禾谷镰孢菌过氧化物酶家族基因的表达规律分析
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摘要
过氧化物酶作为病原真菌抗氧化防御系统的主要组分,能够清除来源于植物体产生的活性氧(reactive oxygen species, ROS),促使病原菌成功侵染宿主植物细胞,禾谷镰孢菌(Fusarium graminearum)过氧化物酶(peroxidase,POX)的相关研究尚未见报道。本研究利用生物信息学手段搜索真菌过氧化物酶基因的数据库,获得了31个禾谷镰孢菌过氧化物酶基因,系统进化分析和保守结构域分析均将其分为18个亚家族。对过氧化物酶家族基因的表达模式进行分析,发现FgPOX (禾谷镰孢菌过氧化物酶, Fusarium graminearum peroxidase)-13、FgPOX-17、FgPOX-16、FgPOX-19等基因在菌丝和分生孢子中均具有较高表达水平,FgPOX-24、FgPOX-29在菌丝中表达水平较高,FgPOX-11、FgPOX-10、FgPOX-4、FgPOX-23在分生孢子中表达水平较高。对过氧化物酶家族基因在侵染过程中的表达规律进行分析,发现FgPOX-13在病菌侵染过程中表达水平明显升高并保持较高水平,FgPOX-17、FgPOX-28、FgPOX-30等基因在病菌侵染的早期表达水平较高,FgPOX-12、FgPOX-10、FgPOX-23等基因在病菌侵染的后期表达水平较高。进一步利用qRT-PCR技术对禾谷镰孢菌过氧化物酶家族基因在H2O2胁迫条件下表达规律进行分析,发现FgPOX-1、FgPOX-4、FgPOX-6、FgPOX-9、FgPOX-10、FgPOX-22、FgPOX-30的表达水平是正常条件下的20倍以上,FgPOX-9、FgPOX-13等随着H2O2胁迫时间的延长,表达水平明显增高,而FgPOX-1、FgPOX-21等随着H2O2胁迫时间的延长,表达水平明显降低。本研究明确了禾谷镰孢菌过氧化物酶家族基因数量、系统进化关系、保守结构域,以及在病菌不同组织、侵染过程和响应H2O2胁迫中的表达规律,为阐明禾谷镰孢菌过氧化物酶家族基因的功能提供了基础资料。
As a main component of the anti-oxidant defense system of pathogenic fungi, peroxidase can remove reactive oxygen species(ROS) derived from plant organisms, and promote the successful infection of host plant cells by pathogens. Related studies on peroxidase(POX) of Fusarium graminearum have not been reported. In this study, 31 Fusarium graminearum peroxidase genes were obtained by searching the database of fungal peroxidase genes using bioinformatics methods, and were divided into 18 subfamilies by phylogenetic analysis and conserved domain analysis. Analysis of the expression pattern of the peroxidase family gene revealed that the FgPOX(Fusarium graminearum peroxidase)-13, FgPOX-17, FgPOX-16, FgPOX-19 and other genes had high expression levels in both mycelium and spore. and the FgPOX-24 and FgPOX-29 had higher expression levels in mycelium, the FgPOX-11, FgPOX-10, FgPOX-4 and FgPOX-23 had higher expression levels in spore. The expression pattern of peroxidase family gene in the process of infection was analyzed. It was found that the expression level of FgPOX-13 was significantly increased and maintained at a high level during the infection of the pathogen. In addition, genes such as FgPOX-17, FgPOX-28 and FgPOX-30 were highly expressed in the early stage of pathogen infection, and genes such as FgPOX-12, FgPOX-10 and FgPOX-23 were highly expressed in the late stage of pathogen infection. Furthermore, qRT-PCR was used to analyze the expression of Fusarium graminearum peroxidase family genes under H2 O2 stress, the expression levels of FgPOX-1, FgPOX-4, FgPOX-6, FgPOX-9, FgPOX-10, FgPOX-22 and FgPOX-30 were found to be20 times higher than that under normal conditions, and FgPOX-9, FgPOX-13, etc. with the prolongation of H2 O2 stress time, the expression level was significantly enhanced, while FgPOX-1, FgPOX-21, etc. with the prolongation of H2 O2 stress time, the expression level was reduced. This study clarified the number of genes,phylogenetic relationships, conserved domains of Fusarium graminearum peroxidase family, and the expression patterns of different tissues, infection processes and H2 O2 stress in pathogens, which could provide basic datas for clarifying the function of Fusarium graminearum peroxidase family genes.
As a main component of the anti-oxidant defense system of pathogenic fungi, peroxidase can remove reactive oxygen species(ROS) derived from plant organisms, and promote the successful infection of host plant cells by pathogens. Related studies on peroxidase(POX) of Fusarium graminearum have not been reported. In this study, 31 Fusarium graminearum peroxidase genes were obtained by searching the database of fungal peroxidase genes using bioinformatics methods, and were divided into 18 subfamilies by phylogenetic analysis and conserved domain analysis. Analysis of the expression pattern of the peroxidase family gene revealed that the FgPOX(Fusarium graminearum peroxidase)-13, FgPOX-17, FgPOX-16, FgPOX-19 and other genes had high expression levels in both mycelium and spore. and the FgPOX-24 and FgPOX-29 had higher expression levels in mycelium, the FgPOX-11, FgPOX-10, FgPOX-4 and FgPOX-23 had higher expression levels in spore. The expression pattern of peroxidase family gene in the process of infection was analyzed. It was found that the expression level of FgPOX-13 was significantly increased and maintained at a high level during the infection of the pathogen. In addition, genes such as FgPOX-17, FgPOX-28 and FgPOX-30 were highly expressed in the early stage of pathogen infection, and genes such as FgPOX-12, FgPOX-10 and FgPOX-23 were highly expressed in the late stage of pathogen infection. Furthermore, qRT-PCR was used to analyze the expression of Fusarium graminearum peroxidase family genes under H2 O2 stress, the expression levels of FgPOX-1, FgPOX-4, FgPOX-6, FgPOX-9, FgPOX-10, FgPOX-22 and FgPOX-30 were found to be20 times higher than that under normal conditions, and FgPOX-9, FgPOX-13, etc. with the prolongation of H2 O2 stress time, the expression level was significantly enhanced, while FgPOX-1, FgPOX-21, etc. with the prolongation of H2 O2 stress time, the expression level was reduced. This study clarified the number of genes,phylogenetic relationships, conserved domains of Fusarium graminearum peroxidase family, and the expression patterns of different tissues, infection processes and H2 O2 stress in pathogens, which could provide basic datas for clarifying the function of Fusarium graminearum peroxidase family genes.
引文
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Mir A A,Park S Y,Abu Sadat M,et al.2015.Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae[J].Scientific Reports,5:11831.
Molina L,Kahmann R.2007.An Ustilago maydis gene involved in H2O2detoxification is required for virulence[J].Plant cell,19(7):2293-2309.
Noguchi M,Yasuda N,Fujita Y.2006.Evidence of genetic exchange by parasexual recombination and genetic analysis of pathogenicity and mating type of parasexual recombinants in rice blast fungus,Magnaporthe oryzae[J].Phytopathology,96(7):746-750.
Onaga G,Wydra K D,Koopmann B,et al.2015.Populationstructure,pathogenicity and mating type distribution of Magnaporthe oryzae isolates from East Africa[J].Phytopathology,105:1137-1145.
Park S Y,Milgroom M,Han S,et al.2008.Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea[J].Phytopathology,98(4):436-442.
Torres M A,Jones J D G,Dangl J L.2006.Reactive oxygen species signaling in response topathogens[J].Plant Physiology,141:373-378.
Tredway L,Stevenson K,Burpee L.2003.Mating type distribution and fertility status in Magnaporthe grisea populations from turfgrasses in Georgia[J].Plant Disease,87(4):435-441.
Wojtaszek P.1997.Oxidative burst:An early plant response to pathogen infection[J].Biochemical Journal,322:681-692.
Yoshida K,Saunders D G,Mitsuoka C,et al.2016.Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements[J].BMC Genomics,17:370.
ZámockyM,Gasselhuber B,Furtmüller P G,et al.2012.Molecular evolution of hydrogen peroxide degrading enzymes[J].Archives of Biochemistry&Biophysics,525(2):131-144.
ZámockyM,Obinger C.2010.Molecular phylogeny of heme peroxidases[J].Springer Berlin Heidelberg,7-35.
Zeigler R,Scott R,Leung H,et al.1997.Evidence of parasexual exchange of DNA in the rice blast fungus challenges its exclusive clonality[J].Phytopathology,87:284-294.
刘星辰,冯胜泽,李学然,等.2017.玉米大斑病菌GATA转录因子家族全基因组鉴定及其表达的初步分析[J].农业生物技术学报,25(12):2018-2026.(Liu X C,Feng SZ,Li X R,et al.2017.Genome-wide identification of GATA transcription factor family and preliminary expression analysis in Setosphaeria turcica[J].Journal of Agricultural Biotechnology,25(12):2018-2026.)
Bao J,Chen M,Zhong Z,et al.2017.PacBio sequencing reveals transposable elements as a key contributor to genomic plasticity and virulence variation in Magnaporthe oryzae[J].Molecular Plant,10(11):1465-1468.
Choi J,Détry N,Kim K T,et al.2014.fPoxDB:Fungal peroxidase database for comparative genomics[J].BMC Microbiology,14:117.
Dean R,Van Kan J A,Pretorius Z A,et al.2012.The top 10fungal pathogens in molecular plant pathology[J].Molecular Plant Pathology,13(4):804-804.
Desjardins A E,Proctor R H,Bai G,et al.1996.Reduced virulence of trichothecene-nonproducing mutants of Gibber?ella zeae in wheat field tests[J].Molecular Plant-Microbe Interactions,9(9):775-781.
Ebbole D J.Magnaporthe as a model for understanding host pathogen interactions[J].Annual Review of Phytopathology,45(1):437-456.
Enjalbert B,Nantel A,Whiteway M.2003.Stress-induced gene expression in Candida albicans:Absence of a general stress response[J].Molecular Biology of the Cell,14(4):1460-1467.
Enjalbert B,Smith D A,Cornell M J,et al.2006.Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans[J].Molecular Biology of the Cell,17(2):1018-1032.
Enjalbert B,Maccallum D M,Odds F C,et al.2007.Nichespecific activation of the oxidative stress response by the pathogenic fungus Candida albicans[J].Infection&Immunity,75(5):2143-2151.
Heller J,Tudzynski P.2001.Reactive oxygen species in phytopathogenic fungi:Signaling,development,and disease[J].Annual Review of Phytopathology,49:369-390.
Hong S Y,Roze L V,Wee J,et al.2013.Evidence that a transcription factor regulatory network coordinates oxidative stress response and secondary metabolism in aspergilla[J].Microbiology Open,2:144-160.
Leung H,Borromeo E S,Bernardo M A,et al.1988.Genetic analysis of virulence in the rice blast fungus Magna?porthe grisea[J].Phytopathology,78(9):1227-1233.
Mehdy M C.1994.Active oxygen species in plant defense against pathogens[J].Plant Physiology,105:467.
Mir A A,Park S Y,Abu Sadat M,et al.2015.Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae[J].Scientific Reports,5:11831.
Molina L,Kahmann R.2007.An Ustilago maydis gene involved in H2O2detoxification is required for virulence[J].Plant cell,19(7):2293-2309.
Noguchi M,Yasuda N,Fujita Y.2006.Evidence of genetic exchange by parasexual recombination and genetic analysis of pathogenicity and mating type of parasexual recombinants in rice blast fungus,Magnaporthe oryzae[J].Phytopathology,96(7):746-750.
Onaga G,Wydra K D,Koopmann B,et al.2015.Populationstructure,pathogenicity and mating type distribution of Magnaporthe oryzae isolates from East Africa[J].Phytopathology,105:1137-1145.
Park S Y,Milgroom M,Han S,et al.2008.Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea[J].Phytopathology,98(4):436-442.
Torres M A,Jones J D G,Dangl J L.2006.Reactive oxygen species signaling in response topathogens[J].Plant Physiology,141:373-378.
Tredway L,Stevenson K,Burpee L.2003.Mating type distribution and fertility status in Magnaporthe grisea populations from turfgrasses in Georgia[J].Plant Disease,87(4):435-441.
Wojtaszek P.1997.Oxidative burst:An early plant response to pathogen infection[J].Biochemical Journal,322:681-692.
Yoshida K,Saunders D G,Mitsuoka C,et al.2016.Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements[J].BMC Genomics,17:370.
ZámockyM,Gasselhuber B,Furtmüller P G,et al.2012.Molecular evolution of hydrogen peroxide degrading enzymes[J].Archives of Biochemistry&Biophysics,525(2):131-144.
ZámockyM,Obinger C.2010.Molecular phylogeny of heme peroxidases[J].Springer Berlin Heidelberg,7-35.
Zeigler R,Scott R,Leung H,et al.1997.Evidence of parasexual exchange of DNA in the rice blast fungus challenges its exclusive clonality[J].Phytopathology,87:284-294.