Gene Expression
Nebulas
Gene Expression NebulasSummary: Cytosine methylation at CG sites (mCG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain mCG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2-/-), which directly segregated from normal heterozygote plants (OsMET1-2+/-), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of mCG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.
Overall Design: mRNA-seq of homozygote, heterozygote and WT
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| Growth Protocol: | The three genotypes of the OsMET1-2 gene in rice (cv. Nipponbare), wild-type (WT, +/+), heterozygote (+/-) and homozygous mutant (-/-) were germinated and grown on the hormone-free, half-strength Murashige & Skoog (MS) medium under 16/8 hrs light/dark at 26/24℃. |
| Treatment Protocol: | - |
| Extract Protocol: | Total RNA from the 11-d-old shoots of the three OsMET1-2 genotypes was isolated for RNA-seq. |
| Library Construction Protocol: | For each genotype, two independent libraries were constructed and sequenced following the standard Illumina sequencing procedure on the HiSeq 2000. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |