Identification Of MiRNA In Tomato Regeneration In Vivo

miRNA is a non-coding small RNA which widely found in eukaryotes and viruses,the length generally between 19 and 24 nt,and expression of the target gene is negatively regulated by cleavage of the target gene mRNA or inhibition target gene mRNA that translation leading to mRNA silencing of the target gene.miRNA is important in the plant,mainly involved in the metabolism,signal transduction,stress response,energy metabolism,material metabolism and other processes.However,there are few studies on miRNA in plant regeneration,and we need further research.In this experiment,tomato was selected as the research materials,and the regenerated system on the callus of tomato was used as the research material to confirm the miRNA.The total RNA was extracted by Trizol method,and the small RNA library of living regeneration was constructed.The results were analyzed by high-throughput sequencing and the related miRNA was found by target gene prediction,The results of bioinformatics analysis were further validated by molecular biology method of RT-PCR and fluorescence quantitative PCR.The main results are as follows:There are 92 known miRNA and 91 novel miRNA were predicted,among the known miRNA,there are 50 conserved miRNA belonging to 18 miRNA families and 42 non-conserved miRNA belonging to 26 miRNA families;Sly-miR482e-3p,Sly-miR171a,Sly-miR164a-5p,Sly-miR156a,novel-56,novel-128 and 6 miRNA related to in vivo regeneration;The six miRNA were successfully identified by RT-PCR;The relative expression level was obtained by fluorescence quantitative PCR.I found that the expression of these six miRNA in vivo was relatively different.In this experiment,miRNA was treated with innovative stem-loop method,successfully solved the problem that miRNA length is too short to be directly amplified.The stem and loop RT-PCR detection system of tomato miRNA was established,which laid the foundation for the study of miRNA research in other growth and development of tomato.