生物技术通报2025，Vol.41Issue(8)：186-196,11.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0084
NbJAZ3在苜蓿花叶病毒侵染本氏烟过程中的作用
The Role of NbJAZ3 in the Infection of Nicotiana benthamiana by Alfalfa Mosaic Virus
摘要
Abstract
[Objective]To explore the effect of NbJAZ3,a gene encoding jasmonate ZIM-domain protein 3(JAZ3),on the infection of Nicotiana benthamiana by alfalfa mosaic virus(AMV),and to provide a theoretical basis for further analyzing the mechanism of AMV infection.[Method]Using N.benthamiana and AMV as materials,AMV was inoculated into N.benthamiana by friction inoculation method.The leaves of N.benthamiana at 1,7,15 and 21 d after inoculation were used for transcriptome sequencing analysis.The differential genes in jasmonic acid(JA)signal transduction pathway were screened,and the expression of NbJAZ3 in AMV infection was detected by real-time fluorescence quantitative PCR(RT-qPCR).NbJAZ3 was cloned,biological information was analyzed,phylogenetic tree was constructed,and multiple sequence alignment was performed.The transient overexpression vector of pCAMBIA1300-NbJAZ3 was constructed.The subcellular localization of NbJAZ3 protein was observed by laser confocal microscopy,and the protein expression was detected by Western blot.NbJAZ3 was transiently overexpressed in N.benthamiana leaves and inoculated with AMV,and the relative expression of AMV CP at 5 d after inoculation was detected by RT-qPCR.The pTRV2-NbJAZ3 silencing vector was constructed by gene silencing technology induced by tobacco brittle fracture virus.After silencing NbJAZ3,AMV was inoculated by friction.The relative expression of AMV CP was detected by RT-qPCR.[Result]Transcriptome sequencing and RT-qPCR further verified that NbJAZ3 was down-regulated on different days after AMV inoculation,and the relative expression of NbJAZ3 was the lowest on the day15,which was 84.43%lower than that of CK.The NbJAZ3 CDS sequence was 1 143 bp in length,encoding 380 amino acids,containing two conserved domains of ZIM and Jas-motif.NbJAZ3 had the highest homology with NsJAZ3 of Nicotiana sylvestris,and the similarity was 96.58%.Microscopic observation showed that NbJAZ3 was localized in the nucleus,cytoplasm and cell membrane of N.benthamiana leaves.Compared with the control group,the relative expression of AMV CP decreased by 83.82%and increased by 78.58%in N.benthamiana with transient overexpression and transient silencing of NbJAZ3 on the day 5 of AMV inoculation,respectively.[Conclusion]AMV infection can cause the down-regulation of NbJAZ3 in N.benthamiana.Transient overexpression of NbJAZ3 can inhibit the infection of AMV,while silencing this gene can promote the infection of AMV,further indicating that NbJAZ3 can be used as a positive regulator of N.benthamiana resistance to AMV infection and play an important role in resisting virus infection.关键词
苜蓿花叶病毒/侵染/本氏烟/NbJAZ3/亚细胞定位/瞬时过表达/基因沉默/生物信息学分析Key words
alfalfa mosaic virus(AMV)/infection/Nicotiana benthamiana/NbJAZ3/subcellular localization/transient overexpression/virus-induced gene silencing(VIGS)/bioinformatics analysis引用本文复制引用
赖诗雨,梁巧兰,魏列新,牛二波,陈应娥,周鑫,杨思正,王博..NbJAZ3在苜蓿花叶病毒侵染本氏烟过程中的作用[J].生物技术通报,2025,41(8):186-196,11.基金项目
国家自然科学基金项目(31860039) （31860039）
