Study Of Broad-Spectrum Antiviral Activity Of GmNH23 Gene By Over-Expression Plant

Most of the resistance?R?genes cloned from plants belong to the NBS-LRR protein-coding gene family.In previous studies,we cloned a soybean NBS-LRR family antiviral gene named GmNH23,which showed resistance toward both TMV and SMV.Until now,we only studied antiviral activity of GmNH23 gene in the transient expression system,but we did not carry out stable expression and functional verification.In order to verify the resistance of GmNH23 after stable expression and to test whether it has broad-spectrum antiviral activity,we obtained GmNH23overexpression transgenic potatoes by using potato susceptible variety Charlotte as materials,and examined their antiviral activities in this paper.A highly efficient potato regeneration and genetic transformation system was establishedsimultaneously.Transformationsystemisdevelopedasfollows.Pre-cultured medium and co-cultured medium for potato leaves were MS+1.6%glucose+5 mg/L NAA+0.1 mg/L 6-BA.The bud induction medium for callus were MS+1.6%glucose+0.02 mg/L NAA+0.02 mg/L GA₃+2.0 mg/L ZTR.The concentration of Agrobacterium tumefaciens infection is determined to be OD₆₀₀=0.7.The infection times are 15 min,and the co-culture time is two days.As regard to the bacteria inhibitor,concentration of temetin and ceftriaxone sodium are 200 mg/L and500 mg/L respectively.Since two types of recombinant vectors with different marker genes such as hygromycin?Hyg?and kanamycin?Kan?were used here,so they were used for resistance screening.Their concentrations in the stages of shoot induction and rooting are 4 mg/L and 50 mg/L respectively.After callus re-differentiation and antibiotic screening,many transformed potato seedlings were obtained.Twenty-one transformed plants were identified by genomic PCR.Further detection by RT-PCR revealed that 16 transgenic lines successfully expressed GmNH23 gene.Western blot experiments also confirmed the expression of GmNH23 protein.PVY?potato virus Y?is a serious disease of potato,and it belongs to the virus family Potyviridae with SMV.Infection of GmNH23 overexpression potato seedlings by PVY showed that the symptoms of transgenic plants were significantly reduced compared with wild type;What's more,the PVX and PVY infection of the overexpressed N.benthamiana seedlings obtained in the previous laboratory showed that the symptoms of the transgenic plants were significantly reduced compared with the wild type plants,and the GmNH23 was transiently expressed and infected by two strains of virus in the wild type N.benthamiana also showed hypersensitivity reaction,indicating that GmNH23 was resistant to both PVX and PVY.At the same time,GmNH23 overexpression transgenic tobacco was obtained.The positive rate of T3 generation was 100%by genomic PCR and 80%by RT-PCR.Transgenic tobacco overexpressing GmNH23 gene was infected by TMV-GFP.It was found that compared with wild-type tobacco,the virus diffusivity of transgenic tobacco decreased by about 5 times,indicating that the stable expression of GmNH23 in tobacco also showed resistance to TMV.Taken together,our results showed that GmNH23 gene,which was originally cloned from soybean,not only had resistance to SMV,but also showed resistance to PVX,PVY and TMV,indicating that it had broad-spectrum antiviral characteristics.Our data lay a theoretical foundation for improving the resistance of crops to virus by using the antiviral gene GmNH23.