调控艾美耳属球虫孢子生殖的转录因子筛选和功能研究

The parasites of genus Eimeria are widespread pathogens which seriously threaten the health of livestock and poultry. The complex life cycle of Eimeria spp. is divided into an endogenous (schizogony and gametogony) and an exogenous (sporogony) stage. The exogenous stage, also called sporulation, which involves maturation of unsporulated oocysts into infectious sporulated oocysts is completed in the suitable environment. However, the molecular mechanisms of initiation and regulation of the sporulation progress are unclear. Combined with the regulatory mechanisms of lower model biological spore reproduction and the applicant's previous research accumulation, this project proposed that transcription factors were the main endogenous factors regulating the sporogony of Eimeria parasites. This proposal aims to select the candidate transcription factors by analyzing and comparing the differential expression genes (DEGs) in various stages in the sporogony in E. tenella from the high-thorough output sequencing results. Furthermore, the function of the candidate transcription factors in regulating the sporogony will be confirmed by constructing the genetic manipulated Eimeria with overexpression and conditional knockout of these genes. These results have important implication for the study of the development of other apicomplexan parasites and lower organisms, and laid a foundation for studying the environmental factors on sporulation. These results will also provide new ideas and insights for the scientific prevention and control of coccidiosis.

艾美耳属球虫是一类严重危害畜禽健康的病原，其生活史复杂，包括宿主体内的裂殖生殖、配子生殖和体外的孢子生殖。孢子生殖需要在适宜的外界环境中完成，是艾美耳属球虫卵囊获得入侵宿主的感染性进行繁衍的无性生殖方式。但是球虫感受外界环境信号启动和调控孢子生殖过程的分子机制尚不清楚。结合低等模式生物孢子生殖的调控机制和申请人的前期研究积累，本项目提出转录因子是调控球虫孢子生殖的主要内源性因素。本项目将以柔嫩艾美耳球虫为模型，利用高通量测序技术对孢子生殖不同阶段的差异基因进行比较分析，筛选出调控孢子生殖的关键转录因子；利用反向遗传操作技术构建候选转录因子过表达或条件性敲除虫株，研究其对孢子生殖的影响，解析转录因子在球虫孢子生殖中的作用。本项目不仅可以推进其他顶复门原虫或低等生物发育调控的研究，同时也为研究环境因素对球虫孢子生殖的影响奠定基础，为畜禽球虫病的科学防控提供新的思路和切入点。

在基金委的资助下，研究了不同温度对孢子生殖的影响，利用光学显微镜及孢子生殖阶段表达荧光蛋白信号的转基因球虫株，观察了艾美耳球虫孢子生殖阶段的形态学变化，发现80%以上的艾美耳球虫孢子生殖同步发育。利用转录组学、蛋白组学技术进行了柔嫩艾美耳球虫孢子生殖启动阶段差异基因的分析，发现孢子生殖启动阶段上调表达基因集中于细胞组分（CC）与分子功能（MF），上调基因富集的通路为蛋白酶体与DNA复制。上调表达蛋白174个，集中于分子功能，主要与丝氨酸肽链内切酶活性、微管结合与GTP酶活性，上调蛋白富集通路与周期节律调节相关。另外，在艾美耳球虫33个AP2转录因子中，孢子生殖启动阶段有16个呈上调表达，7个呈下调表达；另有6个其他家族的转录因子上调表达，4个转录因子下调表达。扩增其中一个转录因子的调控序列，构建荧光标记虫株，观察孢子生殖的规律，为后续筛选核心调控转录因子奠定了基础。分析与预测了显著上调的Top10基因的功能，克隆了相关基因，为后续功能解析奠定了基础。建立与完善了基于CRISPR/Cas9系统的艾美耳球虫基因编辑平台，相关成果发表于《Veterinary Research》杂志，并被遴选为亮点文章（Featured article）。.项目执行期间，负责人获评中国农业科学院优秀博士后，专业技术职称晋升为副高级职称，参加国内学术会议1次，线上国际学术交流多次，发表基金标注SCI论文2篇，另有1篇中文核心期刊已接收，待发表。