水稻立枯病原菌尖孢镰孢菌致病力降低T-DNA突变体的筛选与插入位点鉴定  

Screening and insertion site identification of T-DNA mutants with reduced pathogenicity of Fusarium oxysporum Schelcht

作　　者：张俊华[1] 杨松润 彭莉莉 王亮[1] 杨明秀[1] 徐晓凤 倪哲 李培宁 ZHANG Junhua;YANG Songrun;PENG Lili;WANG Liang;YANG Mingxiu;XU Xiaofeng;NI Zhe;LI Peining(School of Agriculture,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学农学院,哈尔滨150030

出　　处：《东北农业大学学报》2022年第11期10-18,共9页Journal of Northeast Agricultural University

基　　金：黑龙江省自然科学基金项目(C2017032);黑龙江省应用技术研究与开发计划项目(GA19B104);国家重点研发计划项目(2018YFD0300105)。

摘　　要：为深入研究水稻立枯病原菌尖孢镰孢菌(Fusarium oxysporum Schelcht)致病分子机理,利用根癌农杆菌介导遗传转化技术构建尖孢镰孢菌突变体库,筛选致病力降低突变体,检测其T-DNA插入数量和位点。结果表明,691个突变体库构建成功,且突变体可稳定遗传后代。选取34个不同菌落形态变化突变体测定其致病力,筛选到6个致病力降低最严重突变体,分析其菌落形态、菌落生长速率和产孢量。与野生型相比,菌落形态存在差异,其中4个突变体B9、C36、D65、E17生长速率降低,6个突变体产孢数量均降低。通过Southern blot分析发现突变体B92有两个T-DNA插入,其余5个突变体为单个T-DNA插入,利用Hi TAIL-PCR确定6个突变体T-DNA插入位点。结果有助于更好理解尖孢镰孢菌与水稻之间分子相互作用,为进一步确定尖孢镰孢菌致病基因及其功能提供参考。To research the pathogenic molecular mechanism of Fusarium oxysporum Schelcht,the mutants libraries of Fusarium oxysporum were constructed by Agrobacterium tumefaciens-mediated transformation.Then,mutants with reduced pathogenicity were screened and their T-DNA insertion number and sites were detected.The results showed that the 691 mutant libraries were successfully constructed and the mutants could be stably passed on to the offspring.The pathogenicity of the 34 mutants with abnormal phenotype was determined.A total of six mutants with significantly reduced pathogenicity were screened and their colony morphology,growth rate and sporulation quantity were analyzed.Compared with the wild type,the colony morphology was different,the growth rate of the four mutants B9,C36,D65,and E17 decreased,and the sporulation quantities of the six mutants decreased.Southern blot analysis showed that there were two T-DNA insertions in mutant B92,and the other five mutants were single T-DNA insertion,the T-DNA insertion sites of the six mutants were determined by Hi TAIL-PCR.These results contributed to a better understanding of the molecular interactions between Fusarium oxysporum and rice,and provided references for further identification of pathogenic genes and their functions of Fusarium oxysporum Schelcht.

关 键 词：水稻立枯病 尖孢镰孢菌 T-DNA 致病力 插入位点 

分 类 号：S435.111.46[农业科学—农业昆虫与害虫防治]