Functional Characterization Of MicroRNAs Regulated By Transcriptional Factor HY5 In Arabidopsis

microRNAs(miRNAs)are a class of non-coding small RNA molecules of about 21 nt that are widely present in higher eukaryotes.At present,the characterization of miRNA gene expression and function has become a hot topic in recent scientific research,such as the process of miRNA maturation and the fine-tuning of its target gene expression after transcription.In Arabidopsis thaliana,325 miRNA precursors and 427 mature miRNA molecules have been identified,but the structure and function of the vast majority of MIR genes have not been experimentally verified and studied in depth.Here,we preliminary analyzed the function of three miRNAs(miR858a,miR402,and miR777)downstream of HY5,a key regulator of photomorphogenesis in Arabidopsis thaliana,during the growth and development of Arabidopsis seedlings.Here,we predicted that miR858 could target the anthocyanin biosynthesis repressor MYBL2 in Arabidopsis and thus participate in the biosynthesis of anthocyanin.By overexpressing miR858a(miR858a-OX)in wild-type and silencing miR858 using Small Tandem Target Mimic(STTM),we detected anthocyanin content and the expression of anthocyanin synthetic genes inMIR858a-OX and STTM858 transgenic in Arabidopsis thaliana,respectively.In the increase and decrease respectively.Based on bioinformatics analysis results using qRT-PCR,we found no significant changes in the expression of MYBL2 in miR858a-OX and STTM858.We then constructed and obtained proMYBL2:MYBL2-HA/mybl2 transgenic plants and hybridized with MIR858a-OX and STTM858 plants,respectively;in the hybrid plants we detected a decrease and increase in MYBL2-HA protein levels,respectively.Therefore,we conclude that miR858a is involved in the synthesis and accumulation of anthocyanin in Arabidopsis by inhibiting the translation of MYBL2.Then,we verified the expression characteristics of MIR858a and MYBL2 genes dependent on the regulation of "light-HY5" signal,and summed up the molecular regulation model of "light-HY5-miR858a-MYBL2-anthocyanin".This result can provide data support for better understanding of the biosynthesis and regulation mechanism of anthocyanin metabolism,and provide reference for future genetic data to obtain a large number of anthocyanin-producing crops.Besides miR858a,we also explored the function of the MIR402 and MIR777 genes.First of all,to explore their expression characteristics:Through GUS staining we found that MIR402 is mainly expressed in the cotyledon,hypocotyl and apical meristem;HY5 can positively regulate the expression of MIR402,and MIR402 expression in the hypocotyl of the hy5 mutant significantly lower.Similarly,we have concluded that the expression of MIR777 in Arabidopsis seedlings is extremely low,whereas HY5 may negatively regulate the expression of MIR777;in hy5,MIR777 is mainly expressed at the tips of the cotyledon and hypocotyl.Later,we designed and obtained MIR-OX transgenic plants and STTM transgenic plants.In addition to finding that miR777 can inhibit the hypocotyl elongation of the hy5 mutant,there was no significant phenotype difference between the transgenic plants and their respective genetic background plants.In the follow-up study,we will further analyze the molecular mechanism of miR402 and miR777 involved in the regulation of photomorphogenesis in Arabidopsis from defferent expression genes.