关键词: 水稻, 组蛋白H1, CRISPR, 转录组测序, RT-qPCR

Abstract:

【Objective】Histone H1 plays an important role in the maintenance and stabilization of higher chromatin structure. Elucidating the effect of rice histone H1 on gene expressions will help to better understand the regulatory functions of H1 in rice.【Method】Semi-quantitative RT-PCR and real-time quantitative PCR were used to detect the expressions of four H1 genes in rice. CRISPR technology was used to generate h1 mutant plants; subsequently, phenotypic and transcriptomic analysis of the h1 mutant was performed. 【Result】The four rice H1 genes were broadly expressed, and are weakly expressed in root. In T₀ generation of CRISPR-based gene editing plants, multiple mutations in H1.1-H1.4 were identified. In T₁ generation, one Osh1.1 Osh1.3 Osh1.4 homozygous triple mutant was identified, which had various developmental defects and became the material for further transcriptomic sequencing analysis. In T₂ generation, quadruple and triple mutants were acquired, approximate 25% of which were albino seedlings, slow in plant development and defect in drought responses. By transcriptome sequencing of the h1 mutant, 1 055 differentially expressed genes were identified, of which the significantly up-regulated genes were approximately 2.5 times of the down-regulated genes, implying that H1 may inhibit gene expressions at genome-wide scale. 【Conclusion】In the mutant, pathways of photosynthesis, stress responses, amino acid and RNA metabolism were disrupted. The ribosome biogenesis and photosynthesis pathway genes were significantly up-regulated; while the stress-associated dehydrogenase genes were down-regulated. It is presumed that overexpression of ribosome pathway genes resulted in the disorder of protein homostasis, which ultimately caused plant developmental defects.

Key words: rice, histone H1, CRISPR, transcriptome sequencing, RT-qPCR