玉米籽粒大小基因Sem1的分离克隆与分子机制研究

Kernel size is one of the major factors constituting grain yield. Maize kernel mutants are of special importance to study the development process of maize kernel. Cloning and functional analysis of maize kernel mutant genes have deepened our understanding of the molecular mechanisms conferring maize kernel development, which may aid maize high-yield breeding. sem1 kernel defective mutant was identified by the screening of Robertson’s Mutator stocks. Recessive mutants in Sem1 result in small and viable seeds, which can be germinated and grow to mature plant with dramatic altered plant architecture. Previous study indicates that Sem1 can negatively regulate the expression of gnarley1, rough sheath1 and other homeobox genes, through which it modulates the developmental process of seeds (Scanlon et al., 2002). However, Sem1 has not been cloned and the molecular mechanisms underlying kernel development is still elusive. We have isolated and cloned sem1 candidate combining omic techniques (SHOREmap and BSR-Seq) and Mutator transposon tagging. Here, we propose to：1） conduct CRISPR/Cas9 silencing and over-expression of sem1 candidate through transformation, and complementary test of other alleles of sem1 candidate from maize UniformMu or EMS mutant libraries, to functionally validate the causal gene of sem1 mutant. 2） perform Yeast Two-Hybrid, followed by bimolecular fluorescence complementation (BiFC) to screen the interacting proteins of SEM1, further conduct RNA-Seq, coexpression network analysis and H3K14ac ChIP-Seq on the shoot apical meristem and kernels of sem1 mutants and its related wild types for the identification of regulatory network of SEM1. 3) conduct candidate re-sequencing on an association panel of ~500 diverse inbred lines and maize relative-teosinte, followed by association mapping and evolutionary analysis, to identify favorable alleles for high-yield breeding and the selection sites during maize domestication and selection. Our proposal will not only enhance our understanding of the molecular mechanism of maize kernel development, decipher the regulatory network of SEM1 during maize kernel development, but also potentially provide a new functional marker for maize high-yield breeding.

籽粒大小是玉米产量的重要构成因子。突变体基因Sem1可能是一个新的调控玉米籽粒发育的关键基因。但其调控籽粒发育的分子机理仍不清楚。项目组前期通过组学手段与转座子标签法相结合的策略，已鉴定到sem1突变体候选基因-组蛋白乙酰化转移酶复合体基因，在此基础上，本项目拟通过CRIPR/Cas9与超表达转基因实验及等位测验等，验证Sem1候选基因生物学功能；通过酵母双杂技术筛选SEM1互作蛋白，并用双分子荧光互补进行互作验证，进而利用RNA-Seq与ChIP-Seq技术开展籽粒发育不同时期突变体与野生型的转录组比较分析、共表达调控网络分析及全基因组组蛋白乙酰化的比较分析，鉴定Sem1调控网络，解析Sem1影响玉米籽粒发育的分子机制；通过重测序、关联分析与进化分析，鉴定Sem1基因在玉米驯化与育种中的选择位点，挖掘优良等位基因。探索调控玉米籽粒发育的分子机制，为玉米产量分子育种提供理论指导与基因资源。

籽粒大小是决定玉米产量的关键因子。研究人员发现一个玉米籽粒大小变异的经典突变体Sem1。本项目的主要研究内容为：1，定位经典突变体Sem1的功能基因并验证Sem1目标基因的功能；2，鉴定Sem1基因的调控网络，解析Sem1控制玉米籽粒大小的分子机制；3，鉴定Sem1基因在玉米驯化与改良过程中的选择历程，并挖掘可以用于育种的优良等位基因。.本项目结合SHOREmap、BSR-seq、精细定位及表达量分析，将Sem1突变体候选基因锁定为一个编码转录延伸复合物第6号亚基的基因——Elongator complex 6 (Elp6)；创制了Sem1候选基因Elp6的CRISPR敲除及EMS突变体材料，目标基因突变体材料的籽粒大小缩小。原始Sem1突变体与CRISPR敲除及EMS突变体的杂交互补实验进一步证明了Elp6就是Sem1突变体的功能基因。ELP6蛋白在细胞各个位置均有表达，表明其功能的复杂性。差异翻译分析显示突变体籽粒、胚乳中翻译活性明显下降，而胚乳中一些与翻译活动相关的基因翻译丰度的下降可能是导致整个籽粒翻译水平下降的主要原因，而它们翻译丰度的下降主要是转录延伸复合物影响tRNA修饰引起的。差异乙酰化及差异表达分析显示，Elp6突变引起一些胚乳相关基因组蛋白乙酰化水平的下降，从而通过降低转录量间接抑制了它们的翻译。进一步的酵母双杂实验证明，ELP6蛋白与ELP4存在较强互作，说明了转录延伸复合物亚基之间相互协调，行使功能。RNA-seq与CUT&Tag实验表明，ELP通过多个途径影响翻译进而引起多性状表型变异。物种间Elp6基因的进化分析表明，ELP6蛋白在植物中存在多个氨基酸序列一致区域，表现出较高的保守性。玉米种内群体Elp6重测序与关联分析揭示，Elp6在玉米种内存在一定序列变异，且与玉米籽粒大小显著关联，可用于功能性分子标记开发。.本项目克隆了一个新的控制玉米籽粒大小的关键基因Elp6，明确了其通过乙酰化水平的调控影响籽粒发育关键基因的翻译，最终控制玉米籽粒大小。本项目的结果为玉米籽粒产量的定向育种提供了理论支撑与基因资源，具有重要的理论与实践意义。

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