【作者】 陈宗良；

【导师】 赖锦盛；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 玉米籽粒的大型胚乳不仅是胚发育的重要营养器官,而且还是人类最主要的粮食产量和工业原材料的来源,这些特点使得玉米成为最重要的农作物。人们对玉米胚乳发育的各个时期非常清楚,在双受精之后,受精极核进行一段时间的游离核分裂,随后这些游离核经历细胞化形成正常的胚乳细胞,并通过快速的有丝分裂增殖胚乳细胞,之后分化形成四种不同类型的细胞。然而我们对胚乳发育的分子机制目前还了解得非常少。1.本课题分析了Wrinkled kernell(Wrk1)籽粒皱缩突变体的特征。突变体成熟的籽粒表现出胚乳顶部皱缩和籽粒变小,并且这种影响受剂量效应控制；苗期植株虽然相对较小,但是却能正常发育。2.对发育过程中10DAP、12DAP和16DAP籽粒的细胞切片观察发现,Wrkl基因对胚乳发育的影响主要体现在淀粉胚乳细胞和转移层细胞的形成。同时,突变体5DAP的胚乳组织小于野生型的,而7DAP的胚乳则远小于野生型,并表现出不规则的胚乳形态和细胞生长发育严重受阻。这一结果暗示Wrkl基因可能参与胚乳细胞有丝分裂。3.利用α-tubulir抗体对纺锤体进行免疫染色,发现突变体胚乳细胞的姊妹染色单体在有丝分裂后期的分离过程中出现缺陷,主要表现在染色体桥的形成和染色体滞后。4.利用图位克隆法将Wrkl基因定位在3号染色体350-kb的候选区间内,该区间含有三个注释基因,而其中只有编码β-tubulin5蛋白的基因发生突变,该基因的3’端存在一个C到T的改变,这一点突变使该基因提前出现终止密码子。突变体截短的ZmTUBB5蛋白完全缺失了C端尾巴。5.转基因结果表明,含有更多突变型ZmTubb5基因的籽粒顶部会出现皱缩。以上结果表明Wrk1基因编码ZmTUBB5蛋白,该蛋白是胚乳发育和细胞有丝分裂的重要因子。更多还原

【Abstract】 The large endosperm of maize is a nutritive tissue for embryo development, but also important for the primary food products and industrial applications, which make maize the central crop of agriculture. It is clear that maize endosperm develops from the fertilized central cell, originates with repeated free-nuclear divisions followed by cellularization and intensive mitosis, and subsequently forms four differential cell types. Yet the regulation of maize endosperm development remains poorly understood.1. Here, we report the molecular characterization of a Wrinkled kernell (Wrkl) mutant in maize. The endosperm of the Wrkl mutant is deeply furrowed and impacted by gene dosage, whereas the seedling is small but vigorous.2. The section of kernel at10,12and16DAP (day after pollination) showed that the effects of Wrk1mutation are manifest in formation of starch endosperm cells and transfer layer cells. Size of endosperm at5DAP is much smaller than wild type, and the situation is aggravated at7DAP, showing that the endosperm gets defective in morphology and cell growth. The results indicated that Wrk1may be involved in mitosis of endosperm cell.3. Immunostaining of spindle by β-tubulin antibody at anaphase of mitosis in endosperm cells revealed that Wrkl mutant is defective in sister chromatid segregation resulting in chromosome bridge and chromosome lagging.4. Utilized the positional cloning strategy, we mapped the causative mutation to a350-kb region, which contains three predicted gene models. A gene model encoding β-tubulin5protein shows a C to T transition leading to a premature stop codon at the3’end. The truncated ZmTUBB5completely lacks C-terminal tail of isotype-defining region.5. Transformed kernels with exceeded mutation allele of ZmTubb5versus normal allele showed wrinkled crow.These results demonstrate that Wrkl encodes the ZmTUBB5which is critical for endosperm development and mitosis in maize.更多还原