Construction Of CeRNA Regulatory Network And Changes Of Main Physiological And Biochemical Indexes Of Cuticle Cracking In Tomato

Tomato（Solanum lycopersicum L.）is one of the most popular commercial vegetables in the world,but fruit cracking occurs easily in tomato,which affects the appearance quality of fruit and easily causes bacterial infections.This will lead to the reduction or even the losing of commercial value.Therefore,fruit cracking is one of the main obstacles in tomato production.Fruit cracking is influenced more by heredity than by the environments,so it has great significance for molecular design breeding to clarify the molecular mechanisms of fruit cracking.The ceRNA（competing endogenous RNA）theory is a supplement to the traditional miRNA-regulated RNA theory,which reveals the mechanism of interaction between different types of RNA.This hypothesis has been proved to exist widely in animals and plants.In recent years,genes related to fruit cracking have been identified through transcription analysis and QTL mapping.However,there were no reports about ceRNA network associated with tomato fruit cracking.Therefore,we performed whole transcriptome sequencing on the peel of cracking-resistant（CR）and cracking-susceptible（CS）tomato at the red ripen stage,and measured relevant physiological and biochemical indexes.The main findings are as follows:1.Whole transcriptome sequencing was performed on the total peel of CR tomato`NT-251R’ and CS tomato‘NT-54’at the red ripen stage.Through the evaluation of sequencing data quality,identification of RNA,differential expression analysis,identification of miRNA target genes,GO and KEGG functional enrichment analysis,we identified 36 key genes for cuticle cracking of tomato fruits.The key genes can be divided into four categories according to their functions:hormone-related,such as ethylene response factor（Solyc05g052040,Solyc05g051200）;cell wall-related,such as pectinesterase（Solyc03g123620,Solyc03g07810013）,cellulose synthase（Solyc02g089640,Solyc04g071650）;active oxygen metabolism-related such as peroxidase（Solyc04g071900,Solyc05g052280）;and mineral element-related,such as cation/calcium exchanger（Solyc07g006370,Solyc01g098800）,potassium transporter（Solyc02g087000）.2.According to the theory of ceRNA,the expression levels of transcripts that are mutually ceRNA should be positively correlated and have significantly shared miRNA or MRE.Based on this,we constructed a ceRNA regulatory network of tomato fruit cuticle cracking,which includes 73 miRNAs,3 lncRNAs,5 circRNAs and 65 mRNAs.The mutual regulatory relationship between these RNAs may be related to the cuticle cracking of tomato fruit.At the same time,we predict that some ceRNA pairs may affect tomato fruit cuticle cracking by regulating different metabolic pathways.For example,circRNA（novel_circ₀00472）and lncRNA（TCONS₀0043895）competed with mRNAs（Solyc04g049800,Solyc09g007260,Solyc10g084340）to bind miRNAs（sly-miR172b,sly-miR172a）to regulate target gene expression,while Solyc04g049800,Solyc09g007260,Solyc10g084340 encode ethylene-responsive transcription factors,so we speculated that novel circ₀00472,TCONS₀0043895 affects tomato fruit cracking by participating in the ethylene response process.3.In order to investigate the relationship between tomato cell wall components,hydrolase activity,active oxygen metabolism and fruit cracking,’NT-251R’and ’NT-54’in mature green,turning and red ripe stages were used as test materials in this part.And we observed the tissue structure of the peel cell wall,measured the content of pectin,cellulose,hemicellulose,and polygalacturonidase（PG）,β-galactosidase（β-gal）,cellulase（Cx）activities,as well as H2O2 and MDA content,relative electrical conductivity,antioxidant ASA content,antioxidant enzyme（SOD,POD,CAT,GR,APX）activities and soluble protein content of the peel.By analyzing the differences between the two varieties,the relationship between them and fruit cracking has been discovered.The study found that as the fruit matures,the water-soluble pectin showed an upward trend,and the content in CS was significantly higher than in CR fruits in each stage;The protopectin,cellulose and hemicellulose showed a downward trend during fruit maturation,and their content in CR fruits were generally higher than in the CS fruits;The activity of the three hydrolases in CS fruits showed an upward trend during fruit ripening,and the activity in CS fruits were generally higher than in CR fruits.β-gal and Cx activities increase slowly after the turning stage in CR fruits.The content of MDA and relative electrical conductivity showed an upward trend,and the content was generally higher in CS fruit than in CR fruit;The antioxidant ASA activity increased first and then decreased,and compared with CS,the content was higher in CR;The activities of SOD,APX,GR showed a downward trend,and the activties was generally higher in CR than in CS fruits;The activities of POD and CAT showed an upward trend,and compared with the CR,the activities was generally higher in CS;The soluble protein content showed a downward trend,and the content was higher in CS.Overall,the higher content of protopectin,hemicellulose and cellulose,lower hydrolase activity,and higher activity of efficient antioxidant enzymes（APX,GR and SOD）in CR fruits were the reasons for its higher cracking-resistant character.