Mechanism And Antibody Preparation Of NbLTP1 Regulating Tobacco Systemic Resistance Against TMV Infection

Nonspecific lipid transfer proteins(nsLTPs)is a small basic protein rich in cysteine in higher plants,which belongs to the disease-related protein family.It is reported that they play an important role in various physiological processes of plants,such as lipid transmission,signal transduction and pathogen defense.However,the potential molecular mechanism of these proteins in plant immune response is not clear.In order to study the role of nsLTPs in regulating the response of plants to biotic and abiotic stresses,the full-length transcript of nonspecific lipid transfer protein NbLTP1 was successfully cloned from Nicotiana benthamiana.Its open reading frame is 360 bp in length,encoding 119 amino acids Its molecular weight is 12.44 kDa,and its isoelectric point is 7.45.The structure of NbLTP1 protein was predicted and analyzed.NbLTP1 protein contains eight conserved cysteine residues,two conserved pentapeptide domains,a signal peptide at the N-termini and a transmembrane region It also includes seven α-helix,four disulfide bonds and a stable hydrophobic cavity,which is highly conserved in evolution.In addition,N.benthamiana NbLTP1 is highly homologous to the tobacco NaLTP1.The results above show that NbLTP1 belongs to the Type1 class of the nsLTP family.At present,there are relatively few studies on the heterologous expression,purification and antibody preparation of NbLTP1 in tobacco,which restricts the further research of NbLTP1 to some extent.Therefore,we constructed the prokaryotic expression vector pET28a-NbLTP1 to purify the protein and prepared the polyclonal antibody against NbLTP1.The results showed that on the basis of previous studies,firstly,the N-terminal signal peptide sequence was removed,the recombinant pET28a-NbLTP1 prokaryotic expression vector was constructed,and the NbLTP1 protein was successfully expressed in E.coli strain BL21(DE3).The expression conditions of NbLTP1 protein were optimized,in which the expression level of NbLTP1 protein was the highest when the final concentration of IPTG was 1mM and the induction temperature was 37℃.Secondly,the purified NbLTP1 protein was obtained by Ni-NTA column chromatography.Thirdly,the polyclonal antibody against NbLTP1 protein was prepared after New Zealand white rabbits were immunized with the purified protein,and the titer was detected.The results showed that the titer of the antiserum is good,indicating that we have successfully obtained the anti-NbLTP1 serum,which can be used in the follow-up experiment.In this part,the NbLTP1 protein was successfully purified and the serum was prepared,which lays a foundation for further study of its function.In order for further study of the role of NbLTP1 in preventing tobacco mosaic virus(TMV)infection,we carried out a series of experiments.First of all,we detected the expression of NbLTP1 gene in Nicotiana benthamiana infected by TMV,and the results showed that TMV infection induced the expression of NbLTP1 gene.Secondly,we silenced NbLTP1,and performed real-time quantitative PCR in N.benthamiana by virus-induced gene silencing technique(VIGS).The results showed that VIGS could significantly inhibit the expression of NbLTP1 gene.After TMV inoculation,both the accumulation of ROS and the content of MDA in silenced plants increased significantly,and the replication of TMV increased significantly.In silenced plants,the expression of key enzyme genes in salicylic acid(SA)synthesis,signal transuction and genes of SA-mediated disease-related protein were significantly down-regulated in N.benthamiana.The results showed that silenced NbLTP1 inhibited SA signaling pathway.Thirdly,the overexpression vector was successfully constructed by In-Fusion cloning technique.The transgenic lines OE12 and OE14,were obtained by genetic transformation.After inoculation with TMV,the accumulation of ROS in OE12 and OE14 plants decreased,the content of malondialdehyde decreased,and the replication of TMV virus decreased significantly.Therefore,these results show that increasing the expression of NbLTPl in tobacco can reduce the accumulation of ROS caused by TMV infection,reduce the oxidative damage of cell membrane,reduce the accumulation of TMV virus,and enhance the resistance of tobacco to TMV infection.Similarly,the key enzyme genes of SA synthesis,SA signaling pathway and the expression of SA-mediated disease-related protein genes were significantly up-regulated in N.benthamiana,and over-expression of NbLTPl activated salicylate signaling pathway.Fourthly,the results of subcellular localization showed that in the epidermal cells of N.benthamiana,the fluorescence signal of the control vector was mainly concentrated in the nucleus and cell wall,while the green fluorescence signal of NbLTP1 fusion protein was mainly concentrated in the cell wall.Besides,there was no fluorescence in the nucleus,indicating that NbLTP1 may play a major role in the cell wall.To sum up,NbLTP1 induces resistance to TMV infection by activating SA-mediated signal transduction pathway,which improves the resistance of Japanese tobacco to TMV infection.Our study lays a foundation for further study of the molecular mechanism related to disease resistance of NbLTP1 gene.