Functional Identification Of The Key Transcriptional Factor MADS-RIN In Tomato Fruit

Fruit ripening is a complex and developmental process,which is regulated by many factors.Transcriptional factors is one category mainly regulators,and MADS-RIN is a key member.Many studies related to RIN were based on comparasion between AC and rin,because in in rin mutant a DNA fragment deletion leads to fusion of truncated RIN and MC,namely RIN-MC and its minor transcripts and proteins detected.However,obvious high transcripts of newly formed chimeric RIN-MC in rin fruit has been reported lately,which putting forward to an necessity to ensure its proper roles in ripening,if it does,characterization of RIN using traditional AC/rin need re-indentification.In this study,silence of RIN-MC promoted fruit to yellow ripe.Using protein ehrichment assay and transactivate activity assay,it resulted that RIN-MC functioned as a transcriptional factor.Based on RNAi related transgenic technology and comparative transcriptome analysis,the functions of RIN has been re-identified.Main results are as follows:1.High expressions of chimeric RIN-MC in rin mutant affected fruit ripening.High expressions of RIN-MC were detected in rin mutant fruit both at ripening stages and under treatment with ethylene or 1-MCP.Using VIGS technology,silence of RIN-MC promoted fruit to yellow ripe.Expressions of many ripening related genes were affected by silence of RIN-MC.2.Chimeric RIN-MC had obvious protein enrichment in ripening,which holding transcriptional ability.With bioinformatic and experimental assay,CDS of RIN-MC was obtained.RIN-MC protein coud be detected in transient system.In transgenic AC::35S-FM-RIN-MC plants,fruits had yellow ripe phenotype.What's more,endogenous RIN-MC proteins had obvious enrichement in ripening.With CoIP and BiFC assay,RIN-MC could interact with MADS-box factors.Meanwhile,RIN-MC had trans-activity in LUC/REN transient system.All of above results proved RIN-MC functioned as a transcriptional factor.3.Using RNAi-based transgenic materials,comparative trasncriptome of AC/AC::RIN RNAi and AC/rin were obtained,therefore,a global reshaping of RIN functions was implemented.Using DEGs,GO enrichment and KEGG analysis,it proved that description of RIN functions in fruit ripening were reshaped.4.Based on comparative assay in AC/AC::RIN RNAi and AC/rin,functions of RIN were reshaped in carotenoid pathway.Using LC-MS technology and RT-qPCR to analyze content of various carotenoid and related gene expressions,it reshaped the functions of RIN in carotenoid pathway.5.Based on comparative assay in AC/AC::RIN RNAi and AC/rin,interactions with other transcriptional factors of RIN were reshaped.With bioinformatics approaches,analysis related to tomato NF-Y has been implemented.Using VIGS technology and subcellular assay,functions related to tomato NF-Y in ripening were identified.Together,this research characterized functions of chimeric RIN-MC in rin fruit and functions of RIN were reshaped,which helped promoting further studies on RIN related regulating network.