不结球白菜BcCHC1蛋白参与调控TuMV侵染的初步研究
Preliminary studies on the involvement of BcCHC1 in the regulation of TuMV infection in non-heading Chinese cabbage
刘芳 1吕善武 2袁梦果 1杨紫蕊 1侯喜林 1张昌伟1
作者信息
- 1. 南京农业大学园艺学院,南京 210095
- 2. 南京农业大学园艺学院,南京 210095;中国科学院华南植物园,广州 510650
- 折叠
摘要
[目的]为增强不结球白菜对芜菁花叶病素养(TuMV)的抗性,研究探讨了不结球白菜中BcCHC1蛋白与TuMV之间的相互作用机制.[方法]首先从不结球白菜中鉴定出重链网格蛋白(CHC)基因家族的成员BcCHCs,随后克隆了 1个代表性的CHC1基因,并对其进行了亚细胞定位分析.通过双分子荧光互补实验(BiFC)筛选出与BcCHC1蛋白相互作用的TuMV蛋白,并利用病毒诱导的基因沉默技术(VIGS)来沉默BcCHC1基因,以评估其功能.[结果](1)成功克隆了不结球白菜的BcCHC1基因,该基因的编码序列长5 124碱基对,编码1 708个氨基酸.(2)在TuMV感染不结球白菜30 d后,实时荧光定量PCR分析显示,感染TuMV的不结球白菜中BcCHC1基因的表达量显著降低.(3)亚细胞定位研究表明,BcCHC1蛋白主要定位于烟草表皮细胞的细胞膜和细胞核.(4)BiFC实验进一步证实,BcCHC1蛋白能够与TuMV的CI和6K2蛋白发生相互作用,其中与CI的互作主要发生在细胞核内,而与6K2的互作则主要位于细胞膜.(5)通过对BcCHC1基因沉默株系的观察发现,沉默Bc-CHC1基因会导致植株死亡.[结论]BcCHC1蛋白通过与TuMV的CI和6K2蛋白相互作用,影响网格蛋白依赖的内吞作用途径及病毒复制,从而调控TuMV对不结球白菜的侵染.然而,BcCHC1基因调控TuMV侵染不结球白菜的具体机制仍需进一步研究.
Abstract
[Objective]To enhance the resistance of non-heading Chinese cabbage to turnip mosaic virus(TuMV),this study preliminarily explored the interaction mechanism between BcCHC1 protein of non-heading Chinese cabbage and TuMV.[Methods]The study firstly identified members of the heavy chain clathrin(CHC)gene family,BcCHCs,from non-heading Chinese cabbage,and then cloned CHC1 gene.BcCHC1 subcellular localization was analyzed,and bimolecular fluorescence complementation(BiFC)as-say was used to verify the interaction between TuMV and BcCHC1.Finally,virus-induced gene silencing(VIGS)was employed to silence BcCHC1 expression and its function was assessed.[Results]BcCHC1 gene from non-heading Chinese cabbage was cloned,with a coding sequence length of 5 124 bp,encoding 1 708 amino acids.qRT-PCR results showed a significant decrease in the relative expression level of Bc-CHC1 gene in non-heading Chinese cabbage infected with TuMV after 30 days.Subcellular localization re-vealed that BcCHC1 protein was located in the cell membrane and nucleus of tobacco epidermal cells.BiFC assay analysis confirmed that BcCHC1 interacted with CI and 6K2:Interaction with CI was mainly occur-ring in the nucleus,while interaction with 6K2 mainly on the cell membrane.Observations of BcCHC1-si-lenced lines showed that silencing BcCHC1 gene led to plant death.[Conclusion]BcCHC1 regulates the infection of non-heading Chinese cabbage by TuMV through interaction with CI and 6K2,thereby affecting the clathrin-dependent endocytosis pathway and virus replication.However,the mechanism by which Bc-CHC1 regulates TuMV infection in non-heading Chinese cabbage requires further study.
关键词
不结球白菜/BcCHC1/TuMV/互作蛋白/VIGSKey words
non-heading Chinese cabbage[Brassica campestris(syn.Brassica rapa)ssp.chinensis]/TuMV/BcCHC1/protein interaction/VIGS引用本文复制引用
基金项目
江苏省自然科学基金(BK20200560)
江苏省农业科技自主创新项目(CX193038)
出版年
2024
国家科技期刊平台
NSTL
万方数据