Functional Verification Of The Soybean Eukaryotic Initiation Factor EIF4E Gene And Resistance Selection Of Transgenic Progeny

Soybean[Glycine max(L.)Merr.]originated in China and has more than 5,000 years of cultivation history in China.It is one of important oil and food crops in the world.Soybean mosaic virus(SMV)belongs to the genus of potyvirus and is one of the main virus which harm soybeans.SMV widely distributed in the major soybean producing areas in the world.It may cause the decline of soybean yield and the deterioration of quality.The most effective way to prevent and treat SMV is to cultivate resistant varieties.At present,because of the complexity of the SMV strain division and the specificity of soybean resistance to strains of SMV,it is difficult for traditional breeding methods to breed varieties with broad spectrum resistance.Transgenic technology can cultivate soybean varieties with broadspectrum resistance.Eukaryotic translation initiation factor(eIF4E)is a recessive nosotropic gene which is widely present in eukaryotic organisms.It is also one of the essential factors for many RNA viruses to infect plants.Based on the research of interaction between soybean eIF4E protein and virus VPg protein.This article analysed the expression of eIF4E geneand the interaction between the other 10 mature proteins of SMV and eIF4E protein.The results showed that the relative expression level of eIF4E gene of susceptible variety Tianlong No.1 significantly increased at 0-4h after infected SC3,the relative expression level of eIF4E reached the maximum at 4h and significantly decreased from 8h to 12h.However,the relative expression level of eIF4E gene of resistant variety Kefeng No.1have not significantly change during 0～4h after infected bySC3,the relative expression level of eIF4E reached minimum at8 h after infected SC3 and slowly increased from 12h to 72h.The results of tissue expression showed that the order of relative expression level of eIF4E in different tissues,of the resistant cultivar Kefeng No.1 was root,flower,pod mature seed,leaf,stem.The relative expression level of eIF4E was the lowest in the stem.The order of relative expression of eIF4E in different tissues of the susceptible variety Tianlong NO.1 was root,pod,flower,mature seed,leaf,stem.The expression level of eIF4E in the roots of susceptible cultivars was twice more than in disease-resistant cultivars.The results of subcellular localization showed that green fluorescence appeared on the cell membrane and nucleus,indicating that eIF4E gene highly expressed on the cell membrane and nucleus.Protein interaction experiments include yeast two-hybrid and two-molecule fluorescence complementarity.The results showed that NIb,VPg,and NIa-Pro from SMV proteins may interact with eIF4E protein were.The resistance of T3 and T4 generations of agrobacterium-mediated eIF4Ei-transgenic soybeans was identificated.The results showed that positive T3 and T4 transgenic soybean performed high resistance(HR)and delayed disease resistance(DR)to SC3,SC7,SC15and SC18.The qRT-PCR experiments were conducted at 15 days and 30 days after infected SMV,The results showed that the content of SMV virus in the T3 and T4 positive transgenic plants were low and had a slight upward trend,while The contents of virus in the non-transgenic plants were high.DAS-ELISA experiments were performed,the results showed transgenic plants were SMV negation in 3 weeks and 5 weeks after infected SMV.Additionally,Virus-induced seed coat mottling rates were detected in T3 and T4 generation,The average seed coat mottling rates of non-transgenic plant were 51.82%and 38.37%,whereas brown spots have not appeared on the transgenic plants.