摘要
【目的】通过研究辅助成分-蛋白酶(helper component-proteinase,HC-Pro)中第364位保守的赖氨酸(lysine,K364)突变对小西葫芦黄花叶病毒(zucchini yellow mosaic virus,ZYMV)致病力的影响,构建ZYMV弱毒突变体,并评估其遗传稳定性和交叉保护防治效果,为利用弱毒株开发针对ZYMV的绿色防治策略提供理论依据。【方法】利用定点突变的方法,将HC-Pro中K364位点替换为天冬氨酸(aspartic acid,D),研究K364D突变对ZYMV致病力和HC-Pro RNA沉默抑制活性的影响,构建弱毒突变体ZYMV-HC-ProK364D。通过继代接种实验,测试弱毒突变体ZYMV-HC-ProK364D的遗传稳定性;通过交叉保护实验,评估弱毒突变体ZYMV-HC-ProK364D的交叉保护防治作用。【结果】HC-Pro中K364D突变影响了ZYMV致病力,减轻了病毒在甜瓜(Cucumis melo)植株上引起的典型黄化症状,显著降低了病毒积累水平,同时,该突变削弱了ZYMV HC-Pro的RNA沉默抑制活性。ZYMV-HCProK364D-GFP突变体在甜瓜植株中连续3次继代接种后未发生回复突变,保持遗传稳定性。在交叉保护实验中,ZYMV-HC-ProK364D突变体在保护间隔期11 d时,可诱导甜瓜植株产生对野生型ZYMV的显著抗性。【结论】HC-Pro中保守的K364残基是影响ZYMV致病力和HC-Pro RNA沉默抑制活性的关键位点,ZYMV-HC-ProK364D突变体在甜瓜中具有良好的交叉保护作用,可作为弱毒疫苗防治甜瓜ZYMV侵染。
【Objective】By investigating the effects of the mutation at the 364th conserved lysine(K364)in helper component-proteinase(HC-Pro)on the virulence of zucchini yellow mosaic virus(ZYMV),an attenuated ZYMV mutant were developed,and its genetic stability and cross-protection efficacy were assessed.Our study provided the theoretical basis for developing sustainable control strategies against ZYMV using attenuated strains.【Method】Using site-directed mutagenesis,the K364 residue in HC-Pro was substituted with aspartic acid(D)to investigate the effect of the K364D mutation on the virulence of ZYMV and the RNA silencing suppression activity of HC-Pro,thereby constructing the attenuated ZYMV-HC-ProK364D mutant.Furthermore,the genetic stability of ZYMV-HC-ProK364D mutant was assessed through serial passage of progeny viruses.The cross-protection efficacy of ZYMV-HC-ProK364D mutant was evaluated through cross-protection assays.【Result】The K364D mutation in HC-Pro attenuated ZYMV virulence,alleviated typical yellowing symptoms and reduced ZYMV accumulation in Cucumis melo plants.Simultaneously,K364D mutation suppressed the RNA silencing suppression activity of ZYMV HC-Pro.The attenuated mutant ZYMV-HC-ProK364D-GFP maintained genetic stability without reversion mutation after three successive passages in C.melo plants.In cross-protection assays,ZYMV-HC-ProK364D mutant induced significant resistance in C.melo plants against the wild-type ZYMV infection when the protection interval was 11 d.【Conclusion】The conserved K364 residue in HC-Pro was crucial in determining the RNA silencing suppression activity and the virulence of ZYMV.The ZYMV-HC-ProK436D mutant present excellent cross-protection efficacy and is a potential mild vaccine against the wild-type ZYMV infection in C.melo plants.
作者
张悦
任倩
栾雅梦
李翌琛
孙无瑕
任姝颖
许小洁
孙晓辉
ZHANG Yue;REN Qian;LUAN Ya-meng;LI Yi-chen;SUN Wu-xia;REN Shu-ying;XU Xiao-jie;SUN Xiao-hui(Laboratory of plant Virology,The Engineering Research Institute of Agriculture and Forestry,Ludong University,Yantai 264025;Yantai Key Laboratory of Molecular Breeding for High-Yield and Stress-Resistant Crops and Efficient Cultivation,Yantai 264025;Shandong Yantai Acad-emy of Agricultural Sciences,Yantai 265500)
出处
《生物技术通报》
北大核心
2026年第1期271-278,共8页
Biotechnology Bulletin
基金
山东省重点研发计划项目(2023LZGC002)
国家自然科学基金项目(32302310)
山东省自然科学基金项目(ZR2023QC094)
山东省烟台市高校与地方企业合作项目(2021XDRHXMPT09)。
