Stam M, de Bruin R, van Blokland R, van der Hoorn R A, Mol J N, Kooter J M
Department of Developmental Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
Plant J. 2000 Jan;21(1):27-42. doi: 10.1046/j.1365-313x.2000.00650.x.
The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.
反义转基因在植物中的应用是抑制基因表达的有力工具。这种抑制作用的潜在机制仍知之甚少。高水平的反义RNA（as-RNA）有望导致强烈的沉默，但as-RNA水平与沉默程度之间往往没有明显的相关性。为了深入了解这些令人困惑的现象，我们分析了几种矮牵牛转化体，其中色素沉着基因查尔酮合酶（Chs）在花冠中通过反义（as）Chs转基因在转录后被沉默。对这些转化体进行了稳态as-RNA水平、as-转基因的转录水平、整合的T-DNA的重复性和结构以及转基因的甲基化状态的检测。结果表明，这些转化体可分为两类：第一类含有单个拷贝（S）的T-DNA，其as-Chs基因被转录，尽管转录水平比内源性Chs基因低几倍。由于没有足够的as-RNA来降解每一个mRNA，我们推测沉默是由双链RNA诱导的。第二类含有两个T-DNA，它们以反向重复（IR）的形式排列。这些IR位点被严重甲基化，as-Chs转基因的转录几乎没有活性。在as-Chs转基因靠近IR中心的IR位点观察到最强的沉默。由正义Chs转基因组成的IR介导的共抑制也有类似的特征，这表明反义IR介导的沉默也通过共抑制发生，要么通过异位DNA配对，要么通过双链RNA。
