Abstract: 【Objective】Potato Y virus (PVY) is one of the most important viruses harmful to tobacco production in China. NAC transcription factors are closely related to plant disease resistance and stress resistance. This paper clones NbNAC062, analyzes its bioinformatics and studies its role in the process of PVY infection, which provides a target for the development of tobacco antiviral agents. 【Method】Using Nicotiana benthamiana as the material to clone NbNAC062, and using MEGA, UniProt, SMART, TMHMM2.0Server, Sol Genomics Network, PlantCARE and other technologies for bioinformatics analysis; Laser confocal microscope and quantitative real-time PCR (qRT-PCR) are used to clarify the localization of NbNAC062 protein and the change of NbNAC062 mRNA expression before and after PVY infection; Based on virus-induced gene silencing (VIGS) technology and overexpression technology, the pTRV::NbNAC062 silencing vector and the pEarleyGate100::RFP::NbNAC062 overexpression vector are constructed. qRT-PCR and Western Blot are used to detect the changes of PVY accumulation and the expression of unfolded protein response (UPR) related gene BiP after silencing and overexpression in Nicotiana benthamiana.【Result】NbNAC062 encodes 646 amino acids, the N-terminal 28-179 aa is the NAC domain, 129-185 aa is the DNA bonding region, and the C-terminal 621-643 aa is a hydrophobic transmembrane structure. Phylogenetic tree and protein sequence analysis show that Nicotiana benthamiana NbNAC062 is closely related to Nicotiana attenuata NaNAC062. The NbNAC062 promoter contains a variety of cis-acting elements related to abscisic acid, methyl jasmonate, salicylic acid and stress response. PVY infection activates NbNAC062 to transfer from cell membrane to nucleus and induces NbNAC062 up-regulation of expression. For 5 and 7 days after PVY infestation, the NbNAC062 mRNA levels in the treatment group were 2.52 and 1.95 times that of the control group; PVY infection 3 days, the BiP mRNA expression was 2.39 times that of the control group, and PVY infection 7 days. The expression of BiP was significantly lower than that of the control group, down-regulated by 56.77%. Silencing NbNAC062 and inoculating PVY. Compared with the control group, 3, 5, and 7 days after inoculation, the expression of PVY CP mRNA was up-regulated in the silence group, which was 2.12, 2.41, and 1.38 times of that of the control group, respectively. However, the expression of BiP mRNA was down-regulated by 28.19%, 58.11%, and 10.77%, respectively. The PVY CP protein content of the silence group was also significantly higher than that of the control group at 5 and 7 days after vaccination. Overexpressing NbNAC062 and inoculating PVY. 24, 48, 72 hours after inoculation, compared with the control group, the expression of PVY CP mRNA in the overexpression group was down-regulated by 22.60%, 34.51%, 36.21%, and BiP mRNA was up-regulated at 48 and 72 hours after inoculation, which was 1.56 and 1.35 times of that of the control group, respectively. The content of PVY CP in the overexpression group was also lower than that of the control group.【Conclusion】NbNAC062 belongs to the NAC class of membrane-bound transcription factors, which can be activated by PVY infection and transferred to the nucleus. It may regulate the expression of the UPR-related gene BiP to promote cell survival and inhibit early PVY infection.

Key words: NbNAC062, potato virus Y, gene silencing, transient overexpression