原生动物四膜虫有性生殖期新大核发育调控相关基因的鉴定和功能分析

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.

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.

纤毛类原生动物具有独特的核的二态型, 具有体细胞系的多倍体大核和生殖系的二倍体小核。有性生殖过程中，来源于生殖系小核的新大核通过表观遗传调控的机制重塑染色质的组织和结构，产生新的大核. 进化中保守的表观遗传控因子、DNA损伤修复蛋白、转座酶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。本研究通过分析嗜热膜虫基因组数据库和功能基因组基因组数据库，筛选和鉴定新大核发育阶段特异表达的功能基因TCD1，ZFR2，MLH1，MLH3, CYC2等含有保守功能结构域的功能基因，通过构建含有HA标签融合基因的突变体细胞株，免疫荧光定位获得不同基因表达产物的细胞定位模式；通过基因敲除、RNA干扰和过表达技术,分析了不同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 ，通过免疫荧光共定位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,免疫共沉淀和质谱鉴定了不同基因编码蛋白存在直相互作用因子。结果表明Tcd1对新大核发育过程特异DNA序列的删除是必须的，同时对后期DNA的修复也是必须的；MLH1 的敲除突变体不影响生长，但导致小核染色体不稳定，有性生殖不能完成，减数分裂的小核染色体不能正常修复。Mlh3通过金属离子依赖的内切酶活性及与其它因子相互作用在减数分裂错配修复和同源重组过程中发挥重要作用；ZFR2基因的表达缺失导致了H3K27和H3K9三甲基化无法在新发育的大核上富集，无法形成正常的异染色质体和IES序列的特异删除，对四膜虫有性生殖发育的完成是必需的。CYC2表达缺失都严重阻滞了小核在减数分裂前期的延伸，无法形成crescent结构，并最终导致减数分裂发育失败。本研究建立了四膜虫新大核发育过程中的分子调控网络,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。