UeFuz7在菰黑粉菌二型态转换中的作用
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摘要
菰黑粉菌与茭白植株互作形成膨大肉质茎——茭白,已成为我国第二大水生蔬菜。作为一种二态性真菌,菰黑粉菌的二型态转换与侵染茭白密切相关,对茭白孕茭至关重要。本研究基于菰黑粉菌全基因组序列,采用t Blastn进行同源基因查找,克隆得到了玉米瘤黑粉菌Fuz7的同源基因UeFuz7。该基因c DNA全长1 308 bp,无内含子,编码435个氨基酸,在黑粉菌中相对保守。酵母双杂交实验发现,UeFuz7可与UeKpp2蛋白互作。同时其表达模式分析发现,在菰黑粉菌的二型态转换发生时UeFuz7的相对表达量最高。进一步通过PEG介导的原生质体转化获得UeFuz7突变体,表型分析发现:在二型态转换过程中,UeFuz7突变菌株的接合管形成和菌丝生长能力明显减弱。以上结果表明UeFuz7作用于菰黑粉菌的二型态转换。
Jiaobai,the second largest aquatic vegetable in China,was formed because of the interaction of Ustilago esculenta and its host Zizania latifolia. As a dimorphic fungus,the dimorphic transition of U. esculenta is closely related to its early infection,which is very important to formation of Jiaobai. In this study,the Ustilago maydis Fuz7 homologous gene UeFuz7 in U. esculenta was cloned,through homologous gene search by t Blastn based on the whole genome sequence of U. esculenta. The c DNA of UeFuz7 is 1 308 bp,without introns,encoding 435 amino acids,which is relatively conservative in the smut fungi. It was found that UeFuz7 interacted with UeKpp2 by yeast double hybrid experiment. Besides,its expression pattern analysis showed that the relative expression of UeFuz7 was the highest during the dimorphic transition of U. esculenta. Furthermore,PEG mediated protoplast transformation was carried out to construct UeFuz7 mutant,and phenotypic analysis found that UeFuz7 disrupted mutants significantly reduced the ability of conjugation tube formation and hyphal growth during dimorphic transition. The above results indicate that UeFuz7 functions on the dimorphic transition of U. esculenta.
Jiaobai,the second largest aquatic vegetable in China,was formed because of the interaction of Ustilago esculenta and its host Zizania latifolia. As a dimorphic fungus,the dimorphic transition of U. esculenta is closely related to its early infection,which is very important to formation of Jiaobai. In this study,the Ustilago maydis Fuz7 homologous gene UeFuz7 in U. esculenta was cloned,through homologous gene search by t Blastn based on the whole genome sequence of U. esculenta. The c DNA of UeFuz7 is 1 308 bp,without introns,encoding 435 amino acids,which is relatively conservative in the smut fungi. It was found that UeFuz7 interacted with UeKpp2 by yeast double hybrid experiment. Besides,its expression pattern analysis showed that the relative expression of UeFuz7 was the highest during the dimorphic transition of U. esculenta. Furthermore,PEG mediated protoplast transformation was carried out to construct UeFuz7 mutant,and phenotypic analysis found that UeFuz7 disrupted mutants significantly reduced the ability of conjugation tube formation and hyphal growth during dimorphic transition. The above results indicate that UeFuz7 functions on the dimorphic transition of U. esculenta.
引文
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[5]Zhang J Z,Guo D P,Xie G L.Cytology and ultrastructure of interactions betw een Ustilago esculenta and Zizania Latifolia[J].M ycological Progress,2012,11(2):499-508.
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[7]Zhang Y F,Cao Q C,Peng H,et al.Investigation on the differentiation of tw o Ustilago esculenta strainsimplications of a relationship w ith the host phenotypes appearing in the fields[J].BM C M icrobiology,2017,17(1):228.
[8]You W Y,Liu Q,Zou K Q,et al.Morphological and molecular differences in tw o strains of Ustilago esculenta[J].Current M icrobiology,2011,62(1):44-54.
[9]Liu J,Wu Y,Ma A M,et al.Research progress of environmental factors and signal transduction pathw ays of dimorphism in fungi[J].M icrobiology,2008,35(7):1102-1106.
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[12]Chacko N,Gold S.Deletion of the Ustilago maydis ortholog of the Aspergillus sporulation regulator medAaffects mating and virulence through pheromone response[J].Fungal Genetics&Biology,2012,49(6):426-432.
[13]Stasio M D,Brefort T,Mendoza-Mendoza A,et al.The dual specificity phosphatase rok1 negatively regulates mating and pathogenicity in Ustilago maydis[J].M olecular M icrobiology,2010,73(1):73-88.
[14]Zarnack K,Eichhorn H,Kahmann R,et al.Pheromone‐regulated target genes respond differentially to mapk phosphorylation of transcription factor prf1[J].M olecular M icrobiology,2010,69(4):1041-1053.
[15]Muller P,Weinzierl G,Brachmann A,et al.Mating and pathogenic development of the smut fungus Ustilago maydis are regulated by one mitogen-activated protein kinase cascade[J].Eukaryotic Cell,2003,2(6):1187-1199.
[16]Ye Z H,Pan Y,Zhang Y F,et al.Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome[J].DNA Research,2017,24(6):635-648.
[17]Cao Q C.Function of MAP kinase genes UeKpp2 and UeKpp6 in the Ustilago esculenta(in Chinese)[D].China Jiliang University(杭州:中国计量大学),2017.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta c(t))method[J].M ethods,2001,25(4):402-408.
[19]Yu J J,Zhang Y F,Cui H F,et al.An efficient gene-tic manipulation protocol for Ustilago esculenta[J].FEM S M icrobiology Letters,2015,362(12):87.
[20]Banuett F,Herskowitz I.Identification of fuz7,a Ustilago maydis M EK/M APKK homolog required for a-locus-dependent and-independent steps in the fungal life cycle[J].Genes&Development,1994,8(12):1367-1378.
[21]Chen R E,Thorner J.Function and regulation in M APK signaling pathw ays:Lessons learned from the yeast Saccharomyces cerevisiae[J].Biochimica et Biophysica Acta,2007,1773(8):1311-1340.
[22]Sturme M H J,Puccia R,Goldman G H,et al.Molecular biology of the dimorphic fungi Paracoccidioides spp.[J].Fungal Biology Review s,2011,25(2):89-97.
[23]Zhang Y F,Hu P,Cui H F,et al.Cloning and expression of mitogen-activated protein kinase homolog gene UeKss1 in Ustilago esculenta(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(5):634-644.
[24]Kaffarnik F,Müller P,Leibundgut M,et al.PKA and M APK phosphorylation of prf1 allow s promoter discrimination in Ustilago maydis[J].EM BO Journal,2014,22(21):5817-5826.
[25]Brefort T,Doehlemann G,Mendozamendoza A,et al.Ustilago maydis as a pathogen[J].Annual Review of Phytopathology,2009,47(4):423-445.
[26]Larraya L M,Boyce K J,So A,et al.Serial analysis of gene expression reveals conserved links betw een protein kinase A,ribosome biogenesis,and phosphate metabolism in Ustilago maydis[J].Eukaryot Cell,2005,4(12):2029-2043.
[2]Xing A B,Cui H F,Zhang Y F,et al.Current status of cultivation of Ustilago esculenta and its mechanism of pregnancy(in Chinese)[J].Zhejiang Agricultural Science(浙江农业科学),2016,57(10):1616-1621.
[3]Chung K R,Tzeng D D.Nutritional requirements of the edible gall-producing fungus Ustilago esculenta(in Chinese)[J].Journal of Biological Sciences(生命科学杂志),2004,4(2):246-252.
[4]Huang K F,Jiang J Z,Qin Y L,et al.Study on dietary fiber content and physicochemical properties of Zizania Latifolia(in Chinese)[J].Journal of Yangzhou University(扬州大学学报),2007,28(2):88-90.
[5]Zhang J Z,Guo D P,Xie G L.Cytology and ultrastructure of interactions betw een Ustilago esculenta and Zizania Latifolia[J].M ycological Progress,2012,11(2):499-508.
[6]Zhang J Z,Chu F Q,Guo D P,et al.The vacuoles containing multivesicular bodies:A new observation in interaction betw een Ustilago esculenta and Zizania Latifolia[J].European Journal of Plant Pathology,2014,138(1):79-91.
[7]Zhang Y F,Cao Q C,Peng H,et al.Investigation on the differentiation of tw o Ustilago esculenta strainsimplications of a relationship w ith the host phenotypes appearing in the fields[J].BM C M icrobiology,2017,17(1):228.
[8]You W Y,Liu Q,Zou K Q,et al.Morphological and molecular differences in tw o strains of Ustilago esculenta[J].Current M icrobiology,2011,62(1):44-54.
[9]Liu J,Wu Y,Ma A M,et al.Research progress of environmental factors and signal transduction pathw ays of dimorphism in fungi[J].M icrobiology,2008,35(7):1102-1106.
[10]Nadal M,García P M D,Gold S E.Dimorphism in fungal plant pathogens[J].FEM S M icrobiology Letters,2010,284(2):127-134.
[11]Schaeffer H J,Weber M J.Mitogen-activated protein kinases:Specific messages from ubiquitous messengers[J].Molecular&Cellular Biology,1999,19(4):2435-2444.
[12]Chacko N,Gold S.Deletion of the Ustilago maydis ortholog of the Aspergillus sporulation regulator medAaffects mating and virulence through pheromone response[J].Fungal Genetics&Biology,2012,49(6):426-432.
[13]Stasio M D,Brefort T,Mendoza-Mendoza A,et al.The dual specificity phosphatase rok1 negatively regulates mating and pathogenicity in Ustilago maydis[J].M olecular M icrobiology,2010,73(1):73-88.
[14]Zarnack K,Eichhorn H,Kahmann R,et al.Pheromone‐regulated target genes respond differentially to mapk phosphorylation of transcription factor prf1[J].M olecular M icrobiology,2010,69(4):1041-1053.
[15]Muller P,Weinzierl G,Brachmann A,et al.Mating and pathogenic development of the smut fungus Ustilago maydis are regulated by one mitogen-activated protein kinase cascade[J].Eukaryotic Cell,2003,2(6):1187-1199.
[16]Ye Z H,Pan Y,Zhang Y F,et al.Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome[J].DNA Research,2017,24(6):635-648.
[17]Cao Q C.Function of MAP kinase genes UeKpp2 and UeKpp6 in the Ustilago esculenta(in Chinese)[D].China Jiliang University(杭州:中国计量大学),2017.
[18]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta c(t))method[J].M ethods,2001,25(4):402-408.
[19]Yu J J,Zhang Y F,Cui H F,et al.An efficient gene-tic manipulation protocol for Ustilago esculenta[J].FEM S M icrobiology Letters,2015,362(12):87.
[20]Banuett F,Herskowitz I.Identification of fuz7,a Ustilago maydis M EK/M APKK homolog required for a-locus-dependent and-independent steps in the fungal life cycle[J].Genes&Development,1994,8(12):1367-1378.
[21]Chen R E,Thorner J.Function and regulation in M APK signaling pathw ays:Lessons learned from the yeast Saccharomyces cerevisiae[J].Biochimica et Biophysica Acta,2007,1773(8):1311-1340.
[22]Sturme M H J,Puccia R,Goldman G H,et al.Molecular biology of the dimorphic fungi Paracoccidioides spp.[J].Fungal Biology Review s,2011,25(2):89-97.
[23]Zhang Y F,Hu P,Cui H F,et al.Cloning and expression of mitogen-activated protein kinase homolog gene UeKss1 in Ustilago esculenta(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2016,46(5):634-644.
[24]Kaffarnik F,Müller P,Leibundgut M,et al.PKA and M APK phosphorylation of prf1 allow s promoter discrimination in Ustilago maydis[J].EM BO Journal,2014,22(21):5817-5826.
[25]Brefort T,Doehlemann G,Mendozamendoza A,et al.Ustilago maydis as a pathogen[J].Annual Review of Phytopathology,2009,47(4):423-445.
[26]Larraya L M,Boyce K J,So A,et al.Serial analysis of gene expression reveals conserved links betw een protein kinase A,ribosome biogenesis,and phosphate metabolism in Ustilago maydis[J].Eukaryot Cell,2005,4(12):2029-2043.
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