芜菁类黄酮3′-羟化酶基因的功能鉴定及启动子初步分析

类黄酮3′-羟化酶（Flavonoid 3′-hydroxylase，F3′H）是细胞色素P450单加氧酶，在花青素合成途径中催化二氢山奈酚生成二氢槲皮素，进而形成矢车菊色素。利用津田芜菁BrF3′H1和赤丸芜菁BrF3′H2基因构建过量表达载体后遗传转化烟草，转基因植株的花色加深。通过染色体步移法克隆了BrF3′H1和BrF3′H2基因上游846和851 bp的启动子序列。生物信息学分析表明，BrF3′H1P和BrF3′H2P均包含TATA box、CAAT box、光调控元件、MRE、ABRE、ATGCAAAT-motif、ERE、O2-site、RY-element、LTR等多个顺式作用元件；二者的核苷酸序列在7个位点存在差异。利用BrF3′H1P和BrF3′H2P序列替换pCAMBIA1301植物表达载体的35S启动子后遗传转化烟草。GUS组织化学染色结果表明，BrF3′H1P和BrF3′H2P序列均能驱动GUS基因表达。通过PCR方法获得了BrF3′H1P和BrF3′H2P的一系列缺失片段，融合GUS基因后转化烟草。染色结果显示，BrF3′H1P和BrF3′H2P系列缺失片段均具有起始GUS基因表达的活性。BrF3′H1和BrF3′H2基因的功能鉴定及启动子的初步分析将为揭示津田芜菁和赤丸芜菁F3′H基因的光诱导表达调控机理奠定研究基础。

Functional Identification and Promoter Preliminary Analysis of Flavonoid 3′-Hydroxylase Genes in Turnip

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.