Regulation Analysis Of MiR159-MYB65-DFR Involved In Anthocyanin Synthesis In Tomato

Tomato,as a common food and vegetable,is widely cultivated in the world because it is rich in anthocyanins.Among them,Aft(Anthocyanin fruit)type tomato is currently the mainstream tomato test material for studying anthocyanin synthesis.It has been shown that as an antioxidant active substance,anthocyanins can remove excess reactive oxygen species generated by salinity stress;In addition to being regulated by transcription factors and structural genes,the synthesis of anthocyanins in plants is regulated by external salinity stress,but the molecular regulation by which they induce anthocyanin accumulation in tomato is not well defined.In this study,Aft type tomato was used as the main test material.Total RNA was extracted from leaf and root tissues after 100 mmol NaCl treatment,and then Transcriptomics,Small RNA Deep Sequencing and Degradomics analysis were carried out to obtain miRNA159 closely related to salt stress and predicted target genes.This will provide a reference for the following studies on the regulatory role of miR159-Tragets in anthocyanin biosynthesis pathway.The main results are as follows:1.A number of differentially expressed miRNAs and their target genes were identified by multi-omics analysis.miR159 was selected for Degradomics analysis,and a total of 65 target genes were predicted,including 3 MYB-type transcription factors,namely Sl MYB33,Sl MYB65,and Sl MYB104,which had high homology with At MYB3 and were speculated to have similar biological functions.2.Overexpression of miR159 and STTM vector were successfully constructed,transgenic plants were obtained through genetic transformation,and the regulation effect of miR159 expression level on anthocyanin accumulation in tomato fruit was studied.The results showed that the overexpression of miR159 promoted anthocyanin accumulation in tomato fruits,and miR159 may negatively regulate the expression level of target gene Sl MYB65.3.The fusion expression vector pA7-Sl MYB65-GFP containing GFP fluorescent protein was successfully constructed,and the onion epidermis was transformed by vacuum osmosis for subcellular localization.The results showed that Sl MYB65 protein was located in the nucleus and played its biological function.4.The dual luciferase reporter system vectors p GreenⅡ 0800-Luc-SlmiR159 p,p GreenⅡ0800-Luc-Sl DFRp and p35s::Sl MYB65 were constructed successfully,and the target genes regulated by transcription factor Sl MYB65 were verified.The results showed that Sl MYB65 would regulate downstream target genes miR159 and DFR,which is an important structural gene in anthocyanin anabolic metabolism.5.GUS reporter vectors pmiR159p::GUS,pDFRp::GUS and overexpression vector p35s::Sl MYB65 were successfully constructed and transiently transformed into tobacco leaves to explore the possible regulatory role of miR159-MYB65 in anthocyanin synthesis pathway.The results showed that MYB65 negatively regulates the expression levels of DFR and miR159,and was involved in anthocyanin anabolic regulation.The above results showed that external salinity stress induced an increase in miR159 levels in tomatoes,regulated the target gene MYB65 after transcription,negatively regulated DFR expression,and affected anthocyanin anabolism.This study revealed that miR159-MYB65 was involved in regulating anthocyanin anabolic metabolism,providing new insights for broadening the anthocyanin synthesis network of tomato.