Effects Of ?-Man And ?-Hex On Ripening And Softening Of Fruits In Processed Tomato

Processing tomato is one of the pillar industries in Xinjiang.It has a wide planting area and high output.It is an important part of Xinjiang's economic development.Tomato is a climacteric fruit.During the ripening process,ethylene production increases rapidly and the fruit softens rapidly.Therefore,it is particularly important to explore ways to inhibit excessive softening of tomato fruit,extend shelf life,facilitate storage and transportation,and increase sales quality.Free N-glycans are widely present in various organelles of plant cells and participate in the ripening and softening of fruits.The glycoside hydrolase ?-mannosidase(aMan)and ?-D-N-acetylhexosaminidase(?-Hex)belong to different glycosyl hydrolase families,both of which are key to the processing of N-glycans,and specifically expressed during fruit ripening,can promote the production of free N-glycans and accelerate the ripening and softening of tomato fruits.Objective: The CRISPR/Cas9 gene editing system was established in processed tomato,and the tomato endogenous genes a-Man and ?-Hex were Targeted editing to obtain mutant tomato plants with a-Man and ?-Hex gene inactivation and loss of function.Thereby inhibiting the degradation of N-glycoprotein,weakening the softening degree of the ripening fruit of tomato,and providing technical support for prolonging the shelf life of tomato.Methods: The study based on the full length of the ?-Man and ?-Hex gene sequences.The first exon of the ?-Man and ?-Hex genes was edited directly by the hCas9 nuclease combined with 21 bp(19 bp)guide RNA(gRNA)which driven by tomato U6 promoter,and two sgRNAs in tandem,driven by the RNA polymerase II promoter,constructing separately single-target and dual-target plant expression vectors,directing the expression of Cas9 nuclease driven by the 35 S promoter For the target gene;firstly,the wildtype processed tomato plants were used as experimental materials to verify the validity of the double-target editing vector by transient transformation;secondly,the transgenic lines of tomato were obtained by Agrobacterium-mediated genetic transformation.Then the genomic DNA of transgenic tomato leaves was detected and amplified by restriction endonuclease and PCR for the DNA fragments near ?-Man's or ?-Hex's editing site,and the ?-Man or ?-Hex gene mutant was determined by monoclonal sequencing.Results: Plant expression vectors pKAN-sgR-Cas9-Man and pKAN-sgR-Cas9-Hex based on CRISPR/Cas9 system,and vectors pDIRECT-Man and pDIRECT-Hex for dual target editing were constructed;the transgenic lines of tomato were obtained about 14 genetically transformed ?-Man gene independent transformation events and 6 ?-Hex gene independent transformation events by Agrobacteriummediated genetic transformation;The genomic DNA of transgenic tomato leaves was detected and amplified by restriction endonuclease and PCR for the DNA fragments near ?-Man's editing site.The results showed that two strains of transgenic tomato plants were detected mutations.The sequencing results of the ?-Man mutant TA clone suggested that there were two types of editing,one was a 52 bp deletion mutation and the other was a single base mutation,while the ?-Hex transgenic plants were all not detected.In addition,the transient transformation of tomato plants into the double-target editing vector did not detect the mutation phenomenon,and the vector editing effect needs further verification.Conclusions: In this study,the editing of the tomato ?-Man gene was performed,and a frameshift mutant with loss of ?-Man gene function was obtained.