玉米半矮秆基因Sd7和sd8的克隆和功能分析

The plant height is an important agronomic traitin maize. Reducing plant height and increasing planting density reasonably can decrease the risk of lodging and obtain higher grain yield. We identified two novel semidwarf mutant Sd7（Semidwarf 7）and sd8（semidwarf 8）in maize. The mutation of Sd7 was controlled by a dominant locus. Phenotypic comparison indicated that the elongation of internode was restrained in Sd7. Through fine mapping, genomic sequencing, gene expression analysis, measurement of endogenous hormone and treatment of exogenous hormone, we concluded that the expression of ACC synthase gene was changed in Sd7, resulting in the elongation of internode was affected. The sd8 is a recessive mutant. We isolated sd8 gene using map-based cloning, encodes a NAD binding oxidordeuctase. In this present project, we will apply allelic test and transgenic technology to further verify the function of Sd7 and sd8; spatial and temporal expression pattern and protein function will be confirmed by gene expression assays, subcellular localization and measuring enzymatic activity. Interacting proteins (genes) will be identified through yeast-two-hybrid assays, Pull-down assays and EMS mutagenesis. Combined with the analysis of double mutant and RNA-seq, the function of the two genes in the development of maize stem will be illustrated and the primary genetic networks will be constructed. We will also screen for elite alleles of those two genes in the natural population, so as to supply germplasm resources for the breeding of lodging-resistant and high density planting varieties of maize.

株高是玉米的重要农艺性状，适当降低株高，增加种植密度是减轻玉米倒伏、获得高产的重要途径。申请者获得两个玉米半矮秆突变体Sd7（Semidwarf 7）和sd8（semidwarf 8）。Sd7由一对显性核基因控制。其节间细胞伸长受阻，通过定位、表达分析、激素测定实验，推测突变位点改变了候选区间附近ACC合酶基因的转录活性，进而影响Sd7茎秆细胞的伸长。sd8由一对隐性核基因控制，图位克隆推测SD8编码NAD结合的氧化还原酶。在前期研究基础上，申请者将通过等位测验和转基因技术验证Sd7和sd8的候选基因，通过基因表达分析、蛋白定位和酶活分析确定基因时空表达模式和蛋白功能，利用酵母双杂交、Pull-Down和EMS诱变技术探究目的蛋白互作因子；结合双突变体分析和转录组测序技术阐明基因在玉米茎秆发育中的功能，初步构建株高调控网络；同时筛选优异等位基因，为培育抗倒、密植高产玉米新品种提供种质资源。

玉米株高是影响株型的关键性因素，也是连接“源库”的关键桥梁器官，有效降低株高并增加种植密度是减轻玉米倒伏、获得高产的重要途径。本项目克隆了一个玉米半矮秆突变体Sd7。通过扫描电镜观察，发现Sd7茎节间的细胞伸长受到抑制，进而导致节间缩短，造成株高和穗位高显著降低。我们通过图位克隆策略获得候选基因ZmSD7，测序发现ZmSD7基因的第4外显子中插入一个MITE转座子，导致基因发生移码突变。ZmSD7编码了一个ACC合酶，催化乙烯生物合成前体物质ACC的生物合成。转基因验证、内源激素测定和外源激素施加实验结果表明ZmSD7调控玉米茎节间的纵向细胞的伸长，从而调控了玉米的株型变异。研究结果为加速玉米株型的改良提供了强有力的理论基础和技术支撑。另外，我们还克隆了玉米半矮秆基因sd8；解析了DLF1调控玉米株高、叶片数和开花时间的通路机理。在本项目支持下，共发表SCI 论文2 篇，其中包括1篇Plant Physiology，1篇The Plant Journal。