Expression Regulation Analysis Of MiR156 And Its Target Genes In Aft-type Tomato

MicroRNAs are important regulators that widely exist in eukaryotes.Studies in plants and animals have found that their functions are diverse and have important regulatory functions in organisms,including growth and development and stress response.Aft(Anthocyanin fruit)type tomato LA1966 is obtained by crossing the wild tomato species L.chilense native to Chile and the tomato cultivar L.esculentum.Its fruit can produce and accumulate anthocyanins in the immature stage.In this study,Aft-type tomato was selected as the test material,and the tomato fruits at different developmental stages were treated with light and dark.will handle After the transcriptome sequencing and miRNA deep sequencing analysis of the final material,the differentially expressed gene microRNA156 was screened,and its target gene was predicted and its action site and interaction relationship were verified.In order to explore the mechanism of miR156 and its target genes in tomato growth and development and anthocyanin synthesis pathway,miR156-related pCAMBIA1301 recombinant plant expression vector was constructed,and transgenic plants were obtained by infecting tomato through Agrobacteriummediated genetic transformation.This experiment provides a certain reference for the regulatory pathway of miRNA in plant fruit development.The results of this experimental study are as follows:1.Transcriptome sequencing results showed that 23 genes with significant differences were obtained.KEGG enrichment analysis showed that the differential genes under light and dark treatments existed in multiple metabolic pathways,mainly in photosynthesis,ribosome and plant hormone signal transduction.these three metabolic pathways.2.34 differentially expressed miRNAs were obtained from the analysis of small RNA sequencing results,from which miR156was screened,and the degradation group identified the splicing function of miR156 to its target genes.3.Real-time fluorescence quantitative PCR to detect the expression characteristics of miR156 and its target genes in different developmental stages of Aft tomato fruit.4.Construct a recombinant plant expression vector that overexpresses oe-miR156 and suppresses STTM-miR156 expression,Agrobacterium-mediated genetic transformation of tomato,to obtain transgenic plants,and study the expression regulation of miR156 and the developmental phenotype of transgenic plants.5.Construct the recombinant expression vector of the target gene SLSPL15-pA7 of miR156,and use vacuum infiltration of onion epidermal cells to preliminarily identify the function of SlSPL15 in the nucleus.6.The pAD-SPL and pBD-Del vectors were used to construct recombinant expression vectors,and yeast two-hybridization was used to detect the interaction between SPL protein and bHLH protein of miR156 target gene,and to determine the synergistic regulation with transcription factors.