Construction Of Prokaryotic Expression System Of Flammutoxin (FTX) And Mechanism Analysis Of Resistance To TMV

Tobacco mosaic virus(TMV),a plant virus with high infectivity and strong resistance,has many hosts and is very difficult to be controlled.One of the ways to inhibit the virus is using bioactive products because of their advantage of green and environment friendly.Flammutoxin(FTX)and its precursor protein,TEER-decreasing protein(TDP)that only 20 amino acids apart,are produced from the growth stage of Flammulina velutipes fruiting bodies.Studies had found that protein FTX or TDP had anti-TMV properties,but it has not been confirmed yet which protein played a role or main role in the process of anti-TMV.In order to confirm the anti-TMV effect of the proteins and analyze the possible mechanism of disease resistance,we used the Escherichia.coli expression system to construct the FTX and TDP recombinant plasmids successfully,and obtained the recombinant proteins FTX-pET32a and TDP-pET3 2a,and then the TDP-pET32a and FTX-pET32a recombinant proteins were obtained by induction expression and purified the proteins by immobilized metal ion affinity chromatography(IMAC).The results showed that proteins used 1 mM IPTG(inducer)to induce expression for 8 h at 37? can obtain more protein,and the tag protein,recombinant proteins FTX-pET32a and TDP-pET32a can obtain more purifited protein under the elution of 250mM,200mM and 250mM Elution buffer in protein purification.Taking the tobacco Nicotiana tabacum var.K326 as the research object,the antiviral mechanism of FTX and TDP proteins were studied from two aspects of protective effect and curative effect.The experiment of protective effect:the tobacco was sprayed with the proteins FTX and TDP respectively,then inoculated with the TMV 12h later,the defensive enzymes related with induced resistance,such as the peroxidase(POD),phenylalanine ammonia lyase(PAL),chitinase and polyphenol oxidase(PPO)were measured within 1-9 days.The results showed that in 1-9 days,the enzyme activitys of tobacco treated with protein FTX and TDP raised in varying degree comparing the control group.For example,in the 7th day,the enzyme activity of TDP group was 26.8U/g and FTX group was 24.4U/g,which were higher than three control groups(18.6U/g,21.3 U/g and 19.5 U/g).The results indicated that proteins TDP and FTX all can induce the anti-TMV activity of tobacco,and the induced resistance of TDP was higher than that of FTX.The experiment of curative effect:the tobacco was inoculated TMV,and than protein FTX and TDP protein was sprayed on it respectively after 12h.We used the quantitative real-time PCR(qRT-PCR)to detect the change of TMV content in tobacco within 1-9 days,and the ?-actin gene as the internal reference.The results found that the content of TMV treated with protein TDP and FTX were significantly lower than the control groups in 1-9 days,and the TMV content of tobacco treated with protein TDP was the lower than FTX.It indicated that TDP and FTX can inhibit the replication and proliferation of TMV in tobacco,and the rate of inhibition effect to TMV reached 70%in 7th day,and the effect of protein TDP has stronger effect than that of FTX.In this study,prokaryotic expression products of FTX and its precursor TDP were obtained.It was determined that the two prokaryotic expression products have strong anti-TMV effect as same as the direct extracts,and they can be further developed into the low-cost biological pesticides.We speculated that the protein originally purified from Flammulina velutipes may be the mixture of FTX and TDP.The results also showed that protein TDP had stronger antiviral effect than FTX,but whether the protein FTX or TDP,not only they can induce the resistance of the tobacco to inhibit the infection of TMV,but also can directly inhibit the replication and proliferation of TMV in host plant.The anti-TMV mechanism of proteins may be multi-faceted.