尾草履虫接合后体中生殖核选择机制的研究

Ciliated protozoa, Paramecium caudatum has a micronucleus (Mic) and a macronucleus (Mac). Mic is diploid with few gene expression, but undergoes meiosis making a stationary pronucleus (stMic) and a migratory one (miMic), which are equivalent to egg and sperm of metazoan, respectively. Therefore, Mic is truly germinal nucleus. Mac is polygenomic, also called somatic or vegetative nucleus with gene expression necessary for almost all the biological processes including cell division and sexual reproduction (conjugation). During the conjugation of P. caudatum, there are six Mic divisions, three happen before synkaryon (fertilized nucleus) formation( prezygotic ones), and three after (postzygotic ones). Exconjugants of P. caudatum are separated conjugating pairs after synkaryon formation. After the third postzygotic division, there are 4 Mac anlagen (Mac “embryo” of new generation), 4 presumptive Mic (remaining Mic properties) and many old Mac fragments in exconjugants. In fact, there are three stages when germinal nuclear fates are determined during the conjugation. The first is after meiosis, only one meiotic product entering the paroral corn region (PC) survives and finishes the third prezygotic division to form stMic and miMic, the remaining 3 outside PC degenerate. The second is at the telophase of the third postzygotic division, when 4 products locating in the anterior region of the cell become presumptive Mic, the other 4 in the posterior region differentiate into Mac anlagen, which lose Mic properties and will never have the chance to revert back to germinal nuclei. The third is at the first exconjugant division, only one presumptive Mic divides, the other three degenerate finally. So far, it has been clarified that the different fates of the nuclei at the first two stages are closely related with their spatial localizations in the cells. While whether the germinal nuclear fates are related with their spatial localizations in exconjugants is controversial. Our previous studies showed that heat shock disturbed nuclear localization at the end of meiosis and resulted in no PC entrance and multimicronucleates. Heat shock also disturbed antero-posterior nuclear localization at the third postzygotic telophase resulting in exconjugants with extra-number of Mac anlagen but without presumptive Mic. These studies have evidenced the importance of nuclear localization for the germinal nuclear formation in P. caudatum. Failure of germinal nuclear formation means producing offsprings without fertility through the conjugation. Therefore, germinal nuclear formation is one of the most essential issues during conjugation, and clarifying its mechanism becomes indispensable. In our previous study, it was found that germinal nuclear selection does not initiate when exconjugants are kept in starvation. Once exconjugants are provided with culture medium, only one presumptive Mic becomes enlarged and divides along with the exconjugant division. This provides us an excellent system to study the mechanism of germinal nuclear selection in exconjugants. In this proposal, we will study on the relationship between germinal nuclear selection and their localization in exconjugants of P. caudatum mainly by means of immunofluorescence staining. The mechanism for initiating of germinal nuclear selection will be studied by iTRAQ (isobaric tags for relative and absolute quantitation), which provides detail information including different proteins, the same proteins with different modifications such as phosphorylation between the starved and feed conditions of exconjugants, leading us to discover the mechanism of germinal nuclear selection.

在尾草履虫接合生殖过程中，有3个决定小核命运的时期。①减数分裂结束后：在4个分裂产物中，仅口旁锥内1核存活并分裂产生静止原核和迁移原核，口旁锥外3核退化。②配后第三次分裂末期：在8个分裂产物中，细胞前端4核成为预备小核(presumptive micronuclei)，后端4核分化为大核原基(macronuclear anlagen)。③接合后体第一次分裂：在4个预备小核中，仅1核进行有丝分裂，成为新一代的小核，其余3核退化。在前两个时期，小核命运与其空间位置密切相关，而第三时期的小核命运与空间位置的关系尚无定论。项目组前期研究工作表明，饥饿条件下的接合后体中生殖核的选择处于停滞状态，但进食后仅 1个预备小核显著膨大并随着细胞的分裂而分裂，其余3核固缩且不参与分裂。本项目拟通过免疫荧光染色结合蛋白定量分析技术iTRAQ对接合后体中小核命运与其空间位置之间的关系及相关分子生物学机制进行研究。

在草履虫接合生殖过程中，因不同物种，小核经历1~多次选择，被选择的存活或继续发育，未被选择的退化。大核则不同，最终命运都是退化。任一时期的小核选择和大核退化对能否产生正常的后代起着决定性的作用。截至目前的研究表明，多出的减数分裂产物以凋亡的方式退化（吖啶橙和Hoechst33342双染色法使退化核呈浅绿色、黄色、橙色等）。关于亲代大核和多出的预备小核何时以何种方式发生退化尚无定论。在项目实施过程中，除了对尾草履虫接合后体中大核及小核的行为进行了详细的研究，对绿草履虫和多核草履虫也进行了相关研究，得到的主要结果概括如下。①尾草履虫的亲代大核碎片在接合后体分裂6~7次时基本消失，这期间未观察到任何类似于减数分裂产物凋亡核那样的呈浅绿色、黄色或橙色的结构。②尾草履虫接合后体中多出的预备小核未呈现类似于减数分裂产物凋亡核那样的呈浅绿色、黄色或橙色的结构。①和②的结果暗示尾草履虫亲代大核碎片和多出的预备小核以未知的不同于其减数分裂产物凋亡的的方式消失。③绿草履虫中的多次小核分裂产物的退化在消失之前呈黄色。④绿草履虫中的亲代大核在接合后体中消失之前呈黄色。③和④的结果表明绿草履虫亲代大核和受精核形成前后多次的小核退化，其机制可能与尾草履虫减数分裂产物的退化机制相同。⑤蛋白银染色法显示多小核草履虫的接合后体完整口器的形成发生在核决定期，意味着此时已具备摄食能力。由于该时期大大早于接合后体继续发育所需营养的时期，意味着在接合后体获得摄食能力的时期与发育过程中实际需要营养供给的时期之间存在时间差。⑥尾草履虫有接合活性和无接合活性的互补接合型样品的转录组测序，筛选到2个候选基因，为后续阐明接合型物质的本质奠定基础。⑦澄清了扇形游仆虫接合生殖期间小核和大核发育的全过程。⑧发现吖啶橙和Hoechst33342双染色的细胞，其凋亡核依荧光显微镜观察时使用DAPI滤镜还是紫外线滤镜观察到十分不同的结果。紫外线滤镜使凋亡核呈现浅绿色、黄色或橙色，而DAPI滤镜检测不到凋亡后期的核。⑨改良几项常用分子生物学技术方法，包括感受态细胞的制备、PCR的最佳条件、SDS-PAGE制胶技术等，大大提高了实验效率。⑩成功克隆多个活性肽编码基因，为后续开发良好生物新药奠定了基础。