Abstract:

Fusarium verticillioides is a major pathogen responsible for maize ear and stalk rot， posing a serious threat to maize yield and quality. To investigate the gene function and improve frequency of gene knock-out in F. verticillioides， two key genes in the non-homologous end joining （NHEJ） pathway， FvKu70 and FvKu80， were individually knocked out. A comparative analysis was conducted on various aspects， including vegetative growth rate， colony morphology， conidiation， pathogenicity on maize， and knockout efficiency. The results showed that ΔFvKu70 and ΔFvKu80 had no significant differences in their morphological characteristics （mycelium morphology， vegetative growth rate， conidiation， and colony diameter） on the PDA plate. Moreover， they displayed similar pathogenicity in maize stalks compared to the wild-type strain FvLNF15-11. Notably， the frequency of homologous recombination was significantly higher in the deletion mutant strains of ΔFvKu70 and ΔFvKu80 compared to the wild type， and ΔFvKu70 exhibited the highest efficiency of homologous recombination. By successfully constructing ΔFvKu70 and ΔFvKu80 deletion mutants， it becomes feasible to rapidly and effectively achieve gene knockout mutants in F. verticillioides， thereby facilitating the study of key gene functions in this pathogen.

Key words: Fusarium verticillioides, gene knockout, homologous recombination, non-homologous end joining

摘要：

拟轮枝镰孢菌（Fusarium verticillioides）是引起玉米茎基腐病和穗粒腐病的主要病原菌之一，严重威胁玉米的产量和品质。为了深入研究拟轮枝镰孢菌致病基因的功能，对该菌中非同源末端连接（non-homologous end joining，NHEJ）途径中的2个关键基因FvKu70和FvKu80分别进行了基因敲除以创制高效的基因敲除菌株，并比较了野生型菌株和突变体菌株在营养生长速率、菌落形态、产孢量、对玉米的致病力和基因敲除效率等方面的差异。研究结果表明，FvKu70和FvKu80的基因缺失突变体与野生型FvLNF15-11相比，在PDA平板上的形态特征（如菌丝形态、生长速率、菌落直径、产孢量）没有明显差异，对玉米茎秆的致病力也类似。此外，选择尿嘧啶生物合成相关基因FvpyrG作为敲除的靶基因，分析了FvKu70或FvKu80缺失突变体菌株的同源重组效率，结果显示突变体菌株均显著高于野生型，其中ΔFvKu70的同源重组效率最高。综上所述，FvKu70或FvKu80基因缺失突变体可以快速又高效地实现拟轮枝镰孢菌的基因敲除，为进一步研究该菌的功能基因提供了技术支持。

关键词: 拟轮枝镰孢菌, 基因敲除, 同源重组, 非同源末端连接