Genetic Transformation And Transgenic Progenies Resistance To Soybean Mosaic Virus Identification Of GmEF1A And GmVATP Interacting With SMV-P3

Soybean mosaic virus belonging to the genus of Potyvirus is mainly the worldwide pathogen causing soybean disease and jeopardize.Once SMV infects soybean,the yield and quality will deteriorate significantly with mosaic and thanatosis symptoms.Cultivating SMV resistance genotype via traditional methods is the most general control strategy.But the resistance of these varieties is differential to different SMV strains.Moreover,the chromosome of virus is simple and unstable so that it is easy to diversify into many strains.Therefore,the traditional breeding methods is especially with great limitation.The rapidly developing RNA interference technology and transgenosis can break though the barrier,breeding varieties with broad-spectrum and permanent SMV-resistance.The result of yeast two hybrid(Y2H)showed that soybean translation elongation factor(GmEF1A)and soybean vacuolar-ATPase(Gm VATP)can interaction with SMV-P3 protein,and participate in the SMV replication.Then the virus induced gene silencing experiment showed that interfering the function of GmEF1A and GmVATP can improve the soybean resistance to SMV.Until now,it is not reported that transfer GmEF1A and GmVATP into soybean chromosome via Agrobacterium-mediated cotyledonary node transformation system to identify the interacting function during the procedure of SMV infection.Base on it,we used Gateway system to construct RNA interference vector and transformed soybean.We aggregated the screened positive T0 transgenic plants,identified their resistance and analyzed the function of GmEF1A and GmVATP.The result can provide available germplasm resource for research the pathway of resistance and identification the function of candidate genes.We made the nucleotide sequence and amino acid sequence alignment from 5 isoforms of GmEF1A and from 2 isoforms of GmVATP to determine the conserved interval.The RNAi vector pB7GWIWG2(II)-EF1Ai and pB7GWIWG2(?)-VATPi was constructed via Gateway technology,and transformed into soybean genotype Tianlong 1 through Agrobacterium-mediated system.We obtained 8 GmEF1A transgenic seedlings and 37 GmVATP transgenic seedlings,7 plants and 33 plants respectively are positive detected by PCR,herbicide painting and Liberty Link strip.The average of transformed efficiency is 2.75%.The results provided not only materials for functional identification of GmEF1A and GmVATP,but also brand new germplasm resource for soybean pathogen defend breeding.We aggregated 40 T0 positive transgenic plants in isolated net room and obtaining 19 GmEF1A positive seedlings and 160 GmVATP positive seedlings.Southern blot and absolute qRT-PCR analyzed that the exogenous were inserted into soybean chromosome in a low-copy integration pattern.The result of Chi-square(?2)analyses find that there are some linage consistent with the 3:1 or 15:1 ratio of Mendelian inheritance.We screened 11 high resistance(HR)GmEF1A transgenic plants and 41 HR Gm VATP transgenic plants in T1 generation.And their average disease rating in florescence is respectively 1.08 and 1.12,significantly lower than the non-transformed plants.In T2 generation,we detected the expression of GmEF1A and GmVATP and the virus accumulation by quantity real-time PCR and DAS-ELISA.The result showed that GmEF1A and GmVATP were both inhibited under the effect of RNAi vector.The virus accumulation in transgenic plants was gradually decrease in 15 and 30 days post-inoculation(dpi)with SMV while it was obviously increase in nontransfromed plants.We screen 20 HR GmEF1A transgenic plants and 23 GmVATP HR transgenic plants by investigation of resistance.Moreover,the seed coat mottling percentage was 2.09%for GmEF1A and 2.82%for GmVATP,the percentage in nontransformed plant reach up to 87.39%.