Functional Analyses Of SlCHRC In Color Change During Tomato Fruit Ripening Using CRISPR/Cas9 Gene Editing System

The CRISPR/Cas9 system is the most widely used third-generation gene editing technology and has been reported successfully in gene editing and breeding improvement in various models,commercial animals and plants.The appearance color of the commercialized tomato fruit(Solanum lycopersicum.L)is the main factor affecting its commercial value,the color transformation process of thefruitsripening is highly concerned.The post-ripening regulation network of tomato fruit is complicated,and the research on ethylene pathway and carotenoid anabolic pathway involved in color metabolism has been thoroughly studied.However,the mechanism of upstream regulation and intracellar color transformation is still unclear.The currently research have shown that the proteins family with PAP-Fibrillin domain play a regulatory role in the formation of chloroplast membranes,lipid synthesis and carotenoid storage and transportation in fruits.Among them,one of the family’s proteins named Sl CHRC(Chromoplast-associated carotenoid-binding protein)plays a role in the post-fruit color transformation.,while limitly demostrated to the proteomics and tissue expression pattern,and lacks investigation of genetic function.Compared with commercialized tomatoes,Micro-Tom(cv.dwarf/u)has the characteristics of fast growth,short fruit ripening time andobvious transformation process of fruit color,which are conducive to the ideal material for the research of plant fruit development and post-ripening regulation.In this paper,Micro-Tom was used as material,the CRISPR/Cas9 gene editing technology was established,to carry out the function of Sl CHRC.The results as follow:(1)Using Sl PDS as the initial target gene,the CRISPR/Cas9 gene editing technology system was established on Micro-Tom by Agrobacterium-mediated plant transformation;the same phenotypes of bleaching leaves and stems as previous research,and the two mutant phenotypes were analyzed by decoding to be heterozygous mutation.(2)Analysis of family genes of Sl CHRC and other source species by phylogen etic-tree,the Sl CHRC and Chloroplast-associated plastid protein of most Solanaceae species have high homology,The subcellular localization vector At UBQ10-Sl CHRC-e gfp was constructed and transformed by tobacco transient expression system.The ex pressed protein of Sl CHRC was found to be located in the chloroplast.(3)Tissue-specific expression analysis showed that the expression of Sl CHRC was highest in the petals,followed by the leaves,fruits of Mature Green and Turning-Orange stages,not found in the root tissues.(4)Ethylene treatment promoted the chlorophyll degradation and the carotenoids accumulation in fruits,and promoted the up-regulation of Sl CHRC.On the contrary,1-MCP processing suppresses the above process.(5)A multi-target CRISPR/Cas9 editing system for Sl CHRC gene: p YLCRISPR/Cas9-T#Sl CHRC was constructed,three Cas9-slchrc-T0 mutant lines obtained are all heterozygous mutant types,The c DNA clone analysis revealed that the shortened enc oded amino acid chain appeared in Sl CHRC,and the corresponding amino acid seq uence showed a frameshift mutation;The fruits of the three MT tomato mutant line s showed obvious phenotypes: the young leaves were dark green,the compound lea ves were reduced,the plant height decreased;the peel color was greatly delayed,an d the soluble solids and the weight of the bracts were different.There is a phenom enon of seed development defects.In summary,we successfully established the workflows for application of CRISPR/Cas9 gene editing technology on the model tomato Micro-Tom by editing Sl PDS gene,knockouting of the Sl CHRC gene obtained valid mutant lines,the function of items in tomato was re-recognized: the transcript level of Sl CHRC gene is induced by ethylene signal,its protein localization in chloroplast,which may affect the pigment process of tomato fruit,and speculate that it plays an important role in seed development.