| Tobacco mosaic virus(TMV)has been receiving much attention since its discovery in 1883.TMV hosts a wide range and can infect more than 300 plants belonging to more than 30 families such as Cruciferae,Solanaceae,Leguminosae,Asteraceae.Diseases caused by TMV seriously affect the quality and yield of crops,resulting in significant economics loss.The incidence of TMV in tobacco fields is relatively high.The plant will not only appear dwarfed during the seedling stage,but also affect its normal growth,resulting in economic losses of up to 50-70%.So far,very effective control methods have not been developed,so looking for new environmentally friendly antiviral biological pesticides is one of the prevention strategies.Antiviral proteins also belong to biological pesticides,which can not only be directly commercialized for development and application,but also can be expressed in crops for a long time through transgenic technology to produce activity.Flammulina velutipes protein FTX271 can inactivate TMV infection in vitro and can retard and relieve symptoms in K326 tobacco plants,but its mechanism is currently unclear.In this study,the flammutoxin FTX271 was used to introduce the gene into Agrobacterium tumefaciens-mediated transgenic technology.Then,the proteomics technology was used to explore the expression level of FTX271 protein in A.tumefaciens and the inhibition of TMV by the expression products.The FTX271 gene was transformed into Nicotiana benthamiana by A.tumefaciens-mediated method,and 3 transgenic plants obtained successfully though plant tissue culture,named N1?N2?N3.The positive transgenic T1 generation of N1 strain verified by RT-PCR method was subjected to proteome sequencing,and wild type tobacco was used as blank control.Proteomics results show that the FTX271 protein can be expressed in N.benthamiana.Compared with wild-type tobacco,87 differential proteins were up regulated and 79 differential proteins were down regulated in transgenic tobacco,and most of the differential proteins were concentrated in the chloroplast(36.14%).Among the up-regulated differential proteins,there are:mitogen-activated protein kinase(MAPK)and callose,which are involved in plant autoimmunity;defense enzymes,such as superoxide dismutase(SOD)and peroxide,which participate in disease resistance peroxidase(POD),polyphenol oxidase(POL);pathogenesis-related protein(PR)PR-10,chitin involved in systemic acquired resistance(SAR)reaction Enzymes;and trehalose,an emergency metabolite produced by plants under stress,and lipoxygenase(LOX)involved in the synthesis of jasmonic acid(JA).It shows that FTX271 can not only enhance the immunity of transgenic tobacco,promote the expression of disease-resistant defense enzymes,but also induce SAR response.Also,it can induce the up-regulated expression of the key protein LOX in the JA hormone pathway and improve the immunity of transgenic plants.In addition,KEGG signaling pathway analysis of differential proteins revealed that the linolenic acid pathway was significantly up regulated.As a precursor material synthesized by JA,linolenic acid is resistant to biological and abiotic stresses during plant growth and development and it plays an important role.Speculating that the FTX271 protein can promote the expression of JA pathway and strengthen the plant's own defense capabilities.Using expression vector p35s-30B-GFP infected wild-type and T1 generations of transgenic tobacco,respectively.The green fluorescent protein(GFP)was clearly expressed in wild-type tobacco at 3 days postinoculation(dpi),and gradually increased at 3-8 dpi.It was clearly observed in the new leaves and petioles at 8 dpi,indicating that TMV completed the systemic infection at 8 dpi.In the transgenic tobacco,the expression vector was not obvious at 3 dpi,and the fluorescence was clearly observed at 5 dpi.The expression level gradually increased on the 5-8 dpi,and it was found in the new leaves and petioles at 11 dpi.Obvious fluorescence was observed,and TMV completed systemic infection at 11 dpi in transgenic tobacco.It shows that transgenic tobacco can delay the expression and spread of TMV in plants,and further shows that FTX271 can enhance the defense ability of transgenic tobacco.The transient expression system of TMV was used to infect transgenic and wild-type tobacco at the same time,and samples were taken at 8 dpi for proteomic analysis.The expression of FTX271 protein was also detected.Combined with proteomics pathway analysis,it shows that 11 pathways of transgenic tobacco had significant changes.Among them,the up-regulated expression of class? sHSP and class? sHSP was quantified of the endoplasmic reticulum protein processing in the upregulated expression pathway.Small heat shock proteins(sHSP)is a type of protein that mainly functions as a molecular chaperone.It can assist protein renaturation under stress,stabilize protein function,and improve the plant's resistance to stress.It is speculated that FTX271 protein can promote the upregulation of sHSP expression,and this up-regulation may be related to the improvement of TMV resistance of transgenic tobacco.In addition,we also found that the photosynthesis pathway was down-regulated,including the down-regulated expression of the oxygen-evolving enhancer protein 3-1 protein related to the electron transport chain and the photosystem I P700 apoprotein A1 protein that participated in the formation of the PSI reaction center complex.At the 8 dpi,transgenic tobacco may enhance the plant's disease resistance by activating the sHSP pathway to compensate for the impact of TMV on photosynthesis.This study combines transgenic technology and proteomics to successfully obtain FTX271-transgenic tobacco,and establish a platform for studying the mechanism of FTX271 inhibiting viruses.In the transgenic tobacco,we found that FTX271 can activate the induced resistance of tobacco in the ab sence of virus infection and enhance the resistance of transgenes.In addition,after the transient infection with TMV,the symptoms were significantly retard and relieve.Proteomic analysis showed that retarding and delaying TMV expression may be related to the up-regulated expression of sHSP.sHSP may be involved in repairing the activities of TMV-related photosynthesis-related enzymes,and ultimately reduce the mosaic symptoms of transgenic plants after TMV infection.The proteomics results of this study provide a theoretical basis for exploring the effect of transgenes on plant growth and disease resistance.In addition,the successful cultivation of FTX271 transgenic tobacco has laid an important theoretical foundation for the selection and disease control of tobacco resistant varieties. |
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