苦瓜枯萎病菌过氧化氢酶基因FoCat1致病功能分析

 由尖孢镰孢菌苦瓜专化型(Fusarium oxysporum f.sp.momdicae Sun &Huang)引起的苦瓜枯萎病是一种严重影响苦瓜生产的土传维管束病害。本研究利用分割标记的方法构建FoCat1基因的敲除片段,通过PEG介导的原生质体转化方法对FoCat1基因进行敲除,经4对引物验证得到1个阳性转化子ΔFoCat1-3。突变体ΔFoCat1-3在PDA培养基上的菌落大小、菌丝形态、产孢量等生物学特性与野生型相比均无显著性差异,但是其抵抗外源氧胁迫的能力明显减弱,对苦瓜的致病力明显下降。DAB染色结果显示,接种突变体的苦瓜叶片中过氧化氢的含量明显高于接种野生型菌株的苦瓜叶片。研究结果表明,FoCat1可能不参与病菌正常营养生长过程的调控,但是具有清除寄主产生的活性氧,来减弱寄主对病原菌的杀伤作用的功能,从而参与病原菌的致病过程。

Pathogenic function analysis of catalase gene FoCAT1 from Fusarium oxysporum f.sp.momdicae

The wilt of bitter gourd caused by Fusarium oxysporum f.sp. momdicae is one of the soil-borne vascular diseases,which greatly influences the production of bitter gourd.In this study,the Split-Marker strategy was applied to knockout the FoCAT1 gene through PEG-mediated protoplast transformation with the recombinant fragments.The positive mutant candidate (ΔFoCat1-3) was obtained by PCR sceening with four pairs of primers.Compared with the wild strain,the ΔFoCat1-3 mutant showed no significant differences on colony phenotype and conidiation on PDA medium,while the pathogenicity on bitter gourd seedlings was significantly weakened.Further DAB staining of bitter gourd leaves indicated the significant higher level of H₂O₂ after inoculation of the ΔFoCat1-3 mutant compared to that of wild type.These data suggest that the FoCat1 had no obvious functions on the pathogen vegetative growth,while on the ability of exogenous oxygen stress resistance.The FoCat1 might attenuate the killing effect on pathogens by scavenging reactive oxygen species from the host,taking part in the pathogenesis of F.oxysporum f.sp. momdicae.