Inhibition And Application Of Nicotiana Benthamiana Transcription Factor NAC062 On Potato Virus Y Infection

Potato virus Y(PVY)is one of the most destructive viruses in tobacco.NAC transcription factors are closely related to plant growth,development,disease resistance and stress resistance.Clarifying the function of NAC062 in PVY infection can provide a target for research of antiviral agents.In this study,Nicotiana benthamiana is used as the material to explore the regulatory role of Nb NAC062 transcription factor in PVY infection and to develop anti-PVY nano-pharmaceuticals.The main research results are as follows:1.Nb NAC062 is cloned,and it encodes 646 amino acids,the N-terminal 28～179 aa is the NAC domain,and 129～185 aa is the DNA-binding region,C-terminal 621～643 aa is a hydrophobic transmembrane structure.Nb NAC062 is closely related to Na NAC062 of involuntary tobacco.The Nb NAC062 promoter contains phytohormone response elements of ABRE,TGACG-motif,CGTCAmotif,as-1,as well as resistance-related elements of DRE,MYB,and MYC,suggesting that Nb NAC062 is related to plant growth,development and resistance.2.The RFP-Nb NAC062 fusion protein is constructed to infiltrate the leaves.It is observed that Nb NAC062 is localized in the cell membrane under the confocal laser-scanning microscope,and PVY infection activated its transfer from the cell membrane to the nucleus.Quantitative real-time PCR(q RTPCR)analysis of Nicotiana benthamiana inoculation with PVY showed that: the level of Nb NAC062 m RNA in the PVY treatment group is significantly higher than that in the control group at 5 and 7 days,and the expression in the treatment group are 2.52 times and 1.95 times that of the control group.Both protein translocation and up-regulation of m RNA expression show that Nb NAC062 responds to PVY infection.3.p TRV-Nb NAC062 silencing vector and p Earley Gate100-RFP-Nb NAC062 over expression vector are constructed to infiltrate leaves by virus-induced gene silencing(VIGS)and transient over expression.After PVY inoculation,q RT-PCR and Western Blot detection found that: at 3,5,and 7 days after vaccination,PVY CP m RNA in the silenced group is significantly higher than that in the empty vector TRV00 control group,with 2.12 times,2.41 times,and 1.38 times that of the control group,and downregulated expression of Bi P(immunoglobulin-binding protein gene)by 28.19%,58.11%,10.77%;The PVY CP protein content of the silence group is significantly higher than that of the control group at 5～7days,and the fluorescence intensity of PVY-GFP is also higher than that of the control group.Overexpressing Nb NAC062 and inoculating PVY.24,48,72 hours after inoculation,compared with the control group,the expression of PVY CP m RNA in the overexpression group are down-regulated by22.60%,34.51%,36.21%.And Bi P m RNA are up-regulated at 48 and 72 hours after inoculation.They are 1.56 times and 1.35 times that of the control group,respectively.The content of PVY CP in the overexpression group is also lower than that of the control group.The above results show that Nb NAC062 has an inhibitory effect on PVY infection and may play a role in regulating the expression of Bi P.Creating Nb NAC062 knockout and overexpression materials.Under normal conditions,there is no significant difference in seedling emergence between mutants and wild type.4.Nano-drugs are created by coating Nb NAC062 with chitosan quaternary ammonium salt(HACC).The preventive effect of the drugs on PVY infection is detected by using HACC as control.Nicotiana benthamiana are infiltrated with Nb NA062-HACC nanomedicine or HACC,then are inoculated with PVY.q RT-PCR detection showed that the PVY CP m RNA level in the nanomedicine treatment group is decreased by 17.41%,47.81%,13.03% compared to HACC control group,at 5,7,9 days after inoculation,and there is no difference after that.7～8 days after PVY-GFP inoculation in the treatment group,the fluorescence intensity of PVY is significantly darker than that in the control group,and the difference is not obvious after 10 days.In this study,Nb NAC062-HACC nanomedicine is created for the first time,and the prevention effect test shows that nanomedicine could inhibit the infection in the early stage of PVY.Drug preparation and application methods of Nb NAC062-HACC can be optimized to prevent viral disease.In summary,Nb NAC062 is a NAC transcription factor,which is related to plant disease resistance and stress resistance;Nb NAC062 may inhibit the early infection and proliferation of PVY by regulating the expression of Bi P;Nb NAC062 knockout and overexpression materials are created,which can be used for disease resistance,stress resistance,and enrich germplasm resources;Nb NAC062-HACC inhibits the initial infection of PVY and delays the speed of virus proliferation,and can be used for the prevention of PVY in the future.