK J D,CASADEVALL A.The contribution of melanin to microbial pathogenesis[J].Cellular Microbiology,2003,5(2):203-223.
[7]SAPMAK A,BOYCE K J,ANDRIANOPOULOS A,et al.The pbrBgene encodes a laccase required for DHN-Melanin synthesis in conidia of Talaromyces(Penicillium)marneffei[J].PloS One,2015,10(4):1-16.
[8]KAWAMURA C,TSUJIMOTO T,TSUGE T.Targeted disruption of a melanin biosynthesis gene affects conidial development and UV tolerance in the Japanese pear pathotype of Alternaria alternate[J].Molecular Plant-Microbe Interactions,1999,12(1):59-63.
[9]REHNSTROM A L,FREE S J.The isolation and characterization of melanin-deficient mutants of Monilinia fructicola[J].Physiological and Molecular Plant Pathology,1996,49(5):321-330.
[10]WANG Y,CASADEVALL A.Decreased susceptibility of melanized Cryptococcus neoformans to UV light[J].Applied and Environmental Microbiology,1994,60(10):3864-3866.
[11]HYAKUMACHI M,YOKOYMA K,UI T.Role of melanin in susceptibility and resistance of Rhizoctonia solani to microbial lysis[J].Transactions of the British Mycological Society,1987,89(1):27-33.
[12]ROSAS A L,CASADEVALL A.Melanization affects susceptibility of Cryptococcus neoformans to heat and cold[J].FEMS Microbiology Letters,1997,153(2):265-272.
[13]YANGRAE C,AKHIL S,ROBIN A,et al.Transcription factor Amr1induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola[J].PLoS Pathogens,2012,8(10):1-17.
[14]ZHANG C Q,ZHU G N,MA Z H.Isolation,characterization and preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast fungus,Magnaporthe grisea[J].Journal of Phytopathology,2006,154(2):392-397.
[15]ELIAHU N,IGBARIA A,ROSE M S.Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases,Chk1and Mps1,and the transcription factor Cmr1[J].Eukaryotic Cell,2007,6(2):421-429.
[16]NINA H,LAURA-LEENA K,KRISTIINA K.Crystal stucture of a laccase form Melanacarpus albomyces with an intact trinuclear copper site[J].Nature Structural Biology,2002,9(8):601-605.
[17]YOSHIDA H.Chemistry of lacquer(urushi)[J].Journal of the Chemical Society,1883,43:472-486.
[18]WIDSTEN P,Kandelbauer A.Laccase applications in the forest products industry:A review[J].Enzyme and Microbial Technology,2008,42(4):293-307.
[19]BALDRIAN P.Fungal laccases occurrence and properties[J].FEMS Microbiology Reviews,2006,30(2):215-242.
[20]SHEKHER R,SEHGAL S,KAMTHANIA M,et al.Laccase:microbial sources,production,purification,and potential biotechnological applications[J].Enzyme Research,2011,2011:1-11.
[21]GIARDINA P,FARACO V,PEZZELLA C,et al.Laccases:a never-ending story[J].Cellular and Molecular Life Sciences,2010,67(3):369-385.
[22]LV Z,KANG X,XIANG Z,et al.A laccase gene Sh-lac is involved in the growth and melanin biosynthesis of Scleromitrula shiraiana[J].Phytopathology,2017,107(3):353-361.
[23]DURESSA D,ANCHIETA A,CHEN D.et al.RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae[J].BioMed Central Genomics,2013,14(1):607-624.
[24]DOBINSON K F.Genetic transformation of the vascular wilt fungus Verticillium dahliae[J].Canadian Journal of Botany,1995,73(5):710-715.
[25]FAN R,KLOSTERMAN S J,WANG C H,et al.Vayg1is required for microsclerotium formation and melanin production in Verticillium dahliae[J].Fungal Genetics and Biology,2017,98:1-11.
[26]WEI Y,SHEN W,DAUK M,et al.Targeted genedisruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferrednutrient from host plant to fungal pathogen[J].Journal of Biological Chemistry,2004,279(1):429-435.
[27]王光辉.禾谷镰刀菌AMT1基因的功能研究[D].陕西杨凌:西北农林科技大学,2010.WANG G H.Fubctional characterization of AMT1 genes in Fusarium griaminearium[D].Yangling Shaanxi:North West Agriculture and Forestry University,2010.
[28]PAZ Z,GARCíA-PEDRAJAS M D,ANDREWS D L,et al.One step construction of Agrobacterium-recombination-ready-plasmids(OSCAR),an efficient and robust tool for ATMT based gene deletion construction in fungi[J].Fungal Genetics and Biology,2011,48(7):677-684.
[29]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.
[30]彭姗,吕学莲,高峰,等.一种新的棉花黄、枯萎病快速接种方法的研究[J].棉花学报,2008,20(3):174-178.PENG SH,LX L,GAO F,et al.Study on a new rapid inoculation method for Verticillium wilt and Fusarium wilt of cotton[J].Cotton Science,2008,20(3):174-178.
[31]简桂良,孙文姬,马存,等.棉花抗病虫性评价技术规范:第5部分:黄萎病GB/T 22101.3-2009[S].北京:中国标准出版社,2009:2.JIAN G L,SUN W J,MA C,et al.Technical specification for evaluating resistance of cotton to diseases and insect pests:part 5:Verticillium wilt[S].Beijing:Standards Press of China,2009:2.
[32]JANUSZ G,CZURYLO A,FRC M,et al.Laccase production and metabolic diversity among Flammulina velutipes strains[J].World Journal of Microbiology and Biotechnology,2015,31(1):121-133.
[33]TSAI H F,WHEELER M H,CHANG Y C,et al.A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus[J].Journal of Bacteriology,1999,181(20):6469-6477.
[34]TSUJI G,FUJIKAWA J,ISHIDA H.Laccase gene LAC1of Colletotrichum lagenariumis not essential for melanin biosynthesis and pathogenicity[J].Journal of General Plant Pathology,2001,67(3):182-190.
[35]LIN S Y,OKUDA S,IKEDA K,et al.Lac2 encoding a secreted laccase is involved in appressorial melanization and conidial pigmentation in Colletotrichum orbiculare[J].Molecular Plant-Microbe Interactions,2012,5(12):1552-1561.
[36]CAERO D C,RONCERO M I G.Functional analyses of laccase genes from Fusarium oxysporum[J].Phytopathology,2008,98(5):509-518.
[37]KILARU S,HOEGGER P J,KES U,et al.The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[J].Current Genetics,2006,50(1):45-60.
[38]DONG J L,ZHANG Y W,ZHANG R H,et al.Influence of culture condition laccase production and isozyme patterns in the white-rot fungus Trametes gallica[J].Journal of Basic Microbiology,2005,45(3):190-198.
[39]HILDEN K,HAKALA T K,MAIJALA P,et al.Novel thermotolerent laccase produced by the white-rot fungus Physisporinus rivulosus[J].Applied Microbiology and Biotechnolgy,2007,77(2):301-309.
[40]DURRENS P.The phenoloxidases of the ascomycete Podospora anserina:the three forms of the major laccase activity[J].Archives of Microbiology,1981,130(2):121-124.
[41]肖书笑,田呈明,王永林.黄栌枯萎病菌漆酶基因的鉴定及其在微菌核形成过程中的表达研究[J/OL].中国科技论文在线,2013:1-7[2013-12-20].http://www.paper.edu.cn/releasepaper/content/201312-625.XIAO SH X,TIAN CH M,WANG Y L.Genome-wide identification,phylogeny and expression profiling during microsclerotia formation in smoke tree wilt fungus Verticillium dahliae[J/OL].Sciencepaper online,2013:1-7[2013-12-20].http://www.paper.edu.cn/releasepaper/content/201312-625.
[42]莫佳琳,付时雨,詹怀宇.贝壳状革耳菌诱变提高产漆酶能力及其生物漂白的研究[J].中国造纸学报,2006,21(4):29-32.MO J L,FU SH Y,ZHAN H Y.Improving laccase activity of Panus conchatus by mutagenesis and used for biobleaching[J].Transactions of China Pulp and Paper,2006,21(4):29-32.
[43]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):693-703.
[44]赵丹.疣孢漆斑菌漆酶的产生、特性及应用研究[D].哈尔滨:东北林业大学,2012.ZHAO D.The production,properties and applications of Myrothcium verrucaria NF-05[D].Harbin Heilongjiang:Northeast Forestry University,2012.
[45]PERIASAMY R,PALVANNAN T.Optimization of laccase production by Pleurotus ostreatus IMI 395545using the Taguchi DOE methodology[J].Journal of Basic Microbiology,2010,50(6):548-556.
[2]KLOSTERMAN S,JATALLAH Z K,VALLAD G E.Diversity,pathogenicity,and management of Verticillium species[J].Annual Review of Phytopathology,2009,47(1):39-62.
[3]MENZIES J D,GRIEBEL G E.Survival and saprophytic growth of Verticillium dahliae in uncropped soil[J].Phytopathology,1967,57(2):703-709.
[4]POWELSON R L.Significance of Population Level of Verticilliumin Soil[M]∥Root Diseases and Soil-borne Pathogens.University of California Press,Berkeley,USA,1970:31-33.
[5]WILHELM S.Longevity of Verticillium wilt fungus in the laboratory and field[J].Phytopathology,1955,45(3):180-181.
[6]NOSANCHUK J D,CASADEVALL A.The contribution of melanin to microbial pathogenesis[J].Cellular Microbiology,2003,5(2):203-223.
[7]SAPMAK A,BOYCE K J,ANDRIANOPOULOS A,et al.The pbrBgene encodes a laccase required for DHN-Melanin synthesis in conidia of Talaromyces(Penicillium)marneffei[J].PloS One,2015,10(4):1-16.
[8]KAWAMURA C,TSUJIMOTO T,TSUGE T.Targeted disruption of a melanin biosynthesis gene affects conidial development and UV tolerance in the Japanese pear pathotype of Alternaria alternate[J].Molecular Plant-Microbe Interactions,1999,12(1):59-63.
[9]REHNSTROM A L,FREE S J.The isolation and characterization of melanin-deficient mutants of Monilinia fructicola[J].Physiological and Molecular Plant Pathology,1996,49(5):321-330.
[10]WANG Y,CASADEVALL A.Decreased susceptibility of melanized Cryptococcus neoformans to UV light[J].Applied and Environmental Microbiology,1994,60(10):3864-3866.
[11]HYAKUMACHI M,YOKOYMA K,UI T.Role of melanin in susceptibility and resistance of Rhizoctonia solani to microbial lysis[J].Transactions of the British Mycological Society,1987,89(1):27-33.
[12]ROSAS A L,CASADEVALL A.Melanization affects susceptibility of Cryptococcus neoformans to heat and cold[J].FEMS Microbiology Letters,1997,153(2):265-272.
[13]YANGRAE C,AKHIL S,ROBIN A,et al.Transcription factor Amr1induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola[J].PLoS Pathogens,2012,8(10):1-17.
[14]ZHANG C Q,ZHU G N,MA Z H.Isolation,characterization and preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast fungus,Magnaporthe grisea[J].Journal of Phytopathology,2006,154(2):392-397.
[15]ELIAHU N,IGBARIA A,ROSE M S.Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases,Chk1and Mps1,and the transcription factor Cmr1[J].Eukaryotic Cell,2007,6(2):421-429.
[16]NINA H,LAURA-LEENA K,KRISTIINA K.Crystal stucture of a laccase form Melanacarpus albomyces with an intact trinuclear copper site[J].Nature Structural Biology,2002,9(8):601-605.
[17]YOSHIDA H.Chemistry of lacquer(urushi)[J].Journal of the Chemical Society,1883,43:472-486.
[18]WIDSTEN P,Kandelbauer A.Laccase applications in the forest products industry:A review[J].Enzyme and Microbial Technology,2008,42(4):293-307.
[19]BALDRIAN P.Fungal laccases occurrence and properties[J].FEMS Microbiology Reviews,2006,30(2):215-242.
[20]SHEKHER R,SEHGAL S,KAMTHANIA M,et al.Laccase:microbial sources,production,purification,and potential biotechnological applications[J].Enzyme Research,2011,2011:1-11.
[21]GIARDINA P,FARACO V,PEZZELLA C,et al.Laccases:a never-ending story[J].Cellular and Molecular Life Sciences,2010,67(3):369-385.
[22]LV Z,KANG X,XIANG Z,et al.A laccase gene Sh-lac is involved in the growth and melanin biosynthesis of Scleromitrula shiraiana[J].Phytopathology,2017,107(3):353-361.
[23]DURESSA D,ANCHIETA A,CHEN D.et al.RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae[J].BioMed Central Genomics,2013,14(1):607-624.
[24]DOBINSON K F.Genetic transformation of the vascular wilt fungus Verticillium dahliae[J].Canadian Journal of Botany,1995,73(5):710-715.
[25]FAN R,KLOSTERMAN S J,WANG C H,et al.Vayg1is required for microsclerotium formation and melanin production in Verticillium dahliae[J].Fungal Genetics and Biology,2017,98:1-11.
[26]WEI Y,SHEN W,DAUK M,et al.Targeted genedisruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferrednutrient from host plant to fungal pathogen[J].Journal of Biological Chemistry,2004,279(1):429-435.
[27]王光辉.禾谷镰刀菌AMT1基因的功能研究[D].陕西杨凌:西北农林科技大学,2010.WANG G H.Fubctional characterization of AMT1 genes in Fusarium griaminearium[D].Yangling Shaanxi:North West Agriculture and Forestry University,2010.
[28]PAZ Z,GARCíA-PEDRAJAS M D,ANDREWS D L,et al.One step construction of Agrobacterium-recombination-ready-plasmids(OSCAR),an efficient and robust tool for ATMT based gene deletion construction in fungi[J].Fungal Genetics and Biology,2011,48(7):677-684.
[29]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.
[30]彭姗,吕学莲,高峰,等.一种新的棉花黄、枯萎病快速接种方法的研究[J].棉花学报,2008,20(3):174-178.PENG SH,LX L,GAO F,et al.Study on a new rapid inoculation method for Verticillium wilt and Fusarium wilt of cotton[J].Cotton Science,2008,20(3):174-178.
[31]简桂良,孙文姬,马存,等.棉花抗病虫性评价技术规范:第5部分:黄萎病GB/T 22101.3-2009[S].北京:中国标准出版社,2009:2.JIAN G L,SUN W J,MA C,et al.Technical specification for evaluating resistance of cotton to diseases and insect pests:part 5:Verticillium wilt[S].Beijing:Standards Press of China,2009:2.
[32]JANUSZ G,CZURYLO A,FRC M,et al.Laccase production and metabolic diversity among Flammulina velutipes strains[J].World Journal of Microbiology and Biotechnology,2015,31(1):121-133.
[33]TSAI H F,WHEELER M H,CHANG Y C,et al.A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus[J].Journal of Bacteriology,1999,181(20):6469-6477.
[34]TSUJI G,FUJIKAWA J,ISHIDA H.Laccase gene LAC1of Colletotrichum lagenariumis not essential for melanin biosynthesis and pathogenicity[J].Journal of General Plant Pathology,2001,67(3):182-190.
[35]LIN S Y,OKUDA S,IKEDA K,et al.Lac2 encoding a secreted laccase is involved in appressorial melanization and conidial pigmentation in Colletotrichum orbiculare[J].Molecular Plant-Microbe Interactions,2012,5(12):1552-1561.
[36]CAERO D C,RONCERO M I G.Functional analyses of laccase genes from Fusarium oxysporum[J].Phytopathology,2008,98(5):509-518.
[37]KILARU S,HOEGGER P J,KES U,et al.The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[J].Current Genetics,2006,50(1):45-60.
[38]DONG J L,ZHANG Y W,ZHANG R H,et al.Influence of culture condition laccase production and isozyme patterns in the white-rot fungus Trametes gallica[J].Journal of Basic Microbiology,2005,45(3):190-198.
[39]HILDEN K,HAKALA T K,MAIJALA P,et al.Novel thermotolerent laccase produced by the white-rot fungus Physisporinus rivulosus[J].Applied Microbiology and Biotechnolgy,2007,77(2):301-309.
[40]DURRENS P.The phenoloxidases of the ascomycete Podospora anserina:the three forms of the major laccase activity[J].Archives of Microbiology,1981,130(2):121-124.
[41]肖书笑,田呈明,王永林.黄栌枯萎病菌漆酶基因的鉴定及其在微菌核形成过程中的表达研究[J/OL].中国科技论文在线,2013:1-7[2013-12-20].http://www.paper.edu.cn/releasepaper/content/201312-625.XIAO SH X,TIAN CH M,WANG Y L.Genome-wide identification,phylogeny and expression profiling during microsclerotia formation in smoke tree wilt fungus Verticillium dahliae[J/OL].Sciencepaper online,2013:1-7[2013-12-20].http://www.paper.edu.cn/releasepaper/content/201312-625.
[42]莫佳琳,付时雨,詹怀宇.贝壳状革耳菌诱变提高产漆酶能力及其生物漂白的研究[J].中国造纸学报,2006,21(4):29-32.MO J L,FU SH Y,ZHAN H Y.Improving laccase activity of Panus conchatus by mutagenesis and used for biobleaching[J].Transactions of China Pulp and Paper,2006,21(4):29-32.
[43]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):693-703.
[44]赵丹.疣孢漆斑菌漆酶的产生、特性及应用研究[D].哈尔滨:东北林业大学,2012.ZHAO D.The production,properties and applications of Myrothcium verrucaria NF-05[D].Harbin Heilongjiang:Northeast Forestry University,2012.
[45]PERIASAMY R,PALVANNAN T.Optimization of laccase production by Pleurotus ostreatus IMI 395545using the Taguchi DOE methodology[J].Journal of Basic Microbiology,2010,50(6):548-556.