Molecular and cellular study of flavonoid metabolism and detection of relating genes in soybean

抗寒性是大豆（Glycine max (L.)）的重要性状。在凉爽的气候中生产。我们之前分离到了一个与T位点对应的大豆类黄酮3′羟化酶（F3′H）基因，该基因控制着短柔毛和种皮颜色。据报道，T基因与抗寒性之间存在遗传联系，但确切的潜在机制尚不清楚。利用近等基因系（NILs） To7B （TT）和To7G (TT)，研究了15℃低温处理对大豆冷害、抗氧化活性和黄酮类化合物谱的影响。To7G的第二三叶冷害较To7B严重。在To7G中冷却可增强脂质过氧化作用。低温诱导的抗氧化活性增强在To7B中比在To7G中更为明显。高效液相色谱（HPLC）分析表明，冷却处理后的To7B三叶第二叶中槲皮素苷和异鼠李素苷（3′，4′-二羟基黄酮醇衍生物）含量增加，而相同处理后的To7G三叶第二叶中山奈酚苷（4′-单羟基黄酮醇衍生物）含量增加。组织化学染色也显示低温诱导的类黄酮积累。微阵列分析和实时反转录pcr （RT-PCR）结果表明，大豆F3' H转录物水平在低温条件下上调。两种NILs在冷害、抗氧化活性和类黄酮种类上的差异支持了大豆F3' H通过产生3',4'-二羟基化黄酮醇衍生物提高抗氧化活性来影响抗寒性的观点。

Chilling tolerance is an important trait of soybeans (Glycine max (L.) Merr.) produced in cool climates. We previously isolated a soybean flavonoid 3' hydroxylase (F3' H) gene corresponding to the T locus, which controls pubescence and seed coat color. A genetic link between the T gene and chilling tolerance has been reported, although the exact underlying mechanisms remain unclear. Using the soybean near-isogenic lines (NILs) To7B (TT) and To7G (tt), we examined the relationship between chilling injury, antioxidant activity and flavonoid profiles associated with chilling treatment (15℃). Chilling injury was more severe in the second trifoliate leaves of To7G than in those of To7B. Lipid peroxidation was enhanced by chilling in To7G. Chilling-induced enhancement of antioxidant activity was more prominent in To7B than in To7G. High performance liquid chromatography (HPLC) analysis indicated that the contents of quercetin glycosides and isorhamnetin glycosides (3',4'-dihydroxylated flavonol derivatives) increase in the second trifoliate leaves of To7B after chilling treatment, whereas the same treatment increased kaempferol glycoside (4'-monohydroxylated flavonol derivatives) content in the corresponding leaves of To7G. Histochemical staining also demonstrated chilling-induced flavonoid accumulation. Microarray analysis and real-time reverse transcription-PCR (RT-PCR) demonstrated that the transcript levels of soybean F3' H are upregulated by chilling. The differences in chilling injury, antioxidant activity and flavonoid species between the two NILs support the notion that soybean F3' H affects chilling tolerance by increasing antioxidant activity via production of 3',4'-dihydroxylated flavonol derivatives.