Regulating Mechanism Of Ubiquitin Ligase Gene SlSINA4 In Fruit Ripening And UF/SlWOX1 In Flower Organs Development Of Tomato

Fruit is an important part of the human diet structure,which provides necessary nutrients and bioactive substances for human health.As a model climacteric fruits,tomato fruit ripening regulation network has been paid significant experimental attention.Ubiquitination is an important post-translational modification,the embellishment of ubiquitin to target protein is achieved through a sequential action of three enzymes involving ubiquitin activating enzyme(E1),ubiquitin conjugating enzyme(E2),and ubiquitin ligase enzyme(E3).A eukaryotic genome may have one or two E1 genes,dozens of E2 genes,and more than one thousand of E3 genes,can modify numerous proteins.SINA belongs to the RING type E3,which can identify and combine to the target proteins,and transfer the ubiquitin from E2 to the Lys-site of target proteins.In flowering plants,the formation and development of flower organ is the basis of plant breeding,the whole flower is composed of several organs with significant features,including the sepals,petals,stamens,and pistil.WOX is a special Homeobox transcription factors family of plants with Homeodomain.As can be directly involved in the apical meristem,WOX has important function in plant growth and development.In this paper,two studies were carried out.First,SlSINA4 is a protein interacting with MADS-RIN,the fruits ripening with SlSINA4 over-expressed are delayed;further analysis found that SlSINA4 affected the ripening of tomato fruit by interacting with RIN and FRUITFULL protein.Second,mapping the candidate gene UF regulating the flower fusion,and validating the UF gene function.Main results are as follows:1.The results of Y₂H and Co-IP showed that SlSINA4 could interact with MADS-RIN,and SlSINA4 protein was localized in both nucleus and cytoplasm.There are seven putative SINA ubiquitin ligase genes in tomato genome,and homology of c DNA sequences was more than 79%.Tissue expression analysis showed that the SlSINA genes had different expression levels in different tomato tissues.Different expression specificity indicates that the SlSINA genes have spatio-temporal differences in regulating tomato growth and development.Comparing with the WT(AC)fruits,the fruits with SlSINA4over-expression had shallow shoulder and the ripening process started late,from breaker ripening to red ripening needed longer time.Lycopene content in SlSINA4-OE fruits was significantly lower than that in AC fruits of the same period,and as the mature process progressed,the lycopene accumulated slowly.The expression of PDS and ZDS were increased,while PSY1 in SlSINA4-OE fruits was inhibited,resulted in the synthesis of lycopene was hindered.Analysis of ethylene release rate of SlSINA4-OE fruit showed that there is a peak of ethylene in transgenic fruits,but it started later than AC fruit.And the Sl ACO1 expression was significantly decreased in the SlSINA4 over-expressed fruits.After been treated with ethylene,the transgenic fruits still could not restore normal ripening speed,and on the other hand,SlSINA4 expression in the AC fruits treated with ethylene and 1-MCP did not change significantly compared with the control.All above indicated that SlSINA4 played roles on the upstream of ethylene pathway and its expression was not regulated by ethylene.Analyzing the transcription factor regulating tomato fruit ripening,expressions of RIN and FUL1 in transgenic fruits were significantly decreased.And FUL2 in green fruit and red fruit was reduced,while there is no obvious change in the intermediate ripening process.Therefore,SlSINA4 may be regulating tomato fruit ripening via RIN and FRUITFULL.Using yeast two-hybrid,Co-Immunoprecipitation and GST pull-down,resulted that SlSINA4 can interact and combine with RIN and FRUITFULL,the result indicated that SlSINA4 can combine to RIN and FRUITFULL forming a complex,then regulating the ripening gene expression and tomato fruit ripening.2.Analyzing the flower organ morphology of uf mutant,result indicated that petals ray shaped,carpel and style split completely,and the ovule and pollen tubes were exposed,finally there was no fruit.By BSR sequencing analysis and map-based cloning,the UF gene was cloned,then the candidate gene was verified by CRISPR/CAS9.Functional verification results confirmed that UF,encoding WOX1,affected the lateral development of organs.