香蕉枯萎病菌4号生理小种β2-微管蛋白基因敲除与表型分析  

Gene knockout and phenotype analysis of the β2-tubulin in Fusarium oxysporum f. sp. cubense race 4

作　　者：刘远征 漆艳香[1] 曾凡云[1] 丁兆建[1] 何壮 彭军[1] 张欣 谢艺贤[1] LIU Yuanzheng;QI Yanxiang;ZENG Fanyun;DING Zhaojian;HE Zhuang;PENG Jun;ZHANG Xin;XIE Yixian(Chinese Academy of Tropical Agricultural Sciences Environment and Plant Protection Institute, Haikou 571101, Hainan, China;Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, Hainan, China;Haikou Experimental Station Chinese Academy of Tropical Agricultural Sciences, Haikou 570102, Hainan, China)

机构地区：[1]中国热带农业科学院环境与植物保护研究所,海口571101 [2]海南大学热带农林学院,海口570228 [3]中国热带农业科学院海口实验站,海口570102

出　　处：《果树学报》2018年第12期1467-1477,共11页Journal of Fruit Science

基　　金：国家自然科学基金(31471738;31571957;31661143003);现代农业产业技术体系专项(CARS-31-07);农业部南亚办专项(151721301082352712);热科院托举工程项目(1630042018010)

摘　　要：【目的】克隆并鉴定香蕉枯萎病菌(Foc4)β2-微管蛋白(β2-tub)基因,阐明β2-tub在多菌灵的抗药性及在病原菌致病过程中发挥的作用。【方法】采用PCR技术克隆了β2-tub的序列全长,并对其进行生物信息学分析。应用Splitmarker同源重组技术获得Foc4的β2-tub基因敲除突变体,并对其突变体的生物学表型、致病力及其对多菌灵的敏感性进行测定。【结果】生物信息学分析表明,Foc4β2-tub基因全长为1 694 bp,cDNA编码区全长1 347 bp,由4个内含子和5个外显子组成,编码蛋白含448个氨基酸。Foc4对多菌灵表现为敏感(EC50=0.51μg·mL^(-1)),与Foc4相比,β2-tub基因敲除突变体对多菌灵的敏感性(EC50=0.36μg·mL^(-1))表现显著增强,差异显著(p <0.05)。与Foc4相比,β2-tub基因敲除突变体的生物学表型没有变化,对巴西蕉苗的致病力明显减弱。【结论】β2-tub基因具有高度保守性,β2-tub基因敲除突变体对细胞壁选择性压力、氧化压力和渗透压力均没有影响,但致病力下降,同时β2-tub基因的变化会引起Foc4对多菌灵抗性的改变。[Objective]The objective of this study was to clone and identify the β2-tubulin(β2-tub)gene of Fusarium oxysporum f.sp.cubense race 4(Foc4),and reveal the function of β2-tub in resistance of Foc4 to carbendazim and its role in the pathogenic process of Foc4[.Methods]Based on the sequences of β2-tub gene(XM_011327927.1)of Fusarium graminearum and the complete genome sequence of the Foc4 54006 strain(JH658279.1),specific primers were designed.A full-length sequence of β2-tub gene from Foc4 was cloned by PCR amplification,and sequence characterization,phylogenetic clustering and protein domains of β2-tub gene were also analyzed,respectively.The signature domains were analyzed by SMART software and the phylogenetic tree was built by MEGA 6.0.β2-tub knockout mutants of Foc4 were obtained by split-marker homologous recombination technology.Sensitivity of Foc4 and its knockout mutants to carbendazim was tested by inoculating their mycelium onto PDA plates with different concentrations of carbendazim,respectively.The biological phenotypes icluding the growth rate of colonies,the amount of sporulation,the form of mycelium,selective pressure of cell wall,oxidation pressure and osmotic pressure of Foc4 and its knockout mutants were tested.In addition,the pathogenicity of Foc4 and its knockout mutants was tested by pot culture root drenching method.Foc4 and its knockout mutants were treated with 15 Brazil banana seedlings(Cavendish AAA).Each seedling was irrigated with 20mL spores suspension,and aseptic water was used as a control.The corm of Brazil banana was cut longitudinally after 30 d,and the browning degree of the bulb and the condition of the chlorosis of the outer leaves were observed.[Results]Bioinformatics analysis revealed that the complete DNA and cDNA sequences of β2-tub from Foc4 were 1 694 bp and 1 347 bp,respectively.The cDNA of β2-tub included 5 exons and 4 introns and encoded a protein of 448 amino-acids.Moreover,the β2-tub was highly conserved.Compared with other plant pathogenic fungi,the gene w

关 键 词：香蕉枯萎病菌 β2-微管蛋白 基因敲除 致病力 

分 类 号：S668.1[农业科学—果树学]