Cloning And Expression Analysis Of F3’H&MYB4Genes In Lycoris Sprengeri

Lycoris species resources are in rich and widely distributed in China. Lycoris species plants arenamed after “Chinsese Tulip” because of their abundant flower colors and types. Their ornamentalvalue has important significance in Gardens and gardening. Because the petal color of LycorisSprengeri is mixture with red and blue. In this study, in order to study the flower color formationmechisim of Lycoris Sprengeri, we cloned LsF3‘Hand LsMYB4genes related anthocyaninbiosythesis by RT-PCR and RACE technology, analyzed the nucleotide sequences and amino acidsequences by bioinformatics, analyzed the expression moduler in different tissues and organs,different developmental stages, and different clones with different flower color by Real-TimeRT-PCR. The functions of LsF3’H and LsMYB4genes in anthocyanin biosythesis were investigatedthrough constructing over expression vectors, and transgened into tobacco. The main results asfollowed:(1)1587bp LsF3‘H and793bp LsMYB4cDNA complete length were acquired by exploringhomologous genes from NCBI Genebank, designing degenerate primers and combining RACEtechnology. The bioinformatics analysis showed that LsF3‘H and LsMYB4had specific structuraldomains, LsF3‘H had specifically structural domain cytochrome P450, and LsMYB4had highlyconservativestructural domain R2R3-MYB. On the basis of the multiple sequence alignment, weconstructured the phylogenetic tree, and the results showed it is sonsistant with the classification ofplants. LsF3‘H belonged to CYP75subfamily, and LsMYB4had the highest similarity with AtMYB4subfamily of Arabidopsis.(2) The expression of LsF3‘H and LsMYB4genes in petal of Lycoris Sprengeri were studied usingReal Time-qPCR. The results showed that the expression of LsF3‘H and LsMYB4genes weretissue-specific. LsF3‘H gene expressed in different tissues and developmental stages, and therelatived expression quantity of petals was higher10times than that of leaves, the relativedexpression quantity of full bloom stage was higher70times than that of bud period. There weresignificant difference among different clones with different flower color. The relatived expression ofthe clone with light color was higher than the other clones. The expression of LsMYB4gene is higher9-10times than that of leaves, and the relatived expression of full bloom stage was higher20timesthan that of bud period. The relatived expression of different clones was consistant with that of LsF3’H gene.We could guessed that LsMYB4gene had a negative regulation role on flower colorformation of Lycoris Sprengeri.(3) We constructed over expression vectors of LsF3’H and LsMYB4genes by double digestion, andtransformed tobacco by Agarobacterium.19positive transgenetic plants were acquired and verifiedby PCR. But the effect and function of anthocyanin biosythesis of LsF3’H and LsMYB4genes willbe needed to further study.All these results will provide an basis of flower color breeding and molecular modification ofLycoris species.