纤毛虫有性生殖中功能核的分化及其微管结构的作用

Ciliated protozoa (ciliates) are a unicellular eukaryotic group having a germinal micronucleus (Mic) and a somatic macronucleus (Mac), the so-called nuclear dualism. Mic is quite different from Mac in many aspects, whose main function is to produce gametic nuclei during sexual reproduction (refered as "conjugation" in ciliates). So Mic formation is an integral part of ciliate conjugation, which is as important as germ cell formation in multicellular metazoa. In fact, the cultures and conjugation induction of ciliates including P. caudatum, P.tetraurelia and Tetrahymena thermophila are easily done in the lab, and the whole process of conjugation takes as less as 2-3 days. Therefore, ciliates are excellent model organisms to study the mechanisms of germline formation/ protection. During conjugation of P. caudatum, there are three stages closely related with Mic formation. 1) After meiosis, only 1 out of the 4 haploid products locates in the paroral cone region (PC), which survives and divides mitotically yielding a migratory and a stationary pronucleus corresponding to a male and a female gamete of metazoa, respectively, the other 3 outside PC degenerate. 2) At the telophase of the 3rd postzygotic (synkaryon) division, 8 products locate in a pattern of anterior 4 and posterior 4 in the cell. The posterior 4 all differentiate into new Macs, while the anterior 4 remain as presumptive Mics. 3) In exconjugants, only 1 presumptive Mic is selected as the germinal one for next generations, the remaining 3 degenerate. Without specific protecting mechanisms, it will be difficult for the nuclei destined to be the germline ones to escape from either degeneration or macronuclear differentiation. The specific nuclear localizations in the cells might be closely related with Mic formation mechanisms. However, little is clear about this issue. In our previous studies, stage-specific microtubular behaviour related with above three stages have been observed indicating the involvement of microtubules (MTs) in Mic formation. For example, after meiosis, large amounts of cytoplasmic MTs appear and maintain during the 3rd prezygotic division wrapping PC nuclei and two types of prospecitve pronuclei, but never such MTs arround the extra degenerating meiotic products outside PC. As the results, the physical barriers form between the surviving meiotic products and the cytoplasm. Therefore, it is reasonable to hypothesize that MT involved barrier structure plays an important role in Mic formation after meiosis. In current project, we will address the formation and function of the barrier structure, the other components besides microtubules, the spacial relationship with the surviving Mic and PC region by means of microscopic, electromicroscopic and molecular techniques and by the aid of bioinformatic analysis.

纤毛虫有大、小两种细胞核，又称体核、生殖核,无小核纤毛虫不能进行有性生殖，故小核形成是纤毛虫有性生殖的重要组成部分。在尾草履虫有性生殖中，有三个时期与小核形成密切相关。①减数分裂后，仅口旁锥内1核存活，另外3核退化；②核决定期，细胞前方4核保留为预备小核,后方4核以后分化为大核；③接合后体中，仅1个预备小核保留为新一代小核，其余3个退化。项目组前期研究工作揭示，这3个时期均有微管时期特异性活动，提示微管在有性生殖功能核分化过程中的重要作用。特别是减数分裂后，口旁锥内的核及其形成配子核的过程中，核周围出现大量胞质内微管，使核与胞质间形成屏障样结构。本项目拟对该结构的形成、其微管以外的组成成分、与口旁锥和小核间的空间位置关系，以及这些组分在尾草履虫减数分裂产物存活过程中的功能等，从显微、亚显微、分子水平以及利用生物信息学的方法进行研究，以探讨纤毛虫减数分裂产物的分化与微管结构之间的关系。

通过四年努力，未能获得预期实验结果，有关尾草履虫和其他纤毛以及其他动物的研究成果如下。① 35ºC-35.5ºC/ 30 min诱导多小核细胞。为探索多小核细胞最佳诱导条件，对草履虫接合对进行不同温度不同时间处理，35ºC/30 min诱导大量多小核细胞，减数第一次分裂前期III处理产生的多小核在接合后体中不发生分化，IV期热处理产生的多小核发生核分化并发育成为多大核胚基的接合后体。35.5ºC/30 min产生多小核细胞，但不发生核分化。该结果对后续探究核分化的分子机理具有重要意义。② 草履虫接合后体中预小核的退化很可能采取新途径。3个位于口旁锥外的减数分裂产物通过凋亡的方式发生退化，该过程可通过Hoechst 33342和吖啶橙双重活体荧光染色法在活体细胞中进行观察。为研究接合后体中3个预小核的退化是否以相同的方式发生退化，对尾草履虫的接合后体进行单细胞分离培养，然后对接合后体分裂1-6次的细胞进行Hoechst 33342和吖啶橙双重活体荧光染色，但是未能检测到退化的预小核。该结果暗示接合后体中预小核很可能采用不同于减数分裂产物退化的方式进行。③ Bursaria国内新记录种。在浙江农林大学采集一种大型纤毛虫，对其包囊的形成过程进行了初步观察的结果表明，该物种包囊形成过程及特征不同于已报告的其他纤毛虫的包囊，很可能是一种新类型的纤毛虫包囊。④ 明确动物隐性基因的表达。前期研究中发现cDNA的多样性，已通过大量实验证明该多样性系由PCR过程中产生。但是，该现象并不是在检测的所有个体中存在，只能在杂合体基因中显现。这个实验结果揭示了杂合体基因中的来自父本和母本的基因都发生转录。换句话说，至少在转录水平上显隐性基因都是表达的。至于这两种转录子最终是否均翻译成有功能的蛋白质还有待研究。⑤ 成功克隆SCP2、EDF-1、PPDPF等具有重要生物新药研发价值的cDNA。⑥ 优化Gal-3、Mitogalig、Cytogalig、EDF-1和PPDPF等开放阅读框的密码子，构建其原核表达重组质粒，获得大量重组蛋白并制备其高效价的抗血清。⑦ 确立肉源鉴定的分子生物学方法。