胶孢炭疽菌泛素结合酶基因CgUBC2的RNAi突变体获得与功能分析
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摘要
为了研究胶孢炭疽菌泛素结合酶基因CgUBC2的功能以及与致病相关基因间的关联性。利用RNAi技术构建沉默载体pSilent-1:CgUBC2,通过PEG介导法获得突变体菌株,实时荧光定量PCR法分析其侵染寄主过程中的差异表达,以及与PG、ECH、LIP、PKS、HMT、Sin3P、NRPS 7个致病相关基因的关联性。通过特异性引物检测鉴定、表达量分析,获得CgUBC2的RNAi突变体为pSilent-1:CgUBC2-1和pSilent-1:CgUBC2-2,侵染过程中突变体菌株CgUBC2基因表达量、7个致病相关基因的表达量显著低于野生型菌株。成功获得胶孢炭疽菌泛素结合酶基因CgUBC2的RNAi沉默突变体,CgUBC2参与侵染寄主的过程、正向调控7个致病相关基因和致病力。
To study the function of the ubiquitin-conjugating enzyme gene CgUBC2 from Colletotrichum gloeosporioides and its association with pathogenicity related genes, the silence vector(pSilent-1:CgUBC2) was constructed with RNAi and mutant was obtained by PEG-mediated method. Quantitative real-time PCR was used to analyze the differential expression levels of CgUBC2 during the process of infecting host and its association with 7 pathogenic genes, including PG, ECH, LIP, PKS, HMT, Sin3 P, and NRPS. The RNAi mutants of CgUBC2(pSilent-1: CgUBC2-1 and pSilent-1: CgUBC2-2) were obtained by specific primer identification and expression levels analysis. The expression of CgUBC2 gene and 7 pathogenicity-related genes in mutant strains were significantly lower than those in wild-type strains during infection. RNAi mutants of CgUBC2 were successfully obtained. It was found that CgUBC2 participated in the process of infecting host and positively regulated 7 pathogenic genes and pathogenicity.
To study the function of the ubiquitin-conjugating enzyme gene CgUBC2 from Colletotrichum gloeosporioides and its association with pathogenicity related genes, the silence vector(pSilent-1:CgUBC2) was constructed with RNAi and mutant was obtained by PEG-mediated method. Quantitative real-time PCR was used to analyze the differential expression levels of CgUBC2 during the process of infecting host and its association with 7 pathogenic genes, including PG, ECH, LIP, PKS, HMT, Sin3 P, and NRPS. The RNAi mutants of CgUBC2(pSilent-1: CgUBC2-1 and pSilent-1: CgUBC2-2) were obtained by specific primer identification and expression levels analysis. The expression of CgUBC2 gene and 7 pathogenicity-related genes in mutant strains were significantly lower than those in wild-type strains during infection. RNAi mutants of CgUBC2 were successfully obtained. It was found that CgUBC2 participated in the process of infecting host and positively regulated 7 pathogenic genes and pathogenicity.
引文
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Huang W.,2012,Cloning and analysis on bioinformatics of Aspergillus ubiquitin-conjugating enzyme gene,Thesis for M.S.,Northwest Normal University,Supervisor:Wang Y.F.pp.1-2(黄薇,2012,曲霉泛素结合酶基因的克隆与生物信息学分析,硕士学位论文,西北师范大学,导师:王一峰pp.1-2)
Jue D.W.,Sang X.L.,Shu B.,Liu L.Q.,Wang Y.C.,and Shi S.Y.,2017,Functional analysis of a ubiquitin conjugating enzyme gene ZmUBC-76 in maize,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),38(8):1507-1511(决登伟,桑雪莲舒波,刘丽琴,王一承,石胜友,2017,玉米泛素结合酶基因ZmUBC-76的功能分析,热带作物学报,38(8):1507-1511)
Koch A.,Biedenkopf D.,Furch A.,Weber L.,Rossbach O.,Abdellate E.,Linicus L.,Johannsmeier J.,Jelonek L.,Goesmann A.,Cardoza V.,McMillan J.,Mentzel T.,and KarlHeinz.,2016,An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery,PLoS Pathogens,12(10):e1005901
Liu Y.L.,Chen Y.H.,Liu F.Z.,Zhang Y.,and Lian Y.,2015,Cloning and expression analysis induced by verticillium wilt fungus of ubiquitin-conjugating enzyme gene StUBCc from Solanum torvum,Yuanyi Xuebao(Acta Horticulturae Sinica),42(6):1185-1194(刘炎霖,陈钰辉,刘富中,张映,连勇,2015水茄泛素结合酶E2基因StUBCc的克隆及黄萎病菌诱导表达分析,园艺学报,42(6):1185-1194)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Ma C.S.,Li Z.P.,Dai Q.Q.,Han Q.M.,and Huang L.L.,2016,Function of nonribosomal peptide synthetase gene VmN-RPS12 of Valsa mali,Weishengwu Xuebao(Acta Microbiologica Sinica),56(8):1273-1281(马晨琛,李正鹏,戴青青韩青梅,黄丽丽,2016,苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能,微生物学报,56(8):1273-1281)
Oide S.,Moeder W.,Krasnoff S.,Gibson D.,Haas H.,Yoshioka K.,and Turgeon B.G.,2006,Nps6,encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism,is a conserved virulence determinant of plant pathogenic ascomycetes,Plant Cell,18(10):2836-2853
Qi Y.X.,Xie Y.X.,Zhang X.,and Zhang H.Q.,2004,Study of DNA extraction methods in Fusarium oxysporum f.sp.cubense,Shengwu Jishu(Biotechnology),14(6):32-34(漆艳香,谢艺贤,张欣,张辉强,2004,香蕉枯萎菌基因组DNA提取方法的研究,生物技术,14(6):32-34)
Wang A.B.,Jin Z.Q.,Liu J.H.,Jia C.H.,Zhang J.B.,Miao H.X.,and Xu B.Y.,2013,Expression analysis of a banana ubiquitin-conjugating enzyme gene MaUCE2 under abiotic stress,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):77-80(王安邦,金志强,刘菊华,贾彩红,张建斌,苗红霞,徐碧玉,2013,香蕉泛素结合酶基因MaUCE2在非生物胁迫下的表达分析,生物技术通报,(5):77-80)
Wang J.L.,Shi S.Q.,Jia L.Q.,and Jiang Z.P.,2010,Progresson functions of ubiquitin-conjugating enzyme(E2)in plants,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):7-10(王金利,史胜青,贾利强,江泽平,2010,植物泛素结合酶E2功能研究进展,生物技术通报,(4):7-10)
Wei Y.X.,2014,Coloning and functional identification of Laccase gene(Lac1)in pathogenicity from Colletotrichum gloeosporioides the pathogen of mango anthracnose disease,Thesis for M.S.,Hainan University,Supervisor:Liu X.M.,and Pu J.J.,pp.23-25(韦运谢,2014,芒果炭疽病菌漆酶基因Lac1的克隆与致病相关功能鉴定,硕士学位论文,海南大学,导师:刘晓妹,蒲金基,pp.23-25)
Xu C.X.,Jiang J.,Liu T.T.,Wang Y.C.,Liu G.F.,and Yang C.P.,2007,Sequence analysis and function determination of E2s gene from Tamarix androssowii,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),35(11):1-4(徐晨曦,姜静,刘甜甜,王玉成,刘桂丰,杨传平,2007,柽柳泛素结合酶基因(E2s)的序列分析及功能验证,东北林业大学学报,35(11):1-4)
Barreranecha L.L.,Bautistabanos S.,Floresmoctezuma H.E.,and Estudillo A.R.,2008,Efficacy of essential oils on the conidial germination,growth of Colletotrichum gloeosporioides(Penz.)Penz.and Sacc and control of postharvest diseases in papaya(Carica papaya L.),Plant Pathol.J.,7(2):174-178
Chen Y.,Gao Q.,Huang M.,Liu Y.,Liu Z.,and Liu X.,2015,Characterization of RNA silencing components in the plant pathogenic fungus Fusarium graminearum,Scientific Rep.,5:12500
Gai Z.X.,2016,Study on infection process of Colletotrichum gloeosporioides from citrus and main physiological and biochemical changes,Thesis for M.S.,Xi'nan University,Supervisor:Wang R.K.,pp.1-2(盖智星,2016,柑橘胶孢炭疽菌的侵染过程及主要生理生化变化研究,硕士学位论文,西南大学,导师:王日葵,pp.1-2)
He F.,2014,Generation of CgLysM RNAi line of Colletotrichum gloeosporioides and its protein subcellular localization,Thesis for M.S.,Hainan University,Supervisor:He C.Z.,pp.1-2(何芬,2014,橡胶树炭疽病菌效应蛋白基因CgLysM的RNAi突变体构建及其蛋白的亚细胞定位,硕士学位论文,海南大学,导师:何朝族,pp.1-2)
Hu T.L.,Li W.,Liu X.L.,and Dai L.Y.,2014,The role of ubiquitination in plant disease resistance,Weishengwuxue Tongbao(Microbiology China),41(6):1175-1179(胡婷丽,李魏,刘雄伦,戴良英,2014,泛素化在植物抗病中的作用,微生物学通报,41(6):1175-1179)
Huang W.,2012,Cloning and analysis on bioinformatics of Aspergillus ubiquitin-conjugating enzyme gene,Thesis for M.S.,Northwest Normal University,Supervisor:Wang Y.F.pp.1-2(黄薇,2012,曲霉泛素结合酶基因的克隆与生物信息学分析,硕士学位论文,西北师范大学,导师:王一峰pp.1-2)
Jue D.W.,Sang X.L.,Shu B.,Liu L.Q.,Wang Y.C.,and Shi S.Y.,2017,Functional analysis of a ubiquitin conjugating enzyme gene ZmUBC-76 in maize,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),38(8):1507-1511(决登伟,桑雪莲舒波,刘丽琴,王一承,石胜友,2017,玉米泛素结合酶基因ZmUBC-76的功能分析,热带作物学报,38(8):1507-1511)
Koch A.,Biedenkopf D.,Furch A.,Weber L.,Rossbach O.,Abdellate E.,Linicus L.,Johannsmeier J.,Jelonek L.,Goesmann A.,Cardoza V.,McMillan J.,Mentzel T.,and KarlHeinz.,2016,An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery,PLoS Pathogens,12(10):e1005901
Liu Y.L.,Chen Y.H.,Liu F.Z.,Zhang Y.,and Lian Y.,2015,Cloning and expression analysis induced by verticillium wilt fungus of ubiquitin-conjugating enzyme gene StUBCc from Solanum torvum,Yuanyi Xuebao(Acta Horticulturae Sinica),42(6):1185-1194(刘炎霖,陈钰辉,刘富中,张映,连勇,2015水茄泛素结合酶E2基因StUBCc的克隆及黄萎病菌诱导表达分析,园艺学报,42(6):1185-1194)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Ma C.S.,Li Z.P.,Dai Q.Q.,Han Q.M.,and Huang L.L.,2016,Function of nonribosomal peptide synthetase gene VmN-RPS12 of Valsa mali,Weishengwu Xuebao(Acta Microbiologica Sinica),56(8):1273-1281(马晨琛,李正鹏,戴青青韩青梅,黄丽丽,2016,苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能,微生物学报,56(8):1273-1281)
Oide S.,Moeder W.,Krasnoff S.,Gibson D.,Haas H.,Yoshioka K.,and Turgeon B.G.,2006,Nps6,encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism,is a conserved virulence determinant of plant pathogenic ascomycetes,Plant Cell,18(10):2836-2853
Qi Y.X.,Xie Y.X.,Zhang X.,and Zhang H.Q.,2004,Study of DNA extraction methods in Fusarium oxysporum f.sp.cubense,Shengwu Jishu(Biotechnology),14(6):32-34(漆艳香,谢艺贤,张欣,张辉强,2004,香蕉枯萎菌基因组DNA提取方法的研究,生物技术,14(6):32-34)
Wang A.B.,Jin Z.Q.,Liu J.H.,Jia C.H.,Zhang J.B.,Miao H.X.,and Xu B.Y.,2013,Expression analysis of a banana ubiquitin-conjugating enzyme gene MaUCE2 under abiotic stress,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):77-80(王安邦,金志强,刘菊华,贾彩红,张建斌,苗红霞,徐碧玉,2013,香蕉泛素结合酶基因MaUCE2在非生物胁迫下的表达分析,生物技术通报,(5):77-80)
Wang J.L.,Shi S.Q.,Jia L.Q.,and Jiang Z.P.,2010,Progresson functions of ubiquitin-conjugating enzyme(E2)in plants,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):7-10(王金利,史胜青,贾利强,江泽平,2010,植物泛素结合酶E2功能研究进展,生物技术通报,(4):7-10)
Wei Y.X.,2014,Coloning and functional identification of Laccase gene(Lac1)in pathogenicity from Colletotrichum gloeosporioides the pathogen of mango anthracnose disease,Thesis for M.S.,Hainan University,Supervisor:Liu X.M.,and Pu J.J.,pp.23-25(韦运谢,2014,芒果炭疽病菌漆酶基因Lac1的克隆与致病相关功能鉴定,硕士学位论文,海南大学,导师:刘晓妹,蒲金基,pp.23-25)
Xu C.X.,Jiang J.,Liu T.T.,Wang Y.C.,Liu G.F.,and Yang C.P.,2007,Sequence analysis and function determination of E2s gene from Tamarix androssowii,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),35(11):1-4(徐晨曦,姜静,刘甜甜,王玉成,刘桂丰,杨传平,2007,柽柳泛素结合酶基因(E2s)的序列分析及功能验证,东北林业大学学报,35(11):1-4)