The Mechanism Of Transcription Factor NbNAC1 Regulates Plants Systemic Resistance Against Virus Invasion And Preparation Of Its Polyclonal Antibody

In the face of ever-changing environments in plants,the gene expression regulatory network is involved in almost all processes.Moreover,It is well established that NAC transcription factors play essential roles in response to biotic stress,such as drought,high salt,low temperature and other biological stress.However,the molecular mechanism how NAC transcription factors regulate plant hormones signal transduction pathway in response to virus infection is unknown.The first part,we studied that NAC transcription factors regulate plant hormones signal transduction pathway in response to virus infection.In this study,NbNAC1 isolated from Nicotiana benthamiana.We successfully cloned the full-length sequence of NbNAC1 by RT-PCR.The results of phylogenetic tree construction showed that NbNACl is closely related to the stress factor tomato S1NAC1 and pepper CaNAC1.Colocalization experiments indicate that NbNACl is located in the nucleus.Yeast one-hybrid experiments demonstrated transcriptional activation activity at the C-terminus.Expression of NbNAC1 was significantly induced by infection with tobacco mosaic virus(TMV).We use a virus-induced gene-silencing-based genetics approach to investigate the function of NbNAC1.Compared with the control,the virus was abundantly accumulated and the reactive oxygen species erupted in the NbNAC1-silenced.This indicates that restrain expression of NbNAC1 may decrease plant resistance.As a result,the content of salicylic acid(SA)was significantly decreased in the NbNAC1-silenced.And the expression of SA biosynthetic and signaling genes NbICS1,NbNPR1 and defense genes NbPRl,NbPR2 and NbPR5 was significantly decreased.Simultaneous deletion of NAC signal and salicylic acid signal in Nicotiana benthamiana plants increased susceptibility to TMV,Malondialdehyde and conductivity also indicate more serious damage to the cell membrane,indicating that the systemic acquired resistance of plants was significantly weakened.However,SA treatment can partially restore the systemic resistance of tobacco that lacks the NAC signal and enhance the resistance of the plant to viruses.Taken together,our results demonstrate that NbNAC1 regulation the SA in mediating systemic resistance response to virus invasion.Our studies will provide a theoretical foundation for using genetic engineering to improve crop resistance,cultivating good varieties and enhancing the plants resistance to pathogens.In order to study the interaction between NbNAC 1 and SA in detail,we establish two methods of purification of NbNAC1 protein expressed in a prokaryotic system and obtain the purified protein.We constructed pET28a-NbAAC1 and pGEX-4T-1-NbNAC1 vectors and expressed them by E.coil Rosetta(DE3)strain.The target protein was most expressed under induction by 1 mM IPTG,at 37?.SDS-PAGE showed that the pGEX-4T-1-NbNAC1 vector expressed soluble protein,which was mainly concentrated in the supernatant and could be directly purified by Glutathione Resin.The recombinant protein expressed by the pET28a-NbNAC1 vector is present in the inclusion body.This study explored two sets of solutions to the problem of prokaryotic expression of inclusion body proteins.We successfully purified the NbNAC1 protein and prepared its polyclonal antibody.These results lay the foundation for the follow-up research of the interaction between NbNAC and SA,and also provide a new strategy for solving the inclusion problem in prokaryotic expression systems.