玉米胚乳细胞内复制调控的分子机理研究

Endoreduplication was defined as a process in which nucleus DNA were undergoing a normal reduplication but without division and finally endopolyploidized cells with doubled several times nucleus DNA content were prduced. The endoreduplication occurs extensively in some special tissues of plant species, but its regulation mechanism and biology function are not understood very well as so far. In maize, the endoreduplication mainly exists in endosperm cells and its peak period is correspodence with grain fast formation stage. Therefore, the endoredupliction was known as a key dynamic of grain construction and is closely related with formation of economic output of maize. It would be very important signification in theory and practice for discovering systemly essential of endoreduplication regulation, explaining distribution pattern of endoreduplication in endosperm cells and rules of grain formation to compare and analyze expression profile of endosperm cells with different ploidy in maize grain in several levels. Thus, based on previous research, we are going to harvest 8-10 DPA endosperm of maize as materails for isolation of endosperm protoplast through enzyme digest. Then the protoplast with 3C, 6C, 12C DNA content would be purified by flow cytometry for analyzing transcriptom and mapping expression profile of endoreduplication as well as mitosis regulators, on which some predicted genes special for endoreduplication process could be screened through comparing the two expression profiles. After that, some genes with different exprssion between different ploidy protoplast will be checked out. It is anticipted that the molecuar basis and biology function of endoreduplication regulation could be explained systemly.

内复制是指细胞只进行DNA复制，却不分裂，最终形成核DNA含量多次加倍的多倍化细胞的过程。内复制现象普遍存在于植物的特定组织，但其调控机理和功能仍不完全清楚。在玉米中，内复制主要发生于胚乳细胞，其旺盛发生期与籽粒迅速膨大期完全同步，是籽粒建成的关键性动力，与玉米经济产量的形成密切相关。以不同倍性玉米胚乳细胞为对象进行多层次表达比较分析，对系统地揭示内复制发生的内在本质，阐明玉米胚乳细胞的內复制特征和子粒形态建成规律具有重要的理论和实践指导意义。本研究拟在先期研究的基础上，取花后8-10天的玉米胚乳，酶法分离原生质体，再运用细胞流式分选术，分别分离DNA含量为3C、6C和12C的原生质体进行转录组分析，建立有丝分裂和内复制调控的基因表达谱，比较筛选出内复制相关调控基因，以期能够系统地揭示内复制调控的分子基础。

内复制也称内多倍化，是指在有丝分裂过程中，细胞进行数轮DNA复制，却不分裂，最终形成一个具有多倍化核的细胞的过程。在植物中，内复制现象普遍存在，有超过90%的物种的一些特定器官內都存在内复制。但其调控机理和生物学功能仍不十分清楚。由于在基因组较小的葫芦科、十字花科植物中，内复制在各个器官都有发生，很多报道认为内复制水平与植物的基因组大小呈负相关关系。本研究以已知DNA含量水平的鸡红血细胞为内标，通过测定分析相同遗传背景的36个白菜与甘蓝的中间杂种的DNA含量及内复制水平，首次证实了植物的内多倍化水平与基因组大小不显著相关，R值＝－0.0343。在玉米中，内复制主要发生在胚乳细胞之中。通过对不同发育阶段籽粒胚乳细胞进行差异表达分析，则有可能找出內复制代谢通路中各个关键基因以及其对內复制调控的作用机制，为此本研究分别以花后第5、10、15、20天的玉米子粒为材料，进行转录组测序分析，初步分析结果表明，在花后第5天（无內复制发生）和第10天的籽粒胚乳细胞中有2804个基因进行了上调或下调的差异表达，涉及到从DNA复制、RNA转运以及细胞周期调控在内的95个代谢过程，其中有154个基因为尚未注释的新基因。这说明內复制及细胞周期的精确调控是一个非常复杂的过程。当以花后10d的胚乳组织为材料进行原生质体分离及流式核型分析时，结果表明以1%纤维素酶、0.5％离析酶、0.5%半纤维素酶，在渗透压调节剂0.7-0.8 mol L-1和质膜稳定剂0.8 mol L-1的MS培养液内，30℃消解4 h，能够获得大量完整的粗制原生质体，二乙酸荧光素(FDA)染色表明纯化的原生质体仍能保持90%以上的生活力。流式分选术可将有活力原生质体从粗制原生质体悬浮液中富集、纯化出来。当这些原生质体经过流式细胞仪时，共有四个亚群的细胞出现在双参数流式核型图上，这有可能代表了四种不同命运的胚乳细胞，它们在籽粒发育的不同阶段可能经历了不同的分化过程。