纤毛类原生动物具有独特的核二态型,有性生殖过程中来源于生殖小核的新大核维持了亲本大核的染色质的组织和结构. 研究表明进化中保守的表观调控因子、DNA损伤修复蛋白等功能分子参与了纤毛虫新大核发育,然而新大核发育的具体分子机制仍不清楚.本研究拟通过分析嗜热四膜虫基因组不同时期的表达谱,鉴定新大核发育阶段特异表达的功能基因,构建含有HA、V5和GFP等标签融合基因的突变体细胞株, 免疫荧光定位分析基因表达产物的定位模式；通过基因敲除、RNA干扰和过表达技术,分析比较基因的缺失和过表达对四膜虫有性生殖过程中新大核发育的影响,进而通过免疫共定位和FISH技术深入分析新大核发育过程中组蛋白H3K9和H3k27甲基化修饰和DNA序列删除和修复变化,免疫共沉淀分析不同基因编码蛋白是否存在直接相互作用,由此建立四膜虫新大核发育的分子调控网络,为深入理解纤毛虫细胞核发育的表观遗传调控分子机制提供新的数据.

Ciliate harbors two types of nuclei within its single cell: a somatic macronucleus and a germline micronucleus.The diploid germline micronuclei are transcriptionally silent during vegetative growth and undergo meiosis during sexual events. The highly polyploidy somatic macronucleus is responsible for gene expression throughout the life cycle. In each sexual conjugation cycle, the old somatic nucleus disintegrates and a new soma derives from the germline micronucleus. The new macronuclear development is regulated by epigenetic regulated factors and DNA repair proteins which are conserved in evolution. However, the molecular mechanism of new macronuclear development is not clear. To explore molecular mechanism involved in the new macronuclear development in ciliate, the novel genes will be identified by gene expression profile and network analysis in Tetrahymena thermophila.Then, dynamic localization of HA/V5/GFP-tagged proteins in mating Tetrahymena cells will be obtained using specific antibody by immunofluorescence staining. The function of the novel genes will be analyzed by gene knockout, gene knockdown and gene overexpression methods. Furthermore, the change of small RNA, H3K9me3, H3K27me3 and specific internal eliminate sequences (IESs) will be analyzed in these mutant cells. The interaction of the proteins encoded by candidate genes will be analyzed by co-localization or co-immunoprecipitation . Based on the data, the molecular network of the new macronuclear development will be established. The study will strengthen our understanding for epigenetic regulation of the new macronuclear development in ciliate.