Cosuppresion-associated RNA silencing pathway that is different from RNA interference

烟草内质网ω-3脂肪酸去饱和酶(NtFAD3)催化α-亚麻酸(18：3)的形成。将强启动子控制下的NtFAD3基因导入烟草植株，可提高烟草植株的18：3含量。然而，其中一个转基因品系S44表现出共抑制的表型，即18：3含量急剧下降。在S44植株中，内源NtFAD3基因的表达受到Pre-mRNA剪接干扰的抑制。在与内含子6、7和8对应的结构基因序列中观察到了这种错误剪接。事实上，体内剪接试验表明，这些内含子的剪接效率在S44植物中降低。从S44叶片制备的核组分中检测到NtFAD3 siRNAs，表明这些siRNAs参与了剪接的抑制。当另一个NtFAD3转基因拷贝导入S44植株时，在产生的后代中观察到两种表型：一种是共抑制表型，另一种是叶片中18：3含量的增加。后一代植株的18：3含量高于野生型植株，说明NtFAD3基因已成功过表达(突变表型)。由于甲基化敏感的限制性内切酶未能在转基因的启动子区域切割相应的识别位点，因此在S44回复植株中转基因的转录活性应该会下降。然后，通过引入含有启动子序列的发夹结构来诱导转基因启动子区域胞嘧啶残基的甲基化。当这种发夹结构被导入S44植株时，在后代中观察到了回复表型。这些结果表明，共抑制是由转基因的高活性转录诱导的。

A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) catalyzes the formation of α-linolenic acid (18:3). Introduction of the NtFAD3 gene under the control of strong promoter into tobacco plants caused increase of 18:3 content. However, one of the transgenic lines, termed S44 line, showed a cosuppressed phenotype, namely drastic decrease of 18:3 content. In the S44 plants, the expression of endogenous NtFAD3 gene was inhibited by interference of splicing of pre-mRNA. This mis-splicing was observed in the sturctual gene sequence corresponding to the introns 6, 7 and 8. In fact, the splicing efficiencies of these introns are shown to decrease in the S44 plants by in vivo splicing assay. The NtFAD3 siRNAs were detected in the nuclear fraction prepared from the S44 leaves, implying the involvement of these siRNAs in the inhibition of splicing.When another copy of the NtFAD3 transgene was introduced into the S44 plants, two phenotypes were observed in the resultant offsprings; one is cosuppressed phenotype and the other is increase of 18:3 content in leaves. The 18:3 content of latter plants was higher than that of the wild type plants, indicating that the NtFAD3 transgene was successfully overexpressed (revertant phenotype). Since methylation-sensitive restriction enzymes failed to cut the corresponding recognition sites in the promoter region of the transgenes, transcriptional activity of the transgene should be declined in the S44 revertant plants. Then, methylation of cytosine residues in the promoter region of the transgene was induced by introduction of a hairpin construct that harbored the promoter sequences. When this hairpin construct was introduced into the S44 plants, the revertant phenotype was observed in the offsprings. These results indicated that cosuppression was induced by highly active transcription of the transgene.