LcMYB1激活荔枝花色苷生物合成关键基因LcDFR和LcUFGT1的启动子区域分析

Identification of Interaction Region Between LcMYB1 and Promoters of Anthocyanin Biosynthetic Genes LcDFR and LcUFGT1 in Litchi chinensis

花色苷含量是荔枝果实呈现鲜红色的重要次生代谢产物,前人研究结果表明,LcMYB1通过调控关键基因的表达而影响荔枝果皮中花色苷的积累,其中LcDFR和LcUFGT1是荔枝果皮花色苷生物合成的关键基因,但是LcMYB1如何影响基因的表达,是否与结构基因启动子结合以及具体结合区段目前还不清楚。本研究通过克隆和分析LcDFR和LcUFGT1的启动子区域并对其进行分段处理,利用双荧光素酶和酵母单杂交实验,探究LcMYB1与LcDFR和LcUFGT1的启动子的结合区域。结果表明,LcDFR起始密码子上游1927 bp和LcUFGT1起始密码子上游1584 bp的启动子上分别有3个可能的MYB结合位点;双荧光素酶和酵母单杂交实验均显示LcMYB1可以结合LcDFR基因起始密码子上游904 bp到1425 bp包含有1个MYB-CORE元件的区域,LcMYB1与LcUFGT1基因启动子结合的区域是起始密码到上游610 bp,此区域包含有2个MYB-CORE元件。研究结果进一步证实了LcMYB1通过与基因LcDFR和LcUFGT1启动子结合发挥调控作用,并缩小了结合位点的范围。

The accumulation of anthocyanins is one of the most important factors in determining the pericarp coloration of litchi.LcDFR(dihydroflavonol 4-reductase)and LcUFGT1(UDP-glucose:flavonoid 3-O-glucosyltransferase)are two crucial enzymes in the anthocyanin biosynthesis pathway of litchi pericarp,and the activities and gene expressions directly affect litchi fruit coloration.Previous study showed that the expressions of LcDFR and LcUFGT1 were regu-lated by the LcMYB1 transcription factor.However,how LcMYB1 regulates the expression of the target genes,whether LcMYB1 binds to their promoter,which cis-element in the promoter is the binding site of LcMYB1 remain to be un-covered.In this study,the promoters of LcDFR(1927 bp)and LcUFGT1(1584 bp)were cloned and analyzed respec-tively.Six candidate MYB binding sites and some other cis-elements such as light responsive elements and hormone related elements were predicted.The promoters of the two genes were then fragmented and ligated to pGreen-0800-luc plasmid to drive the expression of luciferase(LUC).Dual luciferase assay indicated that LcMYB1 could activate 904 bp to 1425 bp region of LcDFR promoter and 610 bp upstream of LcUFGT1 gene,which contain 1 and 2 candidate MYB-CORE sites,respectively.The interaction was also confirmed by the yeast one hybrid assay.These results sug-gested that LcMYB1 regulated the biosynthesis of anthocyanins by directly targeting the promoters of key structural genes and the binding region was narrowed down.