Abstract:

To elucidate the molecular mechanisms by which mulberry miR408 targets the MnBBP gene to modulate anthocyanin biosynthesis，the effect of mulberry miR408 on anthocyanin metabolism was analyzed by quantitative real-time PCR（qRT-PCR），bioinformatics analysis，5′-rapid amplification of cDNA ends（5′-RACE），and transgenic methodologies. Sequence alignment revealed that the miR408 sequence is identical to that of dicotyledonous plants，while featuring a single nucleotide polymorphism （SNP）at the first nucleotide position in monocotyledons. Expression profiling via qRT-PCR demonstrated differential expression patterns of miR408 across various mulberry tissues，with high expression levels observed in leaves and fruits. Through predictive analysis using the Omicstudio online platform，the mulberry blue-copper binding protein gene（MnBBP）was identified as a target gene of miR408. Degradome sequencing further corroborated that MnBBR is subject to specific cleavage by miR408. 5′-RACE analysis pinpointed the cleavage site between the 9th and 10th bases of the sequence complementary to miR408. Overexpression of mulberry miR408 in Arabidopsis thaliana led to elevated anthocyanin levels in the leaves. Transcriptomic analysis identified differentially expressed genes（DEGs）between the transgenic and wild-type plants，with a notable enrichment of DEGs in the anthocyanin biosynthetic pathway. The up-regulation of five key genes within this pathway was subsequently validated by qRT-PCR. Collectively，this study not only identifies MnBBP as a target gene of miR408 in mulberry，but also elucidates the regulatory roles of miR408 and MnBBP in anthocyanin biosynthesis.

Key words: mulberry, fruit, miR408, MnBBP, transgenic, anthocyanin