中国农业科学2025，Vol.58Issue(19)：3799-3813,15.DOI:10.3864/j.issn.0578-1752.2025.19.001
利用酵母双杂交系统筛选与大豆花叶病毒核内含体蛋白互作的大豆寄主因子
Screening for Soybean Host Factors that Interact with Soybean Mosaic Virus Nuclear Inclusion Proteins Using the Yeast Two-Hybrid System
摘要
Abstract
[Objective]Soybean mosaic virus(SMV)is one of the most damaging viral diseases of soybean,which seriously affects soybean yield and quality.Identification of host proteins interacting with SMV nuclear inclusion proteins(NIa-Pro and NIb)using yeast two-hybrid library screening,aiming to establish a theoretical foundation and propose novel perspectives insights into the molecular mechanisms of SMV infection and soybean resistance.[Method]Firstly,the coding sequences of NIa-Pro and NIb were cloned from the SMV strain SMV-HN and recombined into the pGBKT7 vector to construct the bait plasmids,and then soybean proteins interacting with the two viral functional proteins were identified by yeast library screening.Secondly,the host gene GmOEP16 encoding Outer Envelope Pore Protein 16(OEP16)was cloned,and the interactions of GmOEP16 with NIa-Pro and NIb were clarified by yeast two-hybrid(Y2H)and luciferase complementation assay(LCA).Quantitative real-time PCR(qRT-PCR)was used to analyse the expression pattern of GmOEP16 under SMV treatment and exogenous hormone induction.Finally,virus-induced gene silencing(VIGS)was used to validate the function of GmOEP16 gene in SMV disease response.[Result]pGBKT7-NIa-Pro and pGBKT7-NIb recombinant plasmids were successfully constructed,and 12 soybean host proteins were screened for interactions with NIa-Pro and NIb,respectively.The Y2H assay was further used to verify that NIa-Pro interacted with GmOEP16 and GmDEG5,and NIb interacted with GmOEP16,GmZC3H18 and GmAHP1.The LCA assay was further used to clarify that GmOEP16 interacted with both NIa-Pro and NIb.Expression analysis revealed that GmOEP16 was induced by SMV infection and responded rapidly to salicylic acid(SA)and abscisic acid(ABA)stimuli during early response.The VIGS assay showed that effectively silencing of GmOEP16 resulted in no obvious susceptibility phenotype in leaf tissues relative to the wild-type controls.Meanwhile,the expression of SMV-CP was significantly reduced in the GmOEP16-silenced plants,suggesting that the soybean resistance to SMV was enhanced.Collectively,these findings demonstrated that GmOEP16 could function as a negative regulator of SMV resistance in soybean.[Conclusion]The pGBKT7-NIa-Pro and pGBKT7-NIb bait vectors were successfully constructed,and each 12 soybean host proteins that respectively interacted with pGBKT7-NIa-Pro and pGBKT7-NIb were identified.Among them,GmOEP16 interacted with both NIa-Pro and NIb.GmOEP16 responded to SMV induction and negatively regulated SMV resistance,which promoted SMV infection on soybeans.关键词
大豆花叶病毒/核内含体蛋白/酵母文库筛选/外膜孔蛋白16/病毒诱导的基因沉默Key words
soybean mosaic virus/nuclear inclusion proteins/yeast library screening/outer envelope pore protein 16/virus-induced gene silencing引用本文复制引用
于哲,李海燕,周芳雪,刘润发,田雅琦,吉好木哈,王勇翔,冯雯蜜,牟可欣,井妍..利用酵母双杂交系统筛选与大豆花叶病毒核内含体蛋白互作的大豆寄主因子[J].中国农业科学,2025,58(19):3799-3813,15.基金项目
三亚崖州湾科技城科技专项资助(SCKJ-JYRC-2023-17)、海南省自然科学基金(323RC413,324MS013)、国家自然科学基金(32301921)、海南省研究生创新科研课题(Qhys2024-139) （SCKJ-JYRC-2023-17）
