Analysis of multiple silencing processes of the transgene-induced gene silencing in higher plants

烟草内质网ω-3脂肪酸去饱和酶（NtFAD 3）催化α-亚麻酸的形成。在转基因烟草植物（S44）中，积累了从转基因转录的NtFAD 3 mRNA。我们用蔗糖密度梯度离心法将多聚核糖体分成12个组分。在低蔗糖浓度的组分中发现转基因衍生的NtFAD 3 mRNA，表明NtFAD 3蛋白的合成被部分抑制。另一方面，内源NtFAD 3 mRNA的水平在S44植物中是痕量的，并且检测到来自内源NtFAD 3基因的异常剪接产物。这些异常剪接产物可能来源于内源性NtFAD 3基因第5内含子的剪接被抑制。利用基因枪法将内含子导入烟草细胞后，第5内含子的剪接发生困难，表明该内含子的剪接还需要其他因素，如外显子-内含子或内含子-内含子相互作用。我们的数据表明，在S44植物中，第5内含子的剪接调控将受到转基因诱导的基因沉默的干扰。

A tobacco endoplasmic retliculum ω-3 fatty acid desaturase (NtFAD3) catalyzes the formation of α-linolenate. In a transgenic tobacco plant (S44), the NtFAD3 mRNA transcribed from the transgene was accumulated. We fractionated the polysomes into 12 fractions by sucrose-density-gradient centrifugation. The transgene-derived NtFAD3 mRNAs were found in the fractions with low sucrose densities, indicating that the synthesis of NtFAD3 protein was partially inhibited. On the other hand, the level of endogenous NtFAD3 mRNA was a trace in the S44 plants, and aberrantly spliced products from the endogenous NtFAD3 gene were detected. These aberrantly spliced products would be derived from the inhibition of splicing of the 5th intron of endogenous NtFAD3 gene. The splicing of internal intron (5th intron) was difficult when this intron was introduced into tobacco cells by particle bombardment, suggesting that splicing of this intron would require another factor, such as exon-intron or intron-intron interaction. Our data suggest that in the, S44 plants, the regulation for splicing of the 5th intron would be perturbed by the transgene-induced gene silencing.