桑树MaDFR的克隆及功能分析

【目的】克隆桑树（Morus alba L.）二氢黄酮醇4-还原酶（dihydroflavonol-4-reductase，DFR）基因，并通过烟草过表达研究其对烟草黄酮生物合成的影响，为该基因功能分析及应用提供理论参考。【方法】采用RT-PCR的方法从桑树中克隆出MaDFR，采用农杆菌介导的遗传转化技术，将MaDFR导入模式植物烟草中，获得具有卡那抗性的转基因烟草植株，通过基因组PCR鉴定转基因阳性植株，采用反向PCR的方法分析T-DNA在烟草中的插入位点。通过定量PCR的方法分析MaDFR在烟草中的表达水平，AlCl₃分光光度法测定转基因烟草中总黄酮含量，通过DPPH（1,1-二苯基-2-三硝基苯肼）清除能力分析总黄酮酸水解前后的抗氧化活性，最后通过高效液相色谱（HPLC）分析烟草花冠中花青素的含量及种类。【结果】从中桑5801中克隆并获得了全长为1 026 bp的MaDFR，编码341个氨基酸残基，其蛋白质具有典型的NADPH结合位点。获得7株具有卡那抗性的烟草转基因株系。分子鉴定4株为转基因阳性株系。MaDFR在转基因烟草中能够表达，但不同转基因株系之间MaDFR表达水平存在较大差异。转基因烟草叶片中总黄酮含量明显增加，其中，DFR-4和DFR-7 2个株系分别增加了30.4%和27.6%，DFR-2和DFR-5分别增加了19.8%和14.2%。抗氧化活性分析发现转基因株系叶片中未酸解总黄酮抗氧化活性存在较大差异，DFR-2、DFR-4的抗氧化能力比对照组提高了近5倍，而DFR-5和DFR-7与对照组没有显著性的差异，转基因株系叶片中总黄酮的抗氧化活性与MaDFR的表达水平具有显著的正相关性。转基因株系叶片中酸解总黄酮抗氧化活性与总黄酮的含量具有显著的正相关性。MaDFR在烟草中的过表达能够增加花冠中花青素的含量，DFR-4株系中花青素含量增加了15%，DFR-5和DFR-7 2个株系中花青素的含量增加了一倍，但转基因烟草花冠中花青素种类没有发生改变。【结论】MaDFR在烟草中的过表达能够显著增加烟草的总黄酮含量及抗氧化作用，也可以显著增加烟草花冠中花青素的含量，但不能改变花青素的种类，说明该基因在矢车菊素类花青素的生物合成代谢路径中具有重要作用。

Cloning and Function Analysis of a MaDFR Gene from Mulberry

【Objective】In this study, MaDFR was isolated from mulberry (Morus alba L.) and transformed into tobacco for function analysis of flavonoids biosynthesis and providing application basement. 【Method】Fragment of MaDFR was obtained by RT-PCR and transformed into tobacco by using Agrobacterium tumerficians-mediated transformation approach. Plants with kanamycin resistance were obtained for identification by using genome PCR and insertion sites analysis by reverse PCR. The expression levels were determined by qRT-PCR. The concentration of total flavonoids was analyzed by AlCl₃ and the antioxidant activity of acid hydrolysis and acid hydrolysis was showed by using DPPH scavenging ability. HPLC was used for identification of anthocyanins in tobacco. 【Result】The full-length CDS sequence of MaDFR obtabined from Zhongsang 5801 is 1026 bp in length encoding a putative protein about 341 amino acids with a typical NADPH binding site. A transgenic vector based on pBI121 was constructed and transformed into tobacco by Agrobacterium tumerficians-mediated transformation approach. Seven plants with kanamycin resistance were obtained. Four positive plants were identified by genome PCR and reverse PCR. In transgenic plants MaDFR showed different expression levels. There were significant increases of total flavonoids in transgenic plants, DFR-2(19.8%), DFR-4(30.4%), DFR-5(14.2%), and DFR-7 (27.6%), respectively. There was a large difference in the antioxidant activity between control and transgenic plants, the antioxidant activity of total flavonoids in DFR-2 and DFR-4 showed significant increase by 5 times, while the antioxidant activity of DFR-5 and DFR-7 showed no significant difference compared to control. The antioxidant activity of total flavonoids before acid hydrolysis showed a well correlation with the expression levels of MaDFR in tobacco. The antioxidant activity of acid hydrolysis of total flavonoids in transgenic plants showed a well correlation with the contents of total flavonoids. The overexpression of MaDFR improved the content of anthocyanins in transgenic tobacco corolla, DFR-4(15%), DFR-5(100%) and DFR-7 (100%), respectively, but could not change the components of anthocyanins.【Conclusion】The overexpression of MaDFR in tobacco increased the content and antioxidant activity of total flavonoids. MaDFR also deepened the color of tobacco corolla by improving the content of anthocyanins, and thus playing an important role in biosynthesis of anthocyanins, especially in cyanidin.