Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii

被引:40
作者
Yamasaki, Tomohito [1 ]
Miyasaka, Hitoshi [2 ]
Ohama, Takeshi [1 ]
机构
[1] KUT, Dept Environm Syst Engn, Kochi 7828502, Japan
[2] Kansai Elect Power Co, Environm Res Ctr, Kyoto 6190237, Japan
关键词
D O I
10.1534/genetics.108.092395
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
RNA interferences in the unicellular green alga, Chlangdomonas reinhardtii, call be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi transformed strain with successive cell divisions. Northern Not analyses revealed all inverse correlation between the the copy number Of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest. the existence of all intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent. DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and Suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.
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页码:1927 / 1944
页数:18
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