Application Of Gene Editing Technology To Inhibit Polyphenol Oxidase In Potato

Polyphenol oxidase(PPO)is a kind of copper-containing oxidoreductase,which is widely found in plants,animals and microorganisms.It plays an important role in the process of fruit and vegetable browning.When fruits and vegetables are subjected to mechanical damage,polyphenol oxidase catalyzes the conversion of phenolic compounds to quinones in the presence of oxygen,and quinone reacts with amino acids,proteins and phenols to form brown polymers.As a result,fruits and vegetables are damaged by enzymatic browning.In this study,the polyphenol oxidase genes POT32 and POT33,which mainly exist in potato tubers,were edited by CRISPR-Cas9 technique in order to reduce the incidence of browning induced by mechanical injury in potato tubers and to obtain new browning resistant potato germplasm.This study is of great significance for reducing browning of fruits and vegetables and improving economic efficiency and creating browning resistant germplasm resources.The main findings of the study are as follows:1.The target specific gRNA / Cas9 plant expression vector was successfully constructed and transformed into potatoIn this study,potato Atlantic varieties were used as experimental materials to construct target specific gRNA-Cas9 plant expression vectors,two targets of POT32 and two targets of POT33 gene,according to the target design requirements.SG5797,SG5798,SG5799 and G5800 were recorded respectively.Arabidopsis thaliana U6 promoter was used to activate gRNAs,and enhanced CaMV35 promoter was used to express Cas9 protein and hygromycin resistance gene.54 potato transformed lines were obtained by Agrobacterium tumefaciens genetic transformation,and 9 positive transformation lines were obtained by PCR identification,three are five SG5798 targets,three SG5799 targets and one SG5799 target.Through target sequence sequencing,only two strains were found to have mutations atSG5797 target,and two mutation types were found,one base insertion in plant POT32-2 and one base deletion in plant POT32-5.2.PPO activity decreased after PPO gene editing in potatoIn this study,the PPO activity of potato tubers harvested from two mutant plants and wild type plants was measured and compared.The results showed that the PPO activities of POT32-2 and POT32-5 decreased by 20.77% and 14.88%,and POT32-2 decreased 3.97%compared with wild type browning.The chemical localization of PPO showed that the mutant PPO was mainly located near the vascular,while the wild type distributed uniformly and in a large number on the cut plane,which indicated that the content of PPO was higher.The browning phenotypic observation showed that the browning of wild type occurred earlier,the degree of browning was larger,and the corresponding browning index was higher.In conclusion,we think that PPO gene editing can decrease PPO activity in potato.