Construction Of EIF4E Gene Rnai Vectors And Studies On Soybean Mosaic Virus Resistance Throuth Agrobacterium-mediated Soybean Genetic Transformation

soybean[Glycine max(L.)Merr.]is the important oil crops and food crops,which is native to China and has been cultivated in excess of five thousand years.Soybean mosaic virus(SMV),the most serious hazards soybean virus,is a global disease of soybean,which is widely occured in the word and seriousily effected on the yield and quality of soybean.To preventing and curing the SMV,Cultivation and promotion of resistant varieties is the most economic and effective means.Due to the complex differentiation of SMV strain and the specialized resistance of soybean resistance to SMV strains,traditional breeding method is difficult to develop a broad-spectrum resistance in a short time of soybean varieties.Therefore,the use of transgenic technology has a great significance to cultivating resistant varieties of soybean rapidly that has a broad spectrum of prevention and treatment to soybean mosaic virus.The recessive resistance gene eIF4E(eukaryotic translation initiation factor 4E),which is extensively indwell in eucaryon,is also one of the essential factors in a variety of RNA viruses to infecting plants.The absence or mutation of eIF4E gene can block the interactions between plants and viruses,and affect the replication and infection of viruses,so that the plant can get resistance.In this study,we constructed RNAi vector of eIF4E though GATEWAYTM system.Based on RNAi mechanism can induce the silence of eIF4E specifically,and the cotyledonary node-Agrobacterium-mediated soybean transformation system was used to obtain a broad spectrum of SMV resistant transgenic soybean material.RNAi vector pB7GWIWG2(?)-eIF4Ei of eIF4E was constructed though GATEWAYTM system,and the soybean varity of Tianlong 1 was used in the cotyledonary node-Agrobacterium-mediated soybean transformation system to get transgenic soybean material.Polymerase chain reaction(PCR),herbicides smear and LibertyLink strips were used to identify the T0 generation,and a total of 31 positive T0 plants was obtained,The highest transformation efficiency is as high as 3.83%and the average transformation efficiency rate was 2.23%.The results indicated that target gene has been successfully integrated into the receptor material Tianlongl and expression in protein levels.In the aggregate of 148 positive T1 plants derived from 31 positive T0 plants were obtained after detection and identification.Southern blot hybridization experiments proved that the exogenous gene has inserted into the genomic DNA in a low-copy integration pattern in the recipient soybean material.Chi-square(?2)analyses results showed that there are two T0 lines transmitted the target genes to their T1 progenies in the ratio of 3:1 or 15:1.By identifying the resistance of positive T1 plants to SMV,50 plants of highly resistant(HR)were screened out.The disease rating on average was 1.02 in positive T1 plants,while it was 3 in the nontransformed ones.Additionally,Virus-induced seed coat mottling rate was detected in T1 generation,In the positive plants,the average seed coat mottling rate was 2.35%versus 85.50%for the nontransformed plants.Further detection and identification were performed in the T2 generation,57 HR plants and no susceptible ones were detected.The quantitative real-time PCR indicated that virus accumulation in the positive T2 plants was in a gradual reduction and it was increased rapidly in the nontransformed plants at 15 and 30 days post-inoculation(dpi)with SMV.After 2 months post-inoculation with SMV,doubleantibody sandwich enzyme-linked immunosorbent assays was conducted to test the virus accumulation,and there was no virus detectable in the positive plants.In addition,Virus-induced seed coat mottling was eliminated in the positive T2 plants.