生殖细胞及精细胞的分化与发育

Doubling fertilization in angiosperms requires the normally-developed sperm cells which are the products of generative cell after mitosis. The generative cell is formed at the first division of the microspore. The differentiation and development of the generative and sperm cells are the emphases of plant sexual reproduction research all the time. At the mitosis of microspore, one important cytological event is that plastids are excluded from the generative cell. However, it has never been studied about the relationship between the plastid exclusion and the differentiation of the generative cell. Meanwhile, it is also unclear about the biological significance why the nuclear genes encoding plastid proteins express in the plastid-free sperm cells. Although the generative cell does not have plastids, it contains mitochondria. It has neither been investigated about whether the mitochondria are necessary for the development of the generative and sperm cells. In this project, we plan to use the methods of cytological and molecular biology to survey what roles the plastids and mitochondria play in the differentiation and development of generative and sperm cells. In addition, we also pay attention to two cytological events in sperm cell. One is how the nucleotide sequences around the start codon AUG regulate the efficiency of mRNA translation initiation in sperm cell, and the other is whether the doubling of nuclear DNA content occurs during the maturation of sperm cell. The results will provide more insights into the differentiation and development of generative and sperm cells, and also increase our understanding of sexual reproduction in angiosperms.

被子植物完成双受精需要精细胞的正常发育。精细胞是生殖细胞有丝分裂的产物，而生殖细胞又是小孢子有丝分裂的产物。关于生殖细胞及精细胞的分化与发育，一直以来都是生殖生物学领域的研究重点。小孢子有丝分裂时，质体被排除于生殖细胞之外，但质体排除是否参与生殖细胞的分化以及编码质体蛋白的核基因在无质体精细胞中表达的生物学意义并未被探讨过。尽管生殖细胞不包含质体，但却始终包含线粒体。是否线粒体是生殖细胞及精细胞的发育所必需也未见报道。本项目将利用细胞学及分子生物学方法调查质体和线粒体在生殖细胞及精细胞分化与发育过程中的功能；同时，也将关注精细胞发育过程中另两项细胞生物学事件：起始密码子AUG附近的核苷酸序列在翻译起始效率上对精细胞mRNA蛋白质合成的调控，以及精细胞成熟时细胞核DNA含量是否发生了加倍。这些研究结果有助于理解生殖细胞及精细胞的分化与发育，增加对被子植物生殖生物学的认识。

理解配子的发育是植物生殖生物学的核心问题之一，一直备受关注。长期以来，关于配子发育过程中是否会经历核DNA复制，并无定论。利用EdU染色，我们显示：拟南芥和蓝猪耳精、卵细胞在发育过程中并未发生核DNA复制， 核DNA的复制出现在合子发育过程中。这些结果提示被子植物的成熟配子停滞于细胞周期的G1时相。我们也调查了高度分化的精细胞是否有异于体细胞的翻译起始调控机制。结果表明：在核苷酸水平上，精细胞拥有与体细胞相似的翻译起始调控机制。比如，AUG起始密码子前的4个腺嘌呤核苷酸能够显著提高翻译起始的效率。此外，本项目还探讨了质体和线粒体在生殖细胞及精细胞发育过程中的功能，并建立了一种基于竞争型PCR鉴定单拷贝T-DNA插入片段的方法，为进一步利用该方法测定配子的核DNA倍性提供了技术支持。以上工作为认识配子的发育提供了新的视角。