The Use Of CRISPR/Cas9 Gene Editing Technology To Create Anti-PVY Tobacco Germplasm Research

In order to knock out the eIF4E-6 gene associated with PVY infection and establish the PVY resistance directional improvement system.Two specific targeting eIF4E-6 gene CRISPR-Cas9 expressive vectors,G1 and G2,were constructed.Through tobacco transformation,PCR screening and sequencing of target site,24 mutation plants were obtained.The main results are as follows: 1.Determination of target genes involved in PVY infectionThe eIF4 E or eIF(ISO)4E gene sequence which related to PVY infection in peppers,tomatoes,peas,lettuce and other plants were blasted in China Tobacco Genome Database,and the member of tobacco eIF4 E family highly homologous with the eIF4 E or eIF(ISO)4E genes of above plants was selected to CRISPR/Cas9 target gene.2.The eIF4E-6 target site selectionSeveral CRISPR/Cas genome editing gRNA target sites were designed according to the eIF4E-6 gene sequence.The sequence(20bp)of the gRNA target sites were blasted in China Tobacco Genome database to find homologous sequence.The gRNA target site which homologous sequence(PAM sequence: NGG upstream 16bp)identify matched would be eliminated.The activity of g RNA target was evaluated by Cas9/gRNA restriction fragment DNA in vitro.2 gRNA targets with high specificity and high cutting activity were used in the eIF4E-6 gene CRISPR/Cas9 knockout vector construction.3.Construction of CRISPR/Cas9 expressivevectorThe Oligonucleotide fragments(oligo)for the sense and antisense strands of target sequence were synthesized,adaptor were added on 5' end of each Oligonucleotide fragments alternately.The oligoduplexes with adaptor were formed after annealing;The ligation product of oligoduplexes and CRISPR/Cas9 empty vector was transformed intoDH5 a Escherichia coli competent cells by heat shock.The results of monoclonal colony PCR and sequencingconfirmed thatthe CRISPR/Cas9 expression vector of eIF4E-6 constructed successfully.4.Genetic transformation of tobaccoTobacco cultivar K326 was genetic modified with Agrobacterium mediated leaf discand 256 regenerated plants of T0 generation were obtained.102 positive plants were identified from the PCR screening of hygromycin resistance gene,including45 positive plantsfrom G1 transformation and 57 positive plants from G2 transformation.5.Molecular detection of target gene editingThrough PCR amplification of hygromycin resistant gene by the primer of eIF4 EMutF and eIF4 EMutR,PCR product sequencing and sequence alignment of eIF4E-6 gene,2 nucleotide deletion mutants and 12 nucleotide substitution mutants were identified among the 14 G 1 mutant plants,and all the 10 plants of G2 mutant were nucleotide substitution.