基于致病性丧失突变体的香蕉枯萎镰刀菌致病相关基因克隆及功能鉴定

Banana wilt caused by Fusarium oxysporum f.sp. cubense (Foc) one of the most destructive soil-born vascular diseases of banana worldwide. To date, there is no effective control methods of this disease which has become a major factor limiting the commercial banana production in China. However, the molecular mechanism of the pathogenicity of Foc has not yet to be clarified.Cloning the pathogenenicity- related genes of Foc and identifying their functions is the first step in order to understand the mechanism at the molecular level. We had established T-DNA insertion mutant libraries of Focr1 and Focr4 through Agrobacterium tumefaciens-mediated transformations, and identified respectively,six non-virulent transformants from Focr4 mutants and twenty-one non-virulent transformants from Focr1 mutants based on pathogenicity tests. We have also gained the full genomic data of Focr1 and Focr4 by sequencing strains Focr1-N2 and Focr4-B2. The objective of the proposed project is to clone the pathogenicity-related genes, understand their functions by conducting research on full lenth cDNA clone of the pathogenicity-related gene from Focr1 and Focr4, gene knockout and complement experiments, observation of the bio-phenotype defects, location of the non-virulent gene coding product based on subcellular fluorescence studies, the expression of pathogenicity-related genes in yeasts followed by product purification, analysis of the spectrum sequencing, comparative analysis of the difference chemical compound and extracellular protein of the non-virulent mutants of Focr1 , Focr4 and the wild type. Hopefully, the results of this proposed study will establish a basis for further research to elucidate the molecular regulation network of pathogenicity of Focr1 and Focr4, leading to accelerated progress in the development of new and efficient control measures to solve the banana wilt problem.

香蕉枯萎病是香蕉的毁灭性土传维管束病害，尚无十分有效的防治方法，给我国香蕉产业造成了严重危害，其病原菌的致病分子机理至今不清楚，分离致病相关基因鉴定其功能是厘清香蕉枯萎病菌致病机理的切入点。本项目在前期已建立香蕉枯萎病菌1、4号小种T-DNA突变体库，通过本实验室建立的致病性高效测定方法，筛选获得一批致病性丧失突变体及已完成香蕉枯萎病菌1、4号小种全基因组测序，获得全基因组数据的基础上，通过致病相关基因cDNA全长序列克隆、基因敲除/互补分析、致病性丧失突变体生物学表型缺陷分析、基因编码产物亚细胞荧光定位、比较表达谱测序分析、致病相关基因酵母表达与产物纯化分析、差异化合物比较分析和胞外分泌蛋白比较分析等技术方法，克隆香蕉枯萎病菌1、4号小种中的多数致病相关基因，并明确其具体生物学功能，可为进一步构建香蕉枯萎病菌致病性状调控分子机制网络奠定基础，并为其防控新策略的制定提供理论依据

香蕉枯萎病是香蕉的毁灭性土传维管束病害，尚无十分有效的防治方法，给我国香蕉产业造成了严重危害，其病原菌致病分子机理至今不清楚，分离致病相关基因鉴定其功能是厘清香蕉枯萎病菌（Fusarium oxysporum f.sp.cubense, Foc）致病机理的切入点。本项目通过Focr4-1562、Focr4-1366、Focr4-1701、Focr4-1453等4个致病减弱突变体的T-DNA插入失活的克隆鉴定，分别克隆出4个致病相关基因mpfo1、mpfo2、mpfo3、mpfo4；通过Focr1的致病性丧失突变体Focr1-N2-328克隆出致病相关基因mpfo5。对Focr4-1366P、Focr4-1453P、Focr1-N2-328P及其野生型菌株Focr4、Focr1-N2进行了比较表达谱测序分析，结果表明在Focr4-1366P中有27个基因表达下调、57个基因表达上调，在Focr4-1453P中有13个基因表达下调、40个基因表达上调，在Focr1-N2-328P中有42个基因表达下调、31个基因表达上调。发明了敲除基因原位互补载体构建方法，选取比较表达谱分析结果中上调或下调幅度最大的11个基因构建成功11个基因敲除载体和8个敲除基因的原位互补载体。对香蕉枯萎镰刀菌1、4号小种镰刀菌酸胞内生物合成途径进行了研究，成功推导出镰刀菌酸胞内生物合成途径，找出了镰刀菌酸合成途径中的关键步骤和关键酶，通过对关键酶基因敲除子和互补子的致病性测定，证实了镰刀菌酸是香蕉枯萎镰刀菌1、4号小种的主效毒素和共性终端致病因子，氨基羧基己二烯二酸半醛脱羧酶为合成关键酶，克隆了关键酶基因(暂命名为mpfo6)。上述研究结果明确了香蕉枯萎镰刀菌的共性致病机制，为香蕉枯萎病抗病育种和开发新的防治药物提供了新的靶标，为进一步研究香蕉枯萎镰刀菌不同专化型和小种致病性遗传变异特异机制奠定了基础。