Table_1_GbFLSa overexpression negatively regulates proanthocyanin biosynthesis.docx

Flavonoids are important secondary metabolites with extensive pharmacological functions. Ginkgo biloba L. (ginkgo) has attracted extensive attention because of its high flavonoid medicinal value. However, little is understood about ginkgo flavonol biosynthesis. Herein, we cloned the full-length gingko GbFLSa gene (1314 bp), which encodes a 363 amino acid protein that has a typical 2-oxoglutarate (2OG)-Fe(II) oxygenase region. Recombinant GbFLSa protein with a molecular mass of 41 kDa was expressed in Escherichia coli BL21(DE3). The protein was localized to the cytoplasm. Moreover, proanthocyanins, including catechin, epicatechin, epigallocatechin and gallocatechin, were significantly less abundant in transgenic poplar than in nontransgenic (CK) plants. In addition, dihydroflavonol 4-reductase, anthocyanidin synthase and leucoanthocyanidin reductase expression levels were significantly lower than those of their CK counterparts. GbFLSa thus encodes a functional protein that might negatively regulate proanthocyanin biosynthesis. This study helps elucidate the role of GbFLSa in plant metabolism and the potential molecular mechanism of flavonoid biosynthesis.

黄酮类化合物（Flavonoids）是一类具备广泛药理活性的重要次生代谢产物。银杏（Ginkgo biloba L.）因具有极高的黄酮类药用价值而受到学界广泛关注。然而，目前对于银杏黄酮醇的生物合成机制仍知之甚少。本研究克隆得到全长为1314 bp的银杏GbFLSa基因，该基因编码一个由363个氨基酸残基组成的蛋白质，其序列中包含典型的2-氧代戊二酸（2-oxoglutarate，2OG）-铁(II)加氧酶结构域。我们将分子量为41 kDa的重组GbFLSa蛋白在大肠杆菌（Escherichia coli）BL21(DE3)中成功表达，该蛋白定位于细胞质中。此外，转基因杨树中原花青素（涵盖儿茶素、表儿茶素、表没食子儿茶素与没食子儿茶素）的积累量显著低于非转基因对照（CK）植株；同时，二氢黄酮醇4-还原酶、花青素合酶与无色花色素还原酶的基因表达水平均显著低于对照植株。综上，GbFLSa编码一个具有功能活性的蛋白质，可负向调控原花青素的生物合成过程。本研究有助于阐明GbFLSa在植物代谢中的具体作用，以及黄酮类化合物生物合成的潜在分子机制。