Function Analysis And Regulation To MicroRNA Of Transcription Factor RIN Related With Tomato Fruit Ripening

The development and ripening of fruit is a complex process involving many gene regulatory pathways. Ripening inhibitor (RIN) is a vital transcription factor during fruit ripening in tomato that belongs to the MADS-box family. MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level and play important roles in many physiological and biochemical processes. In this study, deep sequencing approach was employed to explore the interplay among RIN, miRNA and ripening-associated mRNA using the flower and tomato of wild type and rin mutant tomato. Main results were as follows:1. Transcriptome profiling of flower and fruit with differenr developmental stage in AC and rin mutant tomato was studied using high-throughput sequencing approach.638genes that targeted by RIN were identified, of which,372genes were upregulated and266genes were downregulated. The functions of the differently expressed genes were categorized according to molecular function, biological process and cellular structure respectively. The results indicated that RIN was involved in regulating many biological processes and metabolic pathways, including carbohydrate metabolism, lipid metabolism, pigment synthesis, respiration process, the photosynthetic process, cell wall metabolism, signaling, response to stress, seed development.2. miRNAs in flower and fruit of AC and rin mutant tomato were identified using high-throughput sequencing approach, and their expression were profiled during fruit ripening and under ethylene treatment. A total of38conserved families of miRNA and14new families of miRNA were identified. Most families of miRNA had a higher expression in flower than that in fruit, and decreased during fruit ripening.19miRNA families and their corresponding precursor genes were differently expressed between wild-type tomato and rin mutant. Additionally, the expressin of part miRNAs were upregulated under ethylene treatment and the other part miRNAs were downregulated.3. The targets of most families of miRNA were transcription factor, and the targets of several miRNAs were all kind of key enzymes involved in glycometabolism, acidmetabolism and flavor biosynthesis. In addition, the targets of part of the nonconserved miRNAs were genes involved in resistance to stress, suggesting that miRNA may play key roles in process againsting to environmental stress. Furthermore, AGO genes were targeted by miR168and miR403, indicating that miRNAs play an important role in the synthesis process of themselves.4. Full length of RIN gene and part RIN gene that without MADS-box were cloned, and the expression vectors were constructed respectively. Polyclonal antibodies was produced using the protein coding by RIN gene without MADS-box. The results of CHIP and EMSA suggested that RIN could bind to the promoter regions of MIR genes, and play important roles in the regulation of miRNA.