Eukaryotic Expression System Construction And Induced Resistance Mechanism Of Y3 Antiviral Protein

Plants acquire a kind of resistance named systemic acquired resistance?SAR?when they are attacked by some physical,chemical or biological factors and some active substances can increase the resistance.Y3 is a basic glycoprotein extracted from the edible mushroom Coprinus comatus Muell.ex Fr.,and it can inhibit the infection of TMV on tobacco with a high rate reaching to 83%.One of the resistant mechanism may relate to the interaction between TMV and CP,while we aslo assumed that also involved in the SAR.The Y3 protein gene was cloned and ligased with the eukaryotic expression vector,and the Y3 protein was successfully expressed in eukaryotic system of Pichia pastoris.Nicotiana Tabacum CV.,K₃₂₆26 were used in the research to study the systemic induced resistance of Y3 protein to tobacco mosaic virus?TMV?.After Nicotiana Tabacum CV.,K₃₂₆26 were dealt with Y3,some tobacco defense-related enzyme related to SAR activities were measured and the PR1,NPR1,PAL of the signal transduction pathway of SA were detected by real-time fluorescence quantitative PCR.The results are as follows:1.The Y3 protein gene was cloned into vector pPIC-9k and transformed into Pichia pastoris GS115 by electroporation to construct the Y3 protein expression system of Pichia pastoris.Y3 protein was expressed in Pichia pastoris.The optimal effect on the expression of Y3 is that the expression of Y3 protein is the highest induced by 1%methanol.2.After Y3 protein was sprayed on healthy tobacco and tobacco infected with TMV,the enzyme activity of polyphenol oxidase?PPO?,phenylalanine ammonia lyase?PAL?,chitinase and?-1,3 glucanase were increased compared with the control,except peroxidase?POD?.It was demonstrated that Y3 protein may induce SAR.3.Real-time fluorescence quantitative PCR detection showed that after tobacco leaves were sprayed by Y3 protein,the PR1 gene which was the marker gene of SAR was up-regulated;the NPR1 gene which was the key gene of salicylic acid signal transduction pathway was up-regulated;the PAL gene which was the key enzyme gene for SA system was up-regulated.It is further proved that Y3 induces systemic acquired resistance by activating the salicylic acid signalling pathway of tobacco,resulting in a disease-resistant defense response of tobacco.This study enriches the theory of interaction between protein inducers and plants,and it lay a foundation of revealing the acquired resistance and the mechanism of protein inducers in plant systems.