大豆GmF6′H1基因的克隆及功能验证

Molecular Cloning and Functional Characterization of GmF6′H1 in Soybean

以大豆品种‘黑农35号’为材料,利用同源克隆方法获得了一个依赖于Fe(Ⅱ)和2-酮戊二酸的双加氧酶基因,命名为GmF6′H1。荧光定量PCR分析显示GmF6′H1在大豆根中的表达量最高,其次是荚果、叶和茎;2,4-D处理对大豆GmF6′H1的转录水平影响很小,水杨酸和激动素的处理均显著提高了GmF6′H1基因的表达,但随着处理时间的延长表达水平稳定下降,最后接近处理前的水平。带有组氨酸标签的GmF6′H1异源表达的蛋白纯化后,以阿魏酰辅酶A为底物研究了其酶活特性,利用HPLC分析检测到GmF6′H1能够催化阿魏酰辅酶A生成东莨菪素。采用农杆菌介导法将该基因转入拟南芥Atf6′h1突变体,转基因拟南芥与Atf6′h1突变体植株外在表型没有明显的差异,而香豆素的含量分析显示,转基因株系根中香豆素的含量较拟南芥Atf6′h1突变体有所提高,与野生型拟南芥中香豆素的含量接近。

A Fe（ Ⅱ ）- and 2-oxoglutarate-dependent dioxygenase, named as GmF6＇H 1, was cloned from soy- bean variety‘ Heinong 35＇. Expression of GmF6＇H1 in different organs was examined by Quantitative（Q）- PCR analysis. The expression of GmF6＇H1 was the highest in the roots,followed by in the pods,leaves and stems. Its expression showed weakly response to 2,4-D treatment and increased in a time-dependent man- ner after salicylic acid and kinetin treatment separately. With the time of treatment prelonged, GmF6＇H1 expression levels steadily declined, and finally close to the level before the treatments. Purified protein of GmF6＇H1 was able to catalyze feruloyl coenzyme A to scopoletin. By Agrobacterium tumefaciens mediated transformation,GmF6＇ H1 were transformed into the mutant Atf6＇h 1 of Arabidopsis thaliana. Compared with mutant plants, the external phenotype of the transgenic plants had no obvious difference, while its coumarin content in the roots increased the level close to the wild type of A. thaliana.