玉米重要转录因子O2 , PBF和OHPs调控籽粒粒重的分子机理研究

Protein and starch are the two major storage compounds in maize kernels. Generally, high protein leads to low starch in kernel and vice versa. Nutritional quality of kernel is determined by protein and kernel yield is determined by starch content. Quality and yield improvement is always incompatible in the breeding, although its molecular mechanism is still unclear. Reserve synthesis (protein and starch) and the expression of their synthetic enzyme genes are very consistent in the filling stage, demonstrating that the transcriptional control takes a critical role on reserve synthesis. It is known that O2, PBF and OHPs regulated expression of 90% zeins which is the most major seed storage proteins in maize, and therefore controlled its protein nutritional quality. Our previous study in genetics has solidly demonstrated that the transcription factors have a coordinated control for the synthesis of starch and zein protein. Therefore, the project will comprehensively and deeply elucidate the synergistic regulatory mechanism of O2, PBF and OHPs on endosperm starch synthesis by kernel phenotype analysis, determination of storage compounds, expressing analysis of target gene in transcriptional and protein level, molecular and biochemistry assay, etc. Furthermore, the project will employ the strategy of laser microdissection combined with RNA-Seq to elucidate their regulatory mechanism on lipids synthesis in aleurone and subaleurone. This study will provide a molecular basis for breeding high yield and good quality maize.

玉米籽粒储存蛋白和淀粉是最主要的两类储存物质。一般而言，高蛋白就意味着低淀粉，反之亦然。蛋白决定籽粒营养品质，而淀粉含量则影响籽粒产量。品质和产量一直以来是生产育种上的一对矛盾体，从未在分子机制上有突破性研究。这两类储存物质积累及其合成基因的表达高度一致，表明转录调控在其中起关键作用。现已知O2, PBF和OHPs调控了种子最主要储存蛋白——醇溶蛋白90%的表达，进而控制了籽粒营养品质。我们的前期工作在遗传上确凿地证明了这几个转录因子协同调控玉米籽粒储存蛋白和淀粉的合成。为此，本项目拟通过籽粒表型分析、储存物质含量测定、转录组测序分析、基因转录和蛋白水平的检测、分子生化实验验证等手段，全面深入地解析这几个转录因子对淀粉合成的分子调控机理。本项目拟进一步采用激光显微切割与转录组测序相结合的策略，解析这三个转录因子对糊粉层和亚糊粉层脂类合成的调控机制。这一研究将为培育高产优质玉米提供分子基础。

胚乳是玉米种子蛋白和淀粉的主要储存器官，很大程度上影响了玉米的品质和粒重。醇溶蛋白是玉米种子最主要的储存蛋白，在胚乳细胞的内质网内腔合成并沉积形成蛋白体；淀粉则在胚乳造粉体中合成积累形成淀粉粒。成熟玉米胚乳电镜观察显示整个胚乳是由蛋白体和淀粉粒填充形成。发育胚乳的转录组和蛋白数据分析显示所有醇溶蛋白基因和主要淀粉合成酶的编码基因都在授粉后8-10天开始表达，具有高度的时空一致性。目前，所有醇溶蛋白基因表达调控都已不同程度地研究，相关的转录因子被鉴定，其中一些转录因子也同时参与淀粉合成基因的表达。本项目通过研究其中三个胚乳灌浆发育的重要转录因子O2、PBF1和OHPs的调控网络，发现O2是胚乳灌浆储存物质合成的核心调控基因，不仅是醇溶蛋白基因表达的重要转录激活因子，也直接参与多个淀粉合成基因的表达，进而协同控制蛋白和淀粉的合成。在本项目获得资助的当年，我们首先发现O2通过直接结合淀粉合酶SSⅢ基因启动子区的相应顺式元件，调控SSⅢ基因的表达。在o2突变体胚乳中，SSⅢ基因的转录和蛋白水平都显著下降；同时PBF1可以协同O2共同调控SSⅢ的表达。这个工作首次在分子水平上解释玉米籽粒品质和粒重的内在关联（PNAS 2016(113): 10842-10847）。本项目执行期间，我们进一步发现O2也直接参与玉米基因组中的三个蔗糖合酶Sh1, Sus1和Sus2的表达。Sh1在胚乳中的表达最强，蔗糖合酶活性的主要贡献者。这个工作发现O2对这三个蔗糖合酶基因都有转录激活作用，其中对Sus1和Sus2的转录激活要明显强于对Sh1基因的转录激活。o2和sh1双突变的胚乳的蔗糖合酶活性明显比单突下降，淀粉和蛋白合成都受到严重影响。该工作进一步拓展了O2作为胚乳灌浆调控的核心因子的作用（Plant Biotechnology Journal (2020) 18: 1897–1907）。