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（NtFAD3）催化α-亚麻酸盐的形成。在一株转基因烟草植株（S44）中，NtFAD3 mRNA转录积累。我们用蔗糖-密度梯度离心将聚体分成12个部分。在低蔗糖密度的馏分中发现了转基因衍生的NtFAD3 mrna，表明NtFAD3蛋白的合成受到部分抑制。另一方面，内源NtFAD3 mRNA水平在S44植株中是微量的，并且检测到来自内源NtFAD3基因的异常剪接产物。这些异常剪接产物可能源于内源性NtFAD3基因第5内含子剪接受到抑制。通过粒子轰击将内含子（第5内含子）引入烟草细胞时，该内含子的剪接是困难的，这表明该内含子的剪接可能需要其他因素，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内含子的剪接调控会被转基因诱导的基因沉默所干扰。

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.