玉米籽粒形成关键基因的克隆和生物学功能分析

Yield of cereal crops is determined by both seed size and number of seeds per unit area. Seed size is determined by the mechanisms that govern seed development processes. Hence, identification and functional analysis of genes involving in seed size regulation is not only the key to understanding of the underlying mechanisms, but also the foundation to molecular breeding for high yield cereal crops. From double fertilization to formation of mature embryo and endosperm, many genes are required to function. However, only a few of these genes have been revealed and fully characterized in cereal crops. Small seed (kernel) mutants are important genetic materials to gain access to these genes. We have isolated small kernel mutants, smk1 and smk4 in maize by using Mutator transposon mutagenesis. The mutations reduce seed size by 50% to 75%. The two mutants are recessive and monogenic. By using transposon tagging we have cloned Smk1 which encodes a transferase-like protein with unknown function. By using an alternative approach, we have identified the smk4 is tightly linked to ZmNAC1, a transcription factor that presumably functions in cell division and cell cycle regulation. Building upon this foundation, the goals of this study are three folds, 1) to identify the function of these genes; 2) to reveal how they regulate embryo and endosperm development, thus affecting size; 3) to explore the impact on seed development and plant growth in transgenic maize with increased expression of these genes.

种子发育与产量密切相关，因此探讨种子发育的机制，特别是与种子大小相关基因的调控机制不仅是植物学的一个根本问题，也是分子育种的重要基础。从双受精到形成成熟的胚和胚乳，种子发育需要许多基因的参与，而小种子突变体则是发掘和解析调控种子发育关键基因的重要材料。我们用Mu转座子在玉米中分离了小种子突变体smk1和smk4，并进行了初步研究。两个突变体皆为单基因隐性遗传，种子只有野生型的1/2至3/4大小。运用转座子标签技术，我们克隆了Smk1，分别编码一个功能未知的蛋白质；也获得的Smk4候选基因，一个与细胞分裂和细胞周期相关转录因子NAC1。本课题将运用分子生物学和遗传学的方法对其编码蛋白质的功能、表达及如何调节胚和胚乳发育进行深入研究，此外，我们也将通过转基因探索提高表达对种子发育和植株生长的影响。因此，本研究既有理论意义，也有潜在的应用价值。

本课题主要对玉米两个小种子突变体smk1和smk4进行了研究。二者来自于UniformMu突变体库，均为单基因隐性突变，利用转座子标签技术及TAIL-PCR克隆了突变基因，发现二者功能均未知。.Smk1纯合突变胚致死，编码的蛋白定位于细胞质，玉米过表达未发现明显表型，拟南芥同源基因的突变体也没有明显表型。将把转基因植株与突变体进行杂交回补，以明确表型由该基因突变导致。Smk1拟南芥过表达后种子萌发和植株生长受到抑制，但对激素和氧化胁迫没有明显响应。结合RNA-Seq与代谢组学分析，认为该基因可能参与了细胞壁半纤维素阿拉伯糖残基阿魏酸化，正设计实验验证。.对Smk4，发现其纯合突变体能够萌发并结实，植株相对矮小，种子也明显小于野生型。其编码蛋白定位于内含体，突变后内含体运动大大减缓。由于未能发现更多等位突变，因此进行了玉米转化，将利用杂交回补的方法明确表型由该基因突变导致。拟南芥同源蛋白AtSMK4亚细胞定位及突变后的表型与玉米类似，过表达导致拟南芥种子增大，显示该基因有潜在的应用价值。酵母双杂获得了两个AtSMK4的互作蛋白SIP1和SIP2，二者的亚细胞定位与SMK4不完全一致，但有一定程度的重叠。atsmk4 sip1双突变未发现明确的表型。已进行RNA-Seq分析，并将用CoIP获取互作蛋白。