基于全基因组重测序的玉米籽粒突变体smk3候选基因的克隆与功能验证

Maize (Zea mays) is an excellent cereal model for research on seed development, and the underlying molecular mechanisms have always been the hot spot in the research of life science filed.We have a novel single-locus recessive kernel mutant named small kernel 3 (smk3). At the early development stage, mutant kernels are smaller compared to the wild type, and the growth of embryo and endosperm were retarded. Matured mutant kernels have shrunken epidermis and small defective embryo and endosperm, only 5% of them could germinate into seedlings, which died early. In this project, we intend to study the smk3 mutant by observing its phenotype at different development stages first, to clarify the structure variation of smk3. Then isolate the target gene using whole-genome resequencing approach. Functional analysis of the candidate gene will reveal its expression pattern, protein activity and metabolic regulation mechanism. And the gene function will be validated by allelism test or genetic transformation at last. The research will provide scientific evidence toward comprehensive understanding of the molecular regulation mechanism during the early stage of kernel development and establish theoretical basis for molecular manipulation of the maize yield.

玉米籽粒是禾本科种子发育研究的最佳模型，其分化发育的分子机制是生命科学领域研究的热点。本项目获得一个新的隐性单基因控制的玉米籽粒突变体small kernel 3 (smk3)，与正常籽粒相比，突变籽粒发育早期即出现体积变小，胚和胚乳发育迟滞的表型。成熟的突变籽粒种皮皱缩、种子扁小、胚和胚乳结构畸形，仅有5%可以发育成苗，且幼苗早衰。观察结果表明可能是调控籽粒早期发育的关键基因发生了变异。本项目拟以smk3突变体为材料，首先对突变籽粒不同发育时期的表型进行显微观察，明确籽粒结构变异及其产生的时期。然后通过全基因组重测序技术克隆目标基因，采用分子生物学方法对候选基因功能进行分析，揭示其表达调控规律、编码蛋白活性及代谢调控机制。最后通过等位性测验或转基因互补实验进一步验证该基因的功能。本项目的实施将为揭示种子早期发育的分子调控机理提供科学依据，也为玉米产量的分子改良奠定基础。

玉米是全球第一大粮食作物，籽粒是其经济及营养价值的重要体现者。研究玉米籽粒发育过程，克隆关键基因，有助于推进籽粒发育调控网络的深入解析，为玉米籽粒产量和营养性状的遗传改良提供理论支撑。本研究中，玉米EMS诱变突变体smk3具有籽粒发育缺陷表型。对不同发育时期的smk3突变体进行显微观察发现，smk3的胚和胚乳在授粉后11天即出现明显结构缺陷，且胚乳转移层细胞发育异常，表明突变基因在籽粒发育早期起重要调控作用。利用分离群体分组分析法与全基因组测序相结合的策略发现，一个和细胞壁合成有关的甲基转移酶基因（ZmGXM）是我们的候选基因，该基因可能参与了胚乳BETL细胞壁的内生长。组织特异性表达分析发现该基因在玉米授粉后2-10天的籽粒中表达量较高。对野生型和smk3突变体授粉后12天和20天的籽粒进行转录组分析发现，与野生型相比smk3中与细胞壁合成和胁迫响应有关的基因发生了上调表达，而与淀粉、蛋白及脂质合成有关的基因表现为下调表达。综上所述，我们推测ZmGXM基因的突变导致smk3早期胚乳转移层细胞异常发育，BETL细胞壁合成受阻，内生长受抑制，最终导致母体组织中的营养物质不能正常经BETL细胞转移给籽粒的胚和胚乳，籽粒的淀粉、蛋白、脂质合成受抑制，进而形成了发育滞后、组织畸形的籽粒表型。本项目的实施为揭示种子早期发育的分子调控机理提供了科学依据，也为玉米产量和营养性状的分子改良奠定了基础。