The Use Of TALENs Gene Editing Technology To Create Anti-PVY Tobacco Germplasm Research

The interaction between eIF4E-6 which is tobacco translation initiation factor family member and potato virus Y VPg protein determines the infection process of PVY on tobacco.In order to interrupt PVY infection to tobacco cells,enhance the resistance to PVY of tobacco,eIF4E-6 gene in tobacco was targeting modified using TALEN technology.1.Construction of TALENs expression vector of eIF4E-6 geneThe TALENs target sequence were designed combine the specific region predicted from the sequence alignment of eIF4E-6 and tobacco eIF4 E family members with the cutting sites calculated from TALEN targeter online software.The TALENs expression vector was constructed by TALEN module assembly,TALENs activity was evaluated using Luciferase–SSA report vector and two TALENs expression vectors T3 and T4 with ideal DNA cleavage activity in vitro were selected.Construction of TALEN knockout vector and introduction of LBA4404 competent cells by electroporation.2.Tobacco transformationTobacco varieties of K326 susceptible to PVY were genetic modified using the Agrobacterium tumefaciens-mediated leaf disc transformation method and the transformed seedlings were PCR screening for hygromycin B resistance gene,The amounts of T0 positive transformants of T3 and T4 TALENs expression vectors were 40 and 50 plants,respectively.3.Detection of eIF4E-6 gene mutation sitesThrough PCR screening using the primer from eIF4E-6 gene to the positive transformants,inserting of PCR product into T vector and sequencing and the sequence alignment of e IF4E-6 gene of T0 transformants with wild type tobacco,one mutant plant of large deletions in target sites and three mutation plants of nucleotide substitution in target sites were obtained from the transformants of expression vector T3,and five mutant plants of target site nucleotide substitution were found from the expression vector T4 transformants.4.Resistance analysis of mutant materialsAfter inoculation with PVY,the symptoms of mutant materials were decresed than that of wild controls.The results of Real Time RT-PCR showed that the expression of eIF4E-6 gene and the amount of virus were lower than that of non transgenic K326.5.Physiological and biochemical analysis of mutant materialsAfter inoculation with PVY,the activities of PAL,POD and SOD in mutant T0 generation tobacco plants were higher than those of wild type K326 tobacco plants.