组蛋白修饰和DNA甲基化对球虫发育阶段转化和早熟发育的调控机理研究

Eimeria spp., the pathogen of the animal coccidiosis, is an absolute intracellular parasite belong to Apicomplexa phylum. These parasites are highly host-specific, present a monoxenous life cycle in which there are complicated developmental stage coversions, and can be selected as a vaccine candidate by their precocious phenotype. In different developmental stage and precocious line, their morphologies, virulence, and gene expressions will be changed through the gene regulatory machinery. Profiling the regulatory mechanisms for this stage conversion is an interesting and important for deeply understanding the parasite-host interaction, immunoresponses triggering, and finding new validate drug targets. The global genome data analysis reveals that their developmental stage conversion and gene expression could be regulated and controlled by mainly relying on the epigenetic mechanisms like histome modification and DNA methylation, since their DNA-binding cis-transcripting factors coding genes are relatively low. However, relatively rich histone modifying proteins and DNA methylase coding sequences are harbored in their genome data. This gene distribution bias could implicate a novel epigenetic regulatory mechanism is in charge of their gene expression regulation for their stage conversion, differentiation and precocious development. The purpose of this study is to investigate the epigenetic regulatory machineries and mechanisms for their gene expression activation and silencing through the genoem-wide ChIP-Seq, DNA methylation, and RNA-seq sequencing. The sequencing data will be further mined for interpreting and probing the specific genes for controlling the stage conversion, differentiation, and precocious mutation. If funding, our results from this study will be helpful to the Coccidiosis prevention and curing, by the new drug target for chemotherapy, and new vaccine for immunization.

艾美耳球虫具有生活史过程复杂并具有早熟性，生活史各阶段的转化过程中存在形态学、抗原性和毒力因子的明显差异，早熟系毒力减弱但保留免疫原性。探讨阶段转化和早熟形成的调控机理，是深入理解球虫与宿主相互作用及免疫激发机理、药靶筛选和新药开发的理论基础。基因组数据分析表明，球虫相对缺乏与基因表达调控相关的DNA结合的顺式转录因子，而富有组蛋白和DNA甲基化修饰的相关蛋白编码基因，提示表观遗传可能是其主要的基因表达调控方式。本研究拟采用ChIP-Seq和BS-Seq技术对柔嫩艾美耳球虫及其早熟系不同生活史阶段虫体进行基因组水平的表观遗传组测序，以揭示二者在基因组水平上的蛋白质结合位点(转录因子结合位点)和DNA甲基化率与甲基化模式，挖掘与球虫发育阶段转化相关的特定基因，解析组蛋白修饰的染色质重塑和DNA甲基化对球虫生活史中阶段转化和分化及早熟发育的调控机理。研究结果对球虫病防治重要的理论指导意义。

鸡球虫病室危害集约化养鸡业生产的主要疾病之一，在我国常年经济损失高达80亿元。目前球虫病的防治主要依赖于抗球虫药，但由于球虫抗药性的产生，药物效果严重受限，生产实践急需新的抗球虫药物。认识球虫的发育阶段转化及其调控机理对发现新的药物作用靶标至为关键。球虫基因组侧序的研究结果揭示，球虫缺乏与基因表达调控相关的 DNA 结合的顺式转录因子，而富有组蛋白和 DNA 甲基化修饰 的相关蛋白编码基因，提示表观遗传可能是其主要的基因表达调控方式。本研究应用ELISA-like分析技术和 BS-Seq 技术对柔嫩艾美耳球虫未孢子化卵囊、孢子化7h卵囊、孢子化卵囊和子孢子4个阶段的基因组DNA甲基化水平进行分析，结果表明E.tenella和其他3种常见的致病球虫具有低水平的DNA甲基化修饰，且随发育阶段不同而有显著差异，E.tenella的基因组DNA甲基化水平在子孢子阶段最高达0.78 ± 0.10%，而在孢子化7h时最低仅0.09 ± 0.01%。表明DNA甲基化可能是球虫发育调控的一种重要方式。对E.tenella上述不同发育阶段的蛋白质翻译后修饰（乙酰化、磷酸化、泛素化和糖基化）质谱分析发现，不同发育阶段的修饰蛋白质谱、修饰位点和定量修饰组水平也有显著变化，提示蛋白质翻译后修饰是调控球虫发育的另一种机制。对组蛋白去乙酰化酶EtSir2A和鸡ChSIr2A的酶动力学、抑制动力学及其与NAD等小分子化合物的结合热力学比较分析证明，球虫Sir2A的酶学特征显著区别于其宿主鸡的同源酶，具有药靶分子的应用潜力。研究结果证明球虫和弓形虫、隐孢子虫等顶复门原虫均具有低水平的DNA甲基化修饰，修正了既往报道的结果；从分子水平获得了DNA甲基化位点/区域、翻译后乙酰化、磷酸化、泛素化、糖基化等修饰的一系列基础数据，为深入研究这些修饰位点及其具体调控作用奠定了基础。已发表中文文章3篇，一篇SCI论文被接受，3篇SCI轮正在按审稿要求修改和补充数据，申报国家发明专利1项，获得软件著作权16个。