苦瓜枯萎病菌Ⅱ型卤酸脱卤酶基因FoHAD-typeⅡ功能分析  

Function analysis of haloacid dehalogenase typeⅡgene FoHAD-typeⅡin Fusarium oxysporum f.sp.momordicae

作　　者：张园园 文才艺[1] 李凤新 魏晨星 杜红岩 仲荣荣 赵莹 ZHANG Yuanyuan;WEN Caiyi;LI Fengxin;WEI Chenxing;DU Hongyan;ZHONG Rongrong;ZHAO Ying(College of Plant Protection,Henan Agricultural University,Zhengzhou 450002,China;Yulin Customs Comprehensive Technology&Service Center,Yulin 537000,China)

机构地区：[1]河南农业大学植物保护学院,郑州450002 [2]玉林海关综合技术服务中心,玉林537000

出　　处：《植物病理学报》2023年第2期195-206,共12页Acta Phytopathologica Sinica

基　　金：国家自然科学基金青年科学基金项目(31901934);河南省重点研发与推广专项(科技攻关)项目(192102110052)。

摘　　要：苦瓜枯萎病是由尖孢镰刀菌苦瓜专化型(Fusarium oxysporum f.sp.momordicae Sun&Huang)侵染引起的一种严重制约苦瓜安全生产的土传病害。明确苦瓜枯萎病菌的致病机理对苦瓜枯萎病的防治具有重要的指导意义,但目前苦瓜枯萎病菌的致病机理尚不明确。前期分析苦瓜枯萎病菌强致病力菌株SD-1和弱致病力菌株SD-V(携带真菌病毒)转录组差异表达基因时,发现Ⅱ型卤酸脱卤酶(FoHAD-typeⅡ)基因在SD-V菌株中表达量显著下调,推测该基因与苦瓜枯萎病菌的致病性相关。本研究根据同源重组的原理,利用分割标记法(Split-Marker PCR)获得了FoHAD-typeⅡ基因的融合片段,分别通过PEG介导的原生质体转化和农杆菌介导的遗传转化获得该基因的敲除突变体和回补突变体,并对敲除和回补突变体的生物学特性和致病力进行了分析。结果表明,敲除突变体的生长速率和产孢量与野生型菌株相比没有显著差异,菌丝尖端形态和孢子形态也无明显差异,但气生菌丝减少,对渗透胁迫耐受性降低,致病力显著下降;回补突变体的生物学性状和致病力与野生型菌株一致。表明FoHAD-typeⅡ基因在苦瓜枯萎病菌的致病过程中发挥重要作用,但不参与苦瓜枯萎病菌生长和发育的调控。研究结果为后续探究苦瓜枯萎病菌的致病机制提供理论基础,同时,为研究真菌病毒的弱毒机理提供参考。Fusarium wilt of bitter gourd is a soil-borne disease caused by Fusarium oxysporum f.sp.momordicae Sun&Huang(FOM),which seriously affects the production of bitter gourd.Clarifying the pathogenic mechanism of FOM has important guiding significance for the prevention and control of Fusarium wilt of bitter gourd,but the pathogenic mechanism of FOM is still unclear.In the previous analysis of the differentially expressed genes in the transcriptomes of the strong pathogenic strain SD-1 and the weak pathogenic strain SD-V(carrying mycoviruses)of FOM,it was found that the expression level of FoHAD-type II was significantly down-regulated in SD-V strain.Therefore,we speculated that this gene may be involved in the pathogenic process of FOM.In this study,based on the principle of homologous recombination,the fusion fragment of FoHAD-typeⅡwas obtained by Split-Marker PCR,the gene knockout mutant was obtained by PEG-mediated protoplast transformation,and the complementary mutant was obtained by Agrobacterium transformation method.The biological characteristics and pathogenicity of the mutants were tested and analyzed.The results showed that the growth rate and spore production of the FoHAD-typeⅡgene knockout mutant on the PDA medium were not significantly different from the wild-type strain,and there were no significant differences in the morphology of hyphal tips and spores.However,the vegetative growth of the knockout mutant was defective,manifested as different colony morphology,reduced aerial hyphae,significantly reduced virulence,and increased sensitivity to osmotic stress.The pathogenicity of the complemented mutant was consistent with that of the wild-type strain.Therefore,these findings suggested that FoHAD-typeⅡis not involved in the growth and development of FOM,but plays an important role in the pathogenic process of FOM,which may be related to the decline of pathogenicity of FOM caused by mycovirus.The results of this study provide a basis for the subsequent exploration of the pathogenic mechanism of FOM a

关 键 词：苦瓜枯萎病菌 卤酸脱卤酶 基因功能 致病力 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]