大丽轮枝菌致病缺陷型T-DNA插入突变体的筛选与鉴定
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摘要
【目的】从大丽轮枝菌T-DNA突变体库中筛选鉴定致病缺陷型突变体并分析致病相关基因。【方法】将落叶型大丽轮枝菌菌株Vd991的294个T-DNA插入突变体在寄主棉花上进行致病力测定。利用Southern杂交和hiTAIL-PCR(High-efficiency thermal asymmetric interlaced PCR)技术分别对筛选获得的9个致病力显著降低的突变体进行拷贝数和侧翼序列分析。【结果】与接种野生型Vd991的棉花相比,接种突变体的棉花的病情指数极显著降低。Southern杂交表明其中1个突变体中T-DNA为双拷贝插入,其余8个突变体均为单拷贝插入。生物学特性分析发现T-DNA的插入导致这些突变体在生长速率和产孢量等方面与野生型Vd991相比均有不同程度的降低。Blast比对分析明确了这些突变体中T-DNA的插入位置和在基因组上的分布,并从野生型菌株Vd991中成功克隆得到了这些可能影响大丽轮枝菌致病力的相关基因。【结论】筛选和鉴定T-DNA插入突变是在全基因组范围内快速获得致病相关基因信息的1种有效方法,极大的促进了大丽轮枝菌的致病分子机制等研究,为进一步培育抗病棉花品种奠定了基础。
[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes.[Method] In total,294 T-DNA insertion mutants of V.dahliae were tested for their virulence using a cotton infection assay.Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant.DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR.[Result] Based on the virulence assay,the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991.The Southern blot assay revealed that only one mutant contained two T-DNA insertions,while the remaining eight mutants harbored a single T-DNA insert.An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991.The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17.Furthermore,the pathogenicity-related genes were cloned from strain Vd991.[Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V.dahliae on a genome-wide scale.This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V.dahliae.
[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes.[Method] In total,294 T-DNA insertion mutants of V.dahliae were tested for their virulence using a cotton infection assay.Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant.DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR.[Result] Based on the virulence assay,the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991.The Southern blot assay revealed that only one mutant contained two T-DNA insertions,while the remaining eight mutants harbored a single T-DNA insert.An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991.The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17.Furthermore,the pathogenicity-related genes were cloned from strain Vd991.[Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V.dahliae on a genome-wide scale.This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V.dahliae.
引文
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[4]朱荷琴,冯自力,尹志新,等.我国棉花黄萎病菌致病力分化及ISSR指纹分析[J].植物病理学报,2012,42(3):225-235.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
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[12]Maruthachalam K,Klosterman S J,Kang S,et al.Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis[J].Molecular Biotechnology,2011,49(3):209-221.
[13]Zhang D,Wang X,Chen J,et al.Identification and characterization of a pathogenicity-related gene Vd CYP1 from Verticillium dahliae[J].Scientific Reports,2016,6:27979.
[14]李彩红,李志芳,冯自力,等.棉花黄萎病菌致病相关基因Vd MFS1敲除载体的构建[J].中国棉花,2013,40(1):7-12.Li Caihong,Li Zhifang,Feng Zili,et al.The construction of pathogenicity-related gene Vd MFS1 replacement vector of Verticillium dahliae[J].China Cotton,2013,40(1):7-12.
[15]汪佳妮.大丽轮枝菌Vd991 T-DNA插入变体库的构建及致病相关基因的筛选[D].北京:中国农业科学院,2011.Wang Jiani.Verticillium dahliae Kleb.(Vd991)T-DNA mutant library construction and screening of pathogenic-related genes[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[16]王新艳,张丹丹,桂月晶,等.大丽轮枝菌致病性相关突变体快速筛选体系的建立[J].中国农业科学,2015,48(14):2747-2756.Wang Xinyan,Zhang Dandan,Gui Yuejing,et al.Construction of a rapid screening system of pathogenicity-related mutants in Verticillium dahliae[J].Scientia Agricultura Sinica,2015,48(14):2747-2756.
[17]朱荷琴,冯自力,李志芳,等.蛭石沙土无底纸钵定量蘸菌液法鉴定棉花品种(系)的抗黄萎病性[J].中国棉花,2010,37(12):15-17.Zhu Heqin,Feng Zili,Li Zhifang,et al.Evaluating cotton varieties resistant to Verticillium wilt using root-dip method in a bowl without bottom[J].China Cotton,2010,37(12):15-17.
[18]Dobinson K F,Lecomte N,Lazarovits G.Production of an extracellular trypsin-like protease by the fungal plant pathogen[J].Canadian Journal of Microbiology,1997,43(3):227-233.
[19]Liu Y G,Chen Y.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques,2007,43(5):649-650.
[20]刘义杰,李志芳,冯自力,等.棉花黄萎病菌低致病力突变体的表型分析及致病相关基因克隆[J].植物病理学报,2015,45(3):258-269.Liu Yijie,Li Zhifang,Feng Zili,Zhao et al.Phenotypic analysis of low virulent Verticillium dahliae mutant on cotton and cloning of pathogenicity related genes[J].Acta Phytopathologica Sinica,2015,45(3):258-269.
[21]谷素静,汪敏,桑茜,等.棉花黄萎病菌T-DNA插入突变体库的构建及致病缺陷突变体筛选[J].河南农业科学,2014,43(1):69-83.Gu Sujing,Wang Min,Sang Xi,et al.Construction of T-DNA insertional mutant library for Verticillium dahliae and identification of pathogenicity defective mutants[J].Journal of Henan Agricultural Sciences,2014,43(1):69-83.
[22]Sbrissa D,Ikonomov O C,Fenner H,et al.Ar PIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3in a complex to promote PIKfyve activity and functionality[J].Journal of Molecular Biology,2008,384(4):766-779.
[2]Klosterman S J,Atallah Z K,Vallad G E,et al.Diversity,pathogenicity,and management of Verticillium species[J].Annual Review of Phytopathology,2009,47(1):39-62.
[3]赵凤轩,戴小枫.棉花黄萎病菌的侵染过程[J].基因组学与应用生物学,2009,28(4):786-792.Zhao Fengxuan,Dai Xiaofeng.Infection process of Verticillium dahliae Kleb.in Cotton[J].Genomics and Applied Biology,2009,28(4):786-792.
[4]朱荷琴,冯自力,尹志新,等.我国棉花黄萎病菌致病力分化及ISSR指纹分析[J].植物病理学报,2012,42(3):225-235.Pathogenicity differentiation and ISSR fingerprint analysis of cotton Verticillium dahliae in China[J].Acta Phytopathologica Sinica,2012,42(3):225-235.
[5]朱荷琴,冯自力,李志芳,等.分离自棉花的轮枝菌“种”的鉴定[J].中国农业科学,2013,46(10):2032-2040.Zhu Heqin,Feng Zili,Li Zhifang,et al.Identification of isolates of Verticillium species from cotton[J].Scientia Agriculture Sinica,2013,46(10):2032-2040.
[6]O'Donnell K,Cigelnik E,Weber N S,et al.Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis[J].Mycologia,1997,89(1):48-65.
[7]Frandin E F,Thomma B P H J.Physiology and molecular aspects of Verticillium wilt diseases caused by V.dahliae and V.alboatrum[J].Molecular Plant Pathology,2006,7(2):71-86.
[8]Pegg G F,Brady B L.Verticillium wilts[M].Wallingford,UK:CABI Publishing,2002.
[9]Mishra N C,Tatum E L.Non-mendelian inheritance of DNA-induced inositol independence in Neurospora[J].Proceedings of the National Academy of Sciencesof the United States of America,1973,70(12):3875-3879.
[10]Mullins E D,Chen X,Romaine P,et al.Agrobacterium-mediated transformation of Fusarium oxysporum:An efficient tool for insertional mutagenesis and gene transfer[J].Phytopathology,2001,91(2):173-180.
[11]Chen X L,Yang J,Peng Y L.Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation[M]//Xu J R,Bluhm B.Fungal Genomics.New York:Humana Press,2011:213-224.
[12]Maruthachalam K,Klosterman S J,Kang S,et al.Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis[J].Molecular Biotechnology,2011,49(3):209-221.
[13]Zhang D,Wang X,Chen J,et al.Identification and characterization of a pathogenicity-related gene Vd CYP1 from Verticillium dahliae[J].Scientific Reports,2016,6:27979.
[14]李彩红,李志芳,冯自力,等.棉花黄萎病菌致病相关基因Vd MFS1敲除载体的构建[J].中国棉花,2013,40(1):7-12.Li Caihong,Li Zhifang,Feng Zili,et al.The construction of pathogenicity-related gene Vd MFS1 replacement vector of Verticillium dahliae[J].China Cotton,2013,40(1):7-12.
[15]汪佳妮.大丽轮枝菌Vd991 T-DNA插入变体库的构建及致病相关基因的筛选[D].北京:中国农业科学院,2011.Wang Jiani.Verticillium dahliae Kleb.(Vd991)T-DNA mutant library construction and screening of pathogenic-related genes[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[16]王新艳,张丹丹,桂月晶,等.大丽轮枝菌致病性相关突变体快速筛选体系的建立[J].中国农业科学,2015,48(14):2747-2756.Wang Xinyan,Zhang Dandan,Gui Yuejing,et al.Construction of a rapid screening system of pathogenicity-related mutants in Verticillium dahliae[J].Scientia Agricultura Sinica,2015,48(14):2747-2756.
[17]朱荷琴,冯自力,李志芳,等.蛭石沙土无底纸钵定量蘸菌液法鉴定棉花品种(系)的抗黄萎病性[J].中国棉花,2010,37(12):15-17.Zhu Heqin,Feng Zili,Li Zhifang,et al.Evaluating cotton varieties resistant to Verticillium wilt using root-dip method in a bowl without bottom[J].China Cotton,2010,37(12):15-17.
[18]Dobinson K F,Lecomte N,Lazarovits G.Production of an extracellular trypsin-like protease by the fungal plant pathogen[J].Canadian Journal of Microbiology,1997,43(3):227-233.
[19]Liu Y G,Chen Y.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques,2007,43(5):649-650.
[20]刘义杰,李志芳,冯自力,等.棉花黄萎病菌低致病力突变体的表型分析及致病相关基因克隆[J].植物病理学报,2015,45(3):258-269.Liu Yijie,Li Zhifang,Feng Zili,Zhao et al.Phenotypic analysis of low virulent Verticillium dahliae mutant on cotton and cloning of pathogenicity related genes[J].Acta Phytopathologica Sinica,2015,45(3):258-269.
[21]谷素静,汪敏,桑茜,等.棉花黄萎病菌T-DNA插入突变体库的构建及致病缺陷突变体筛选[J].河南农业科学,2014,43(1):69-83.Gu Sujing,Wang Min,Sang Xi,et al.Construction of T-DNA insertional mutant library for Verticillium dahliae and identification of pathogenicity defective mutants[J].Journal of Henan Agricultural Sciences,2014,43(1):69-83.
[22]Sbrissa D,Ikonomov O C,Fenner H,et al.Ar PIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3in a complex to promote PIKfyve activity and functionality[J].Journal of Molecular Biology,2008,384(4):766-779.