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5124碱基对,编码1708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BcCHC1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。
[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)assay 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 5124 bp,encoding 1708 amino acids.qRT-PCR results showed a significant decrease in the relative expression level of BcCHC1 gene in non-heading Chinese cabbage infected with TuMV after 30 days.Subcellular localization revealed 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 occurring in the nucleus,while interaction with 6K2 mainly on the cell membrane.Observations of BcCHC1-silenced 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 BcCHC1 regulates TuMV infection in non-heading Chinese cabbage requires further study.
作者
刘芳
吕善武
袁梦果
杨紫蕊
侯喜林
张昌伟
LIU Fang;LYU Shanwu;YUAN Mengguo;YANG Zirui;HOU Xilin;ZHANG Changwei(College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;Guangdong Provincial Key Laboratory of Applied Botany and South China Key Laboratory of Molecular Analysis and Genetic Improvement of Agricultural Plants,South China Botanical Garden,Chinese Academy of Sciences,Guangzhou 510650,China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2024年第8期1229-1238,共10页
Acta Botanica Boreali-Occidentalia Sinica
基金
江苏省自然科学基金项目(BK20200560)
江苏省农业科技自主创新资金项目[CX(19)3038]。
关键词
不结球白菜
BcCHC1
TUMV
互作蛋白
VIGS
non-heading Chinese cabbage[Brassica campestris(syn.Brassica rapa)ssp.chinensis]
TuMV
BcCHC1
protein interaction
VIGS
