概要: In our study, we identified a new transgenerational epigenetic mutant in rice, Epi-LF (Epi-LongFeng). The metastable phenotype of Epi-lf resembles that of rice Epi-d1, and the inheritance is largely similar to that of the agoutiAy mouse mutant. We found that in Epi-lf,mCHH methylation was present in a 155-bp sequence of DNA in close proximity to the 3' end of the LF gene, but the abundance of the siRNAs corresponding to this region was not significantly different from that of the wild type (WT), suggesting that this is a mCHH island like modification. In addition, ectopic mCHH modifications accumulated in the sporophytic tissues and embryos of the Epi-lf mutants. The majority of these ectopic CHH regions did not fit with the OsDRM2 (Domain Rearranged Methylase2)-mediated CHH methylation of genomic regions, suggesting that unknown genetic factors may co-ordinate DNA demethylation and methylation, especially for non-CG methylation.
项目整体设计: We sequenced transcriptome, methylome and small RNA in different tissues of Eip-lf mutants and wild-type rice
The Epi-lf mutant was identified in a screen of an anther-cultured population of Japonica rice (Oryza sativa ssp. japonica; variety ZhongHua11; ZH11) in 2005. The mutant phenotype of Epi-lf was stably inherited over more than 20 generations. The mutant and WT rice plants were planted in paddy fields under natural conditions or in glasshouses with a day temperature of 30°C and a night temperature of 24°C.
处理方案:
The ambient temperature above 35 ℃ was about 6 h a day and the highest temperature was about 40 ℃ during almost the whole growth period of the natural heat stressed plants, the ambient temperature above 35 ℃ was only 3 days and the mean daytime temperature was about 30 ℃ during the growth period of the normal cultivation condition plants.
提取方案:
Total RNA was extracted from different tissues using TRIzol reagent (Thermo Fisher Scientific). The first-strand cDNA was synthesized using 1 μg RNA and ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo), according to the manufacturer’s instructions, using 29 PCR cycles to amplify the LF transcripts and 23 PCR cycles for Actin1. The RT-PCR analysis was repeated three times with similar results. Genomic DNA was isolated from the flag leaves of WT and tillers with different phenotypes of Epi-lf using the CTAB method. Briefly, 1 μg of genomic DNA was treated with sodium bisulphite using the Qiagen EpiTect Bisulfite kit, according to the manufacturer’s instructions. The treated DNA was dissolved in 15 mL distilled water, and 3 μL of this solution was used as the template in a 50-μL PCR (94°C for 4 min; followed by 40 cycles of 94°C for 30 s , 55°C for 30 s and 60°C for 30 s; with a final 60°C for 10 min).The PCR products were purified and cloned into pEASY-T5-Blunt Zero Cloning Kit vectors.
建库方案:
RNA libraries were prepared for sequencing using standard Illumina protocols.
