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
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摘要
大丽轮枝菌(Verticillium dahliae)侵染引起的棉花黄萎病是棉花生产上的重要病害之一,而其致病的分子机制尚不清楚,为此,以强致病力落叶型黄萎病菌FGH2为材料,利用农杆菌介导转化技术构建棉花黄萎病菌T-DNA插入突变体库并进行致病缺陷突变体的筛选。当农杆菌与黄萎病菌分生孢子浓度比为250∶1、承载介质为NC膜、乙酰丁香酮浓度400μmol/L、共培养时间48h时,转化效率高达每106个分生孢子产生612.5个转化子。通过采用优化后的转化体系,构建了含1 200个T-DNA插入转化子的棉花黄萎病菌突变体库。利用苗期分生孢子蘸根法测定300个转化子在棉花幼苗上的致病力,获得3个致病力显著降低的突变体181、546、1009,其中546的微菌核形成能力丧失,1009的微菌核形成能力明显下降。黄萎病菌T-DNA插入突变体库的构建,为棉花黄萎病菌致病及微菌核形成机制的研究奠定了基础。
The fungus Verticillium dahliae causes cotton Verticillium wilt,one of the most devastating diseases of cotton,however,the molecular basis of pathogenicity of V.dahliaeis unclear.In this study,Agrobacterium tumefaciens-mediated transformation(ATMT)was applied for insertional mutagenesis of V.dahliae FGH2,a highly virulent and defoliating strain.The collection of 1 200T-DNA random insertion transformants of V.dahliae was generated,using the optimized cocultivating conditions of V.dahliae conidia and Agrobacterium(1∶250ratio)for 48h,with the AS concentration of 400μmol/L and the nitrocellulose filter as the filter type in the co-cultivation,resulting in 612.5transformants per 106 conidia.Three pathogenicity defective V.dahliae mutants were obtained through testing the pathogenicity of 300T-DNA transformants on the cotton variety Yinshan 1seedling by dipping roots with spore suspension,including one T-DNA in-sertion mutant losing the ability for forming microsclerotia and one T-DNA insertion mutant defective of microsclerotia development capability.The T-DNA insertional mutagenesis library of V. dahliae lays a foundation for researching molecular mechanisms of pathogenicity and microsclerotia development of V.dahliae.
The fungus Verticillium dahliae causes cotton Verticillium wilt,one of the most devastating diseases of cotton,however,the molecular basis of pathogenicity of V.dahliaeis unclear.In this study,Agrobacterium tumefaciens-mediated transformation(ATMT)was applied for insertional mutagenesis of V.dahliae FGH2,a highly virulent and defoliating strain.The collection of 1 200T-DNA random insertion transformants of V.dahliae was generated,using the optimized cocultivating conditions of V.dahliae conidia and Agrobacterium(1∶250ratio)for 48h,with the AS concentration of 400μmol/L and the nitrocellulose filter as the filter type in the co-cultivation,resulting in 612.5transformants per 106 conidia.Three pathogenicity defective V.dahliae mutants were obtained through testing the pathogenicity of 300T-DNA transformants on the cotton variety Yinshan 1seedling by dipping roots with spore suspension,including one T-DNA in-sertion mutant losing the ability for forming microsclerotia and one T-DNA insertion mutant defective of microsclerotia development capability.The T-DNA insertional mutagenesis library of V. dahliae lays a foundation for researching molecular mechanisms of pathogenicity and microsclerotia development of V.dahliae.
引文
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[12]Li K N,Rouse D I,German T L.PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticilliumspp.[J].Applied and Environmental Microbiology,1994,60:4324-4331.
[13]石磊岩,王波,文学.我国棉花黄萎病菌类型分化及培养特性研究[J].植物保护学报,1993,20(3):247-252.
[14]Betts M F,Tucker S L,Galadima N,et al.Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae[J].Fungal Genetics and Biology,2007,44(10):1035-1049.
[15]Jeon J,Park S Y,Chi M H,et al.Genome-wide functional analysis of pathogenicity genes in the rice blast fungus[J].Nature Genetics,2007,39(4):561-565.
[16]Xu L S,Chen W D.Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum[J].Molecular Plant-Microbe Interactions,2013,26(4):431-441.
[17]徐荣旗,汪佳妮,陈捷胤,等.棉花黄萎病菌T-DNA插入突变体表型特征和侧翼序列分析[J].中国农业科学,2010,43(3):489-496.
[18]高峰,彭姗,彭晓玲,等.棉花黄萎病菌插入突变体库的构建及致病相关基因DVK1的克隆与鉴定[J].棉花学报,2011,23(1):64-68.
[19]Meyer V,Mueller D,Strowig T,et al.Comparison of different transformation methods for Aspergillus giganteus[J].Current Genetics,2003,43(5):371-377.
[20]Takahara H,Tsuji G,Kubo Y,et al.Agrobacterium tumefaciens-mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii[J].Journal of General Plant Pathology,2004,70:93-96.
[2]马存,简桂良.我国棉花抗黄萎病育种现状、问题和对策[J].中国农业科学,1997,30(2):58-64.
[3]赵丽芬,李增书,张寒霜,等.棉花黄萎病种质资源鉴定及抗性品种选择[J].华北农学报,2007,22(B10):254-256.
[4]杨秀荣,刘水芳,孙淑琴.棉花枯、黄萎病的诊断与防治[J].天津农业科学,2010,16(2):51-53.
[5]Michielse C B,Hooykaas P J,van den Hondel C A,et al.Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J].Current Genetics,2005,48:1-17.
[6]Dobinson K F,Grant S J,Kang S.Cloning and targeted disruption,via Agrobacterium tumefaciens-mediated transformation,of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae[J].Current Genetics,2004,45:104-110.
[7]Rauyaree P,Ospina-Giraldo M D,Kang S,et al.Mutations in VMK1,a mitogen-activated protein kinase gene,affect microsclerotia formation and pathogenicity in Verticillium dahliae[J].Current Genetics,2005,48:109-116.
[8]Li G T,Zhou X Y,Xu J R.Genetic control of infectionrelated development in Magnaporthe oryzae[J].Current Opinion in Microbiology,2012,15(6):678-684.
[9]Klosterman S J,Subbarao K V,Kang S,et al.Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens[J].PLoS Pathogens,2011,7(7):e1002137.
[10]桑茜,袁虹霞,王振跃,等.河南省不同地区棉花黄萎病菌分离物致病性及其毒素致萎活性测定[J].棉花学报,2010,22(4):333-338.
[11]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,2001,91:173-180.
[12]Li K N,Rouse D I,German T L.PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticilliumspp.[J].Applied and Environmental Microbiology,1994,60:4324-4331.
[13]石磊岩,王波,文学.我国棉花黄萎病菌类型分化及培养特性研究[J].植物保护学报,1993,20(3):247-252.
[14]Betts M F,Tucker S L,Galadima N,et al.Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae[J].Fungal Genetics and Biology,2007,44(10):1035-1049.
[15]Jeon J,Park S Y,Chi M H,et al.Genome-wide functional analysis of pathogenicity genes in the rice blast fungus[J].Nature Genetics,2007,39(4):561-565.
[16]Xu L S,Chen W D.Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum[J].Molecular Plant-Microbe Interactions,2013,26(4):431-441.
[17]徐荣旗,汪佳妮,陈捷胤,等.棉花黄萎病菌T-DNA插入突变体表型特征和侧翼序列分析[J].中国农业科学,2010,43(3):489-496.
[18]高峰,彭姗,彭晓玲,等.棉花黄萎病菌插入突变体库的构建及致病相关基因DVK1的克隆与鉴定[J].棉花学报,2011,23(1):64-68.
[19]Meyer V,Mueller D,Strowig T,et al.Comparison of different transformation methods for Aspergillus giganteus[J].Current Genetics,2003,43(5):371-377.
[20]Takahara H,Tsuji G,Kubo Y,et al.Agrobacterium tumefaciens-mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii[J].Journal of General Plant Pathology,2004,70:93-96.