Development Of Processing Tomato Mutants In EIF4E And EIF(iso)4E Genes And Investigation Of Their PVY Resistance

The eukaryotic translation initiation factor(eIF4E)is a large family of genes encoded by multiple genes that perform important biological functions in living organisms.There are three genes encoding eIF4 E identified in tomato,namely eIF4E1,eIF4E2,and eIF(iso)4E.Previous studies have found that the VPg protein of potato virus Y can selectively use eIF4 E to assist in the translation of viral mRNA and the diffusion between host cells.Objective: The vectors that can specifically target the processing of tomato eIF4E1,eIF4E2,and eIF(iso)4E genes was constructed by CRISPR/Cas9 technology,on this basis,the vector’s validity was quickly obtained through transient transformation method and then genetic transformation of tomato was carried out.The CRISPR/Cas9 system provides technical support for the development of PVY-resistant plants,while laying the foundation for improved varieties of processed tomato in Xinjiang.Methods: The target sequence of eIF4E1,eIF4E2,and eIF(iso)4E genes was designed based on the original CRISPR/Cas9 system originally constructed in the laboratory and the vector NTI biological software was used.Based on this,a CRISPR/Cas9 system expression vectors that can target-to-process tomato for the eIF4E1,eIF4E2,and e IF(iso)4E genes was constructed;Taking the constructed CRISPR/Cas9 targeted editing vector of the eIF4E1 gene as an example,the Agrobacterium-mediated transient transformation was used to quickly obtain the efficiency of the vector constructed by the CRISPR/Cas9 system applied in this study.Six vectors constructed using two CRISPR/Cas9 systems were genetically transformed to process tomato;the resulting transgenic plants were identified by restriction enzyme analysis for mutations.Results and conclusions:(1)It consists two parts that The eIF4 E targeted editing vector constructed using the CRISPR/Cas9 editing system in this study.The expression frame containing the target sequence sgRNA was initiated by the RNA polymerase type III tomato U6 promoter.Since the transcription initiation site of tomato U6 promoter is usually G,the target sequence,the first base of 5′sequence is G,designed by Vector NTI software was used in this study.The Cas9 protein was initiated by the 35 S promoter of cauliflower mosaic virus(CaMV)and nucle localization signal(NLS)was added at the N-terminus.In this study,a restriction enzyme assay was used for the detection of mutants.Therefore,the designed target sequence contains specific restriction endonuclease recognition sites.(2)In this study,the vacuum transmigration transient transformation method was used to evaluate the effectiveness of the constructed CRISPR/Cas9 system vectors.The nucleotide sequence comparison results showed that all the 9 positive clones that were sequenced had mutations,and all the mutations were all single-base substitutions that resulted in the substitution of a single amino acid in the polypeptide chain at the processed tomato eIF(iso)4E target sequence.(3)The electrophoretograms identified by restriction analysis of the mutations after transient transformation showed that the uncleaved bands were relatively bright,indicating that the proportion of transformed cells was larger during the transient transformation and also explained on the other hand that the vacuum infiltration transient conversion method this study used has high conversion efficiency and could be used for target sequence evaluation experiments in the subsequent application of CRISPR/Cas9 systems.