玉米Edk1(Early delayed kernel 1)基因的克隆及其在胚乳早期发育中的功能研究

Seed size is a critical agronomic trait related to the maize yield. Although regulatory mechanisms of maize filling were widely studied, researches on the early development stage of maize endosperm were less reported. Using EMS mutagenesis, we screened a small seed mutant edk1 showing delayed development at early stage and reduced endosperm cells. By MBS (Mapping-by-sequencing) and Map-based cloning, we cloned this gene and determined that the mutation in edk1 resulted in a CGA → TGA transition, leading to a premature stop codon in the coding sequence. Spatial and temporal expression analyses revealed that Edk1, coding a new microtubule binding protein, was most abundantly expressed in endosperm cells at 2-3 DAP (syncytium to cellularization). In addition, EDK1 was determined to be located on microtubule system. These results indicate that EDK1 plays an important role in nuclear divisions and cellularization at early stage probably by influencing the microtubule formation or stability. Therefore, the biological function and regulation network of EDK1 in the early development of endosperm will be clarified in this project by performing allelic test and CRISPR to verify the gene; applying subcellular localization and BiFC (Bimolecular Fluorescence Complementation) assay to identity the binding domain of EDK1; using TUB-CYP transgenic maize, tobacco suspension system and fluorescence immunohistochemistry to study the effect of EDK1 on microtubule system, and utilizing the transcriptome analysis and yeast single hybrid to exploring the upstream and downstream networks of EDK1. In conclusion, this project will not only reveal the molecular mechanism of early development of maize seeds, but also lay a foundation for the genetic improvement of maize seed size and yield.

籽粒大小是决定玉米产量的一个重要因素。玉米籽粒的研究大多集中在灌浆及其调控，而胚乳早期发育的研究很少。我们通过EMS诱变获得一个胚乳早期发育迟滞、胚乳细胞数目减少的小粒突变体edk1；通过MBS和图位克隆发现突变体中Edk1编码区由于一个C→T的突变产生一个终止密码子；Edk1编码一个微管结合蛋白，在授粉后2-3天的胚乳细胞中（合胞体-细胞化）高表达；该蛋白定位于微管系统中，这些结果表明EDK1可能通过影响微管的形态或稳定来影响胚乳早期发育。本项目将通过等位测试和CRISPR验证该基因；亚细胞定位和BiFC研究该蛋白的功能结构域；用TUB-CYP转基因玉米、烟草悬浮系统和荧光免疫组化研究EDK1对微管系统的影响；用转录组学和酵母单杂研究其调控网络，从而阐明EDK1在胚乳早期发育中的生物学功能。本项目不仅可以揭示玉米胚乳早期发育的分子机理，同时也为玉米种子大小和产量的遗传改良提供理论基础。

玉米胚乳早期发育影响胚乳的细胞数目进而影响种子的大小和产量。由于缺乏胚乳早期特异的突变体和相关细胞学实验技术，玉米胚乳早期发育的研究鲜有报道。我们通过EMS诱变获得一个胚乳早期发育迟滞、胚乳细胞数目减少的小粒突变体edk1（后命名为vks1，varied-kernel-size）。通过MBS和图位克隆发现突变体中Vks1编码区由于一个C→T的突变产生一个终止密码子；Vks1编码一个驱动蛋白ZmKIN11。通过RT-qPCR和原位杂交的方法发现Vks1在授粉后2-3天的胚乳细胞中（合胞体-细胞化）高表达；通过GFP融合蛋白的亚细胞定位及免疫组化的方法发现VKS1蛋白定位于细胞核和微管系统。同时经过一年多的摸索建立了玉米胚乳细胞的实时荧光免疫组化技术，发现VKS1蛋白动态定位于胚乳有丝分裂过程中的微管系统（纺锤体、成膜体）。Vks1突变体中由于ZmKIN11蛋白的缺失，导致胚乳有丝分裂过程中微观系统发生紊乱，使合胞体期游离核核质域发生改变，早期胚乳细胞快速分裂过程中纺锤体组装、姐妹染色单体分离和成膜体形成出现异常，从而影响了早期发育胚乳细胞的快速分裂，导致胚乳细胞数量减少，最终导致种子变小。这项研究首次通过正向遗传学、细胞学和分子生物学的方法揭示了玉米胚乳早期发育的新机制，为今后研究玉米胚乳早期发育打开一个突破口，也为玉米种子大小和产量的遗传改良提供理论基础。这项工作以封面文章于2019年发表在The Plant Cell上。