Molecular cloning and functional analysis of a flavanone 3-hydroxylase gene from blueberry

<i>Vaccinium corymbosum</i> (blueberry) is touted as a superfood with numerous health benefits due to its high levels of flavonoids. Flavanone 3-hydroxylase (F3H) is a key regulatory enzyme of the flavonoid pathway. In this study, we cloned the full-length cDNA of <i>F3H</i> (designated <i>VcF3H</i>) from young blueberry leaves using rapid amplification of cDNA ends (RACE). The cDNA contained a 1080-bp open reading frame that encoded a 359-amino acid protein. The deduced VcF3H protein showed high similarities to other plant F3Hs. Conserved amino acid motifs required for ferrous iron binding (HXD) and 2-oxoglutarate binding (RXS) were identified in VcF3H, VcFLS (flavonol synthase), and VcANS (anthocyanidin synthase). Quantitative RT-PCR analysis demonstrated that <i>VcF3H</i> was expressed in all tissues tested, with particularly high expression in young leaves, fruits (pink and blue), and stems. Anthocyanins accumulated mainly in fruits, whereas flavonols were found mainly in leaves and stems. Furthermore, the expression pattern of <i>VcF3H</i> was similar to that of <i>VcCHS</i>, <i>VcDFR</i>, and <i>VcANS</i> in various tissues. Heterologous expression of <i>VcF3H</i> in <i>Arabidopsis thaliana</i> increased the anthocyanin content in leaves, but did not affect the flavonol content. Thus, <i>VcF3H</i> seems to be involved in anthocyanin synthesis in the flavonoid biosynthetic pathway when ectopically expressed in <i>Arabidopsis</i>.

伞房花越橘（*Vaccinium corymbosum*，蓝莓）因其富含类黄酮而被誉为具有多种健康益处的超级食物。黄烷酮3-羟化酶（Flavanone 3-hydroxylase，F3H）是类黄酮生物合成通路的关键调控酶。本研究采用cDNA末端快速扩增技术（rapid amplification of cDNA ends，RACE），从蓝莓幼叶中克隆得到F3H的全长cDNA，将其命名为VcF3H。该cDNA包含一个长度为1080 bp的开放阅读框，编码一个含359个氨基酸残基的蛋白质。推导得到的VcF3H蛋白与其他植物的F3H蛋白具有较高的序列相似性。在VcF3H、VcFLS（黄酮醇合酶，flavonol synthase）与VcANS（花青素合酶，anthocyanidin synthase）中，均鉴定到了亚铁离子结合（HXD）及2-氧戊二酸结合（RXS）所需的保守氨基酸基序。实时定量RT-PCR分析结果显示，VcF3H在所有检测的组织中均有表达，其中在幼叶、粉色果实、蓝色果实以及茎中的表达量尤其高。花青素主要在果实中积累，而黄酮醇则主要分布于叶片和茎部。此外，在不同组织中，VcF3H的表达模式与VcCHS（查尔酮合酶，chalcone synthase）、VcDFR（二氢黄酮醇4-还原酶，dihydroflavonol 4-reductase）及VcANS均较为相似。将VcF3H在拟南芥（*Arabidopsis thaliana*）中进行异源表达，可提升其叶片中的花青素含量，但对黄酮醇含量无显著影响。综上，当VcF3H在拟南芥中异位表达时，其似乎参与了类黄酮生物合成通路中的花青素合成过程。