关键词: 芜菁, 类黄酮3′-羟化酶基因, 遗传转化, 启动子, 功能分析

Abstract: Flavonoid 3′-hydroxylase(F3′H) is a cytochrome P450 monooxygenase, it can convert dihydrokaempferol to dihydroquercetin in the pathway of anthocyanin biosynthesis, and the cyanidin can be formed. The overexpression vectors of BrF3′H1 and BrF3′H2 genes cloned from ‘Tsuda’ turnip and ‘Yurugi Akamaru’ turnip, respectively, were constructed, and then were transformed into tobacco. The flower color of transgenic tobacco plants was darker. The 846 and 851 bp promoter fragments upstream of BrF3′H1 and BrF3′H2 genes were obtained by genome walking method. Many cis-acting elements in BrF3′H1 and BrF3′H2 promoters were identified by bioinformatics analysis, such as TATA box, CAAT box, light responsive elements, MRE, ABRE, ATGCAAAT-motif, ERE, O2-site, RY-element and LTR. The nucleotide sequences of BrF3′H1P and BrF3′H2P had 7 differences. The 35S promoter of pCAMBIA1301 plant expression vector was substituted by BrF3′H1P and BrF3′H2P, respectively, and then the recombinants were introduced into tobacco through the mediation of Agrobacterium tumefaciens. Histochemical staining result of GUS indicated that the expression of GUS reporter gene could be driven by BrF3′H1P and BrF3′H2P sequence. A series of 5′-end deleted fragments of BrF3′H1P and BrF3′H2P were obtained by PCR, which were fused with GUS reporter gene and then were transformed into tobacco. The stain result showed that the deleted fragments of BrF3′H1P and BrF3′H2P had starting activity that could initiate the expression of GUS gene. The functional identification of BrF3′H1 and BrF3′H2 genes and the preliminary analysis of their promoters would lay a foundation for clarifying the mechanism on light inducible expression regulation of F3′H genes in ‘Tsuda’ turnip and ‘Yurugi Akamaru’ turnip.

Key words: Turnip, fvonoid 3′-hydroxylase gene, genetic transformation, promoter, functional analysis