轮枝镰孢菌FvST12基因敲除载体的构建及功能分析

Gene Knockout Vector Construction and Function Assay of FvST12 in Fusarium verticillioides

【目的】鉴定并分析轮枝镰孢菌中STE12类转录因子的基因功能。【方法】以尖孢镰孢菌转录因子Fost12蛋白为靶序列,在轮枝镰孢菌基因组数据库中进行BlastP搜索,发现一个与Fost12蛋白同源性高达98%的基因,命名为FvST12。基于Double-Joint PCR技术,构建该基因的敲除载体,通过真菌原生质体转化及筛选获得基因敲除突变体,并对突变体的表型及致病性进行系统分析,以明确该基因的功能。【结果】突变体在生长速率、菌落形态、产孢量以及高渗和氧化等逆境胁迫反应上与野生型无显著差异;致病性分析表明,突变体致病力明显下降。【结论】轮枝镰孢菌FvST12对致病性起重要调控作用,但不参与营养生长、产孢和高渗和氧化胁迫等逆境反应。

【Objective】The objective of this study is to identify and analyze the STE12 homolog transcription factor gene in Fusarium verticillioides.【Method】 The F.oxysporum Fost12 transcription factor was used as a target sequence to search its homolog by BlastP in F.verticillioides genome sequence.FVEG_05267.3,a predicted gene in F.verticillioides,was identified and it showed 98% similarity to Fost12.It was called FvST12 in this study.Based on the Double-Joint PCR,the knockout vector was constructed and null knockout mutants were generated through fungal protoplast transformation.The gene knockout mutants were confirmed by PCR and Southern blot.Furthermore,the function of the gene in F.verticillioides was systematically characterized 【Result】Compared with the wild type,Fvst12 mutant has normal growth rate,condiation,colony morphology,and stress responses（osmotic and oxidative stress） tested in this study.However,the mutant reduced the virulence on maize ear and stalk.【Conclusion】The transcription factor FvST12 is an important virulence factor but it isn＇t involved in the vegetative growth,conidiation,osmotic and oxidative stress responses in F.verticillioides.