香蕉枯萎病菌内源报告基因Foc4carS的鉴定及其应用

Identification and Applification of an Endogenous Reporter Gene Foc4carS in Fusarium oxysporum f.sp.cubense race 4

香蕉枯萎病是由尖孢镰刀菌古巴转化型(Fusarium oxysporum f. sp. cubense, Foc)引起的香蕉毁灭性土传病害,其中4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。carS基因通过调控下游car结构基因参与调控镰刀菌类胡萝卜素的生物合成,本研究克隆鉴定了Foc4carS基因(FOIG_05085),Foc4carS蛋白具有典型的RING-finger蛋白结构域。利用分割标记法(Split-marker PCR)获得Foc4carS基因的融合片段,同时构建含有Foc4carS基因sgRNA591序列的pUC-fFuCas9-HTBNLS-hph-Foc4carS基因编辑载体,通过PEG介导的原生质体转化获得该基因的敲除突变体、回补突变体以及基因编辑敲除体,并对敲除和回补突变体的生物学特性和致病力进行分析。结果显示:ΔFoc4carS突变体的菌落直径、产孢量和致病力等生物学表型与野生菌株Foc4无显著差异,而ΔFoc4carS突变体菌落颜色呈深橙色,Foc4carS基因的缺失影响了次生代谢产物类胡萝卜素的生物合成;基因编辑的ΔFoc4carS(HDR)突变体不论是再生筛选板还是继代后的PDA平板,其菌落均出现典型的深橙色,表明Foc4carS可作为内源报告基因,在香蕉枯萎菌Foc4中进行基因质粒型CRISPR/Cas9编辑可行。

Fusarium oxysporum f.sp.cubense(Foc),the causal agent of Fusarium wilt(Panama disease),is one of the most devastating diseases of banana(Musa spp.).The Foc race 4(Foc4)is currently known as a major concern in global banana production.carS is involved in the biosynthesis of Fusarium carotenoid by regulating downstream car structural genes.In this study,Foc4carS(FOIG_05085)was cloned and identified.Foc4carS had a typical RING-finger protein domain.The fusion fragment of Foc4carS was obtained by using Split-marker PCR,and the pUC-fFuCas9-HTBNLS-hph-Foc4carS gene editing vector containing the sequence of sgRNA591 of Foc4carS was constructed,the PEG-medi-ated protoplast transformation was used to obtain the knockout mutants,the complement mutants and the gene-editing mutants.The biological characteristics and pathogenicity of the knockout mutants and the complement mutants were analyzed.The results showed that the colony diameter,sporulation and pathogenicity ofΔFoc4carS mutants were not significantly different with wild strain Foc4,but the colony color ofΔFoc4carS mutant was dark orange,deletion of the Foc4carS gene affected the biosynthesis of the secondary metabolite carotenoid,and gene-editedΔFoc4carS(HDR)mutants showed typical dark orange coloration in both regenerative screening plates and subcultured PDA plates.The results indicated that Foc4carS could be used as an endogenous reporter gene to perform gene plasmid CRISPR/Cas9 editing in F.oxysporum f.sp.cubense Foc4.