Abstract：Lycoris sprengeri is a kind of non-model perennial bulbous plant of Lycoris, whose flower color is bicolor with red and blue, and which is great ornamental value. It is of great significance to explore the mechanism of flower color formation by using genetic engineering technology for the breeding of new varieties of L. sprengeri. Virus-induced gene silencing (VIGS) is a new reverse genetics technology, which could be used to study the gene function of non-model plants. In this study, LsMYBs VIGS vectors were constructed by seamless cloning technology , the buds of L. sprengeri were infected using vacuum permeation method, and the expression of LsMYBs and anthocyanin contents of the petals in L. sprengeri after infection were detected, which would establish the VIGS technology system in L. sprengeri and analyzed the gene function of LsMYBs. The results showed that the expression of LsMYBs genes decreased significantly and the total contents of anthocyanin increased significantly after LsMYBs genes silencing. The relative expression of LsMYBs gene was relatively stable by 400 bp insertion fragment in VIGS vector, while the relative expression of LsMYBs was unstable after inserting a full length fragment of cDNA. The relative expression of structural genes ((chalcone synthase (LsCHS), chalcone isomerase (LsCHI), flavanone 3-hydroxylase (LsF3H), flavonoid 3'-hydroxylase (LsF3'H), dihydroflavonol-4-reductase (LsDFR), anthocyanidin synthaes (LsANS), flavonoid 3-o-glycosyltransferase (LsUFGT)) related to anthocyanin formation was affected after LsMYB4 gene silencing, the relative expression of all of the structural genes increased significantly during the whole anthocyanin biosynthesis process (early biosynthesis genes (EBGs) and late biosynthesis genes (LBGs)) and the degree of change was varying; while the expression of LBGs genes (LsANS, LsDFR3 and LsUFGT) increased significantly when LsMYB5 silencing, however, the expression of other genes did not change significantly. It could be inferred that LsMYB4 and LsMYB5 might play a negative regulatory role in the regulation of anthocyanin formation, while the target genes may be different. The results in this study provide a technical and theoretical basis for further analysis of the regulatory mechanism of LsMYBs transcription factors on the biosynthesis of anthocyanin in L. sprengeri.
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