Puf1在弓形虫速殖子与缓殖子转化中的作用及机制研究

T.gondii is an opportunistic pathogen and the biological basis is tachyzoite-bradyzoite interconversion, post-transcriptional regulation might play a critical role in it. RNA binding proteins mediating translational repression is an important form of the transcription regulation, and the PUF family is an important member of the RNA binding proteins, but its role in T.gondii tachyzoite-bradyzoite stages conversion is not clear. For the first time, we studied T.gondii Puf1(TgPuf1) protein, the results showed that TgPuf1 has strong RNA binding affinity, and it appeared to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating genes expression during tachyzoite-bradyzoite stages conversion. We propose the RNA-Protein Pull-down and RNA-EMSA technologies to find and identify the target mRNAs of TgPuf1, determine TgPuf1 associtating proteins through co-immunoprecipitation, and explore the function of TgPuf1 protein through construction TgPuf1 gene disruption strain, and then observe the changes in the phenotype and analyze the changes in the expression profile. The objective is to clarify the function and mechanism of TgPuf1 in tachyzoite-bradyzoite conversion.

弓形虫速殖子和缓殖子期特异基因的转录后调控，在其相互转化过程中起着重要作用。RNA结合蛋白介导的翻译抑制是转录后调控的重要形式，PUF家族是RNA结合蛋白的重要成员，但PUF蛋白在弓形虫速殖子与缓殖子转化中的作用尚不明确。申请者前期工作已证实弓形虫RNA结合蛋白TgPuf1具有RNA结合活性，并发现TgPuf1的表达及分布在速殖子与缓殖子间有显著差异。因此，推测TgPuf1蛋白可能参与速殖子与缓殖子转化的基因表达调控。在此基础上，本项目拟①采用RNA-Protein Pull-down和RNA-EMSA技术，寻找并验证TgPuf1的靶mRNA；②通过Co-IP技术，筛选并分析与TgPuf1蛋白相互作用的蛋白分子；③采用CRISPR/CAS9技术，构建TgPuf1基因敲除虫株，观察其对虫体转化及表达谱的影响。研究目标是阐明TgPuf1在速殖子与缓殖子转化中的作用和机制。

弓形虫速殖子和缓殖子期特异基因的转录后调控，在其相互转化过程中起着重要作用。RNA结合蛋白介导的翻译抑制是转录后调控的重要形式，PUF家族是RNA结合蛋白的重要成员，但PUF蛋白在弓形虫速殖子与缓殖子转化中的作用尚不明确。我们的研究发现弓形虫RNA结合蛋白TgPuf1具有RNA结合活性，TgPuf1的表达及分布在速殖子与缓殖子间有显著差异。因此，推测TgPuf1蛋白可能参与速殖子与缓殖子转化的基因表达调控。在此基础上，本项目①通过构建TgPuf1-HA转基因弓形虫虫株，用抗HA标签的单抗与弓形虫的裂解产物进行免疫共沉淀(Co-Immunoprecipitation, Co-IP)，纯化提取与TgPuf1相互结合的蛋白质进行质谱分析；②构建TgPuf1基因缺陷(∆TgPuf1)虫株，通过与野生型虫株比较，观察∆TgPuf1虫株表型的改变，结果显示∆TgPuf1缺陷虫株经体外缓殖子诱导速殖子向缓殖子转化的效率显著低于野生型虫株，推测TgPuf1可能参与了速殖子与缓殖子相互转化的调控。