Kumagai Hirotaka, Kouchi Hiroshi
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan.
Mol Plant Microbe Interact. 2003 Aug;16(8):663-8. doi: 10.1094/MPMI.2003.16.8.663.
We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express beta-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.
我们利用发根农杆菌介导的毛状根转化，研究了自互补发夹RNA（hpRNA）表达在模式豆科植物百脉根的根和根瘤中诱导RNA沉默的效果。通过组成型或根瘤特异性启动子表达β-葡萄糖醛酸酶（GUS）的转基因株系，被携带与GUS编码区序列互补的hpRNA表达构建体的发根农杆菌感染，从而进行超转化。超过60%的毛状根中的GUS活性降低或几乎完全沉默。在根瘤器官发生的早期和晚期，在毛状根上形成的共生根瘤中也观察到GUS基因的沉默。这些结果表明，通过毛状根转化实现的瞬时RNA沉默为在根和根瘤中起作用的基因的功能缺失分析提供了一个强大的工具。
