The Prokaryotic Expression And Resistance Characteristics Of Plant Virus Inhibitor Y3

Tobacco mosaic virus?TMV?is an RNA single-stranded virus,which has a wide host range and can infect critical crops such as Cruciferae,Cucurbitaceae,Solanaceae and causes serious damage to it.Prevention of leaf virus disease has become very important.Compared with the environmental pollution caused by the use of chemical agents and the occurrence of phytotoxicity to crops,the exploration and use of natural products against TMV is clearly a sustainable development approach.Recent studies have shown that plant virus inhibitor protein not only have anti-TMV activity,but also may increase the host plants'resistance to pathogenic bacteria,which is of potential application value.The Y3 protein is a basic protein extracted from the fruit body of the coprinus comatus?Coprinus comatus?and has the ability to inhibit tobacco mosaic virus.Studies have shown that Y3 protein can interact with the coat protein of tobacco mosaic virus?TMV CP?.In vitro and in vivo interactions to inhibit the synthesis of viral proteins,thereby reducing the viral infection rate.In addition,the properties of Y3 were expressed as Ribosome inactivating proteins?RIPs?,in order to confirm the interaction sites of Y3 protein and TMV CP and to determine whether they are RIPs,and the relationship between antiviral activity and RIPs.In this study,site-directed mutagenesis and prokaryotic expression systems were constructed for Y3,and DNA-nuclease activity,RNA N-glycosidase activity,and anti-TMV activity of each recombinant protein obtained from the prokaryotic expression system were examined.?1?After structural prediction,the mutation site was determined by fixed point mutation technique mediated by PCR.The 128th base bases of the gene y3t1 was mutated from thymine?T?to guanine?G?,and the corresponding amino acid was transformed from phenylalanine?Phe?to cysteine?Cys?;the 165th base in gene y3t2was mutated from adenine?A?to cytosine?C?,the corresponding amino acid was mutated from arginine?Arg?to serine?Ser?.After the mutant of Y3 gene was obtained,the prokaryotic expression system of Y3 and its mutants was constructed.The results showed that the Y3 gene and its mutants were successfully incorporated into the expression vector pET32a and were successfully expressed in the Rosetta strain.After optimizing the expression temperature of each protein,the recombinant protein,P-Y3,P-Y3T1 and P-Y3T2,with a molecular weight of 35kDa,was purified by nickel column affinity chromatography.?2?The DNA-nuclease activity of the Y3 protein and the recombinant protein obtained from the hair of the hair was detected.The results showed that all the proteins could cut the plasmid pET28a,pET32a and pUC19 from the super spiral conformation to linear or open conformation,which indicated that each protein had the activity of DNA-nuclease.The RNA N-glycosidase assay of each protein showed that the 28S rRNA in the total RNA of tobacco treated with each protein did not release specific Endo fragments,indicating that each protein does not have RNA N-glycosidase activity,so that,the detection of DNA-nuclease activity and RNA N-glycosidase activity of each protein revealed that Y3 protein is not a RIPs.?3?In the detection of anti TMV activity,both in vivo and in vitro indicated that the protein obtained from prokaryotic expression system could inhibit the infection of TMV to tobacco.In vitro,the OD₄₅₀ value of the TMV content in the 9 days after tobacco inoculation with P-Y3T1-treated TMV was 1.205,which was higher than that in the same period after tobacco inoculation with TMV treated with Y3,P-Y3 and P-Y3T2.The TMV OD₄₅₀ values were 0.892,0.848,and 0.742.In the in vivo test,the OD₄₅₀value of the TMV content in the samples treated with P-Y3T1 9 days after inoculation with TMV was 1.211,which was higher than that treated with Y3,P-Y3,and P-Y3T2.After the tobacco was inoculated with TMV,the TMV OD₄₅₀ values were 0.687,0.832,and 0.672 in the samples at the same time,indicating that the mutation site in y3t1 may be the protein resistance site.