Based On The Overexpression Strategy To Verify The Function Of Tobacco PVY Resistance Candidate Gene EIF4E

Potato virus Y(Potato virus Y, PVY) is one of the main members of the potato virus Y group.Mechanism of potato Y virus infected host is associated with a translation initiation factor e IF4 E in plants, e IF4 E interacted with viral RNA5’ end similar to the cap structure connection protein VPg, to speed up the translation of viral RNA.In pepper and tomato have been confirmed, in order to verify in tobacco and PVY resistance related details of which members of the e IF4 E family, a series of experiment design.This study first referred to the pea, chili, tomato, lettuce and tobacco e IF4 E and/or e IF(iso)4E m RNA sequence in the genome database(tobacco,Honghuadajinyuan) in comparison,obtained 11 e IF4 E sequences in tobacco. Through the analysis of homology, 2 PVY candidate resistance genes-e IF4E-6, e IF4E-10.Specific primers were designed and e IF4E-6, e IF4E-10, cloned from susceptible material LJ925, e IF4E-6, e IF4E-10 gene 750 bp of the same size, construction of expression vector of e IF4E-6 gene and e IF4E-10 gene in plants, the e IF4E-6 gene and e IF4E-10 gene into tobacco resistance cultivar VAM by Agrobacterium mediated method, through the training of 2-3 d,agrobacterium 6min, co culture 3 d, 100 mg/L kanamycin resistant screening, hardening and transplanting a series of steps, obtained the e IF4E-6 300 transformation seedlings and e IF4E-10 280 transformation seedlings,PCR method for identifying positive plants obtained from each selected 120 transformation seedlings strain was amplified by PCR detection, e IF4E-6 into the positive tobacco transformation rate was 88.3 % after transformation into tobacco e IF4E-10 gene, positive rate was 84.2 %. EIF4E-6 and e IF4E-10 were elected for each of 60 transgenic strains and 10 control plants using friction inoculation method of virus inoculation on plant, the statistical results of the onset of e IF4E-6 transformed seedling rate reached 81.7 %, the incidence of e IF4E-10 transformed seedling rate of only 5 %, individual transformation seedlings resistance breakthrough, preliminary results indicated that e IF4E-6 and PVY resistance is strong correlation. e IF4E-10 and PVY resistance is not related.