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

与 RNA 干扰不同的共抑制相关 RNA 沉默途径

基本信息

批准号：
17570030
负责人：
KODAMA Hiroaki
金额：
$ 2.24万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570030/
关键词：
RNA silencing fatty acid desaturase α-linolenic acid splicing

项目摘要

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.

烟草内质网ω-3脂肪酸去饱和酶（NtFAD 3）催化α-亚麻酸（18：3）的形成。将强启动子控制下的NtFAD 3基因导入烟草植株，引起18：3含量的增加。然而，其中一个转基因株系，称为S44株系，表现出共抑制表型，即18：3含量急剧下降。在S44植株中，NtFAD 3基因的表达受到前体mRNA剪接干扰的抑制。在对应于内含子6、7和8的结构基因序列中观察到这种错误剪接。事实上，通过体内剪接测定，这些内含子的剪接效率在S44植物中显示出下降。在S44植株的细胞核中检测到NtFAD 3 siRNA，表明这些siRNA参与了剪接的抑制，当将NtFAD 3转基因导入S44植株时，在所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。后者植物的18：3含量高于野生型植物的含量，表明NtFAD 3转基因成功地过表达（回复突变体表型）。由于甲基化敏感的限制性内切酶不能切割转基因启动子区的相应识别位点，因此转基因在S44回复突变体中的转录活性会下降。然后，通过引入携带启动子序列的发夹构建体来诱导转基因启动子区中胞嘧啶残基的甲基化。当将该发夹结构导入S44植物中时，在后代中观察到回复突变表型。这些结果表明，共抑制诱导的高活性转录的转基因。