甘蓝型油菜矮杆基因ED1的图位克隆与功能分析

Plant height, as one of the most important agronomic traits of rapeseed (Brassica napus L.), is related to plant lodging resistance. Appropriate dwarfing can improve yield by enhancing lodging resistance and optimizing mechanized harvesting. Therefore, isolation of new genes regulating plant height is crucial for improving rapeseed yield. ed1 (extremely dwarf1), a rapeseed dwarf mutant with plant height of about 24cm, was discovered to be stably inherited. Genetic analysis indicated that one pair of semi-dominant gene controlled the dwarf phenotype. We genotyped the F2 individuals using 60k SNP array for preliminary mapping, and genome-wide association study (GWAS) showed that the major locus related to dwarf phenotype is on the chromosome C05. We further narrowed the ED1 to a 350-kb region (contained 14 ORFs), and gene prediction showed that no reported genes governing plant height were found in this region. Based on the previously study, the objectives of this proposal is to isolate the ED1 gene by map-based cloning method and characterize the target gene by phenotypic observations of transgenic plants, expression analysis, subcellular localization assays and yeast-two hybrid assays. Meanwhile, the molecular mechanism for dwarfism will be further validated, as well as the value of the dwarf gene in hybrid breeding.

株高是油菜重要的农艺性状之一，与植株抗倒伏性有关。植株适当矮化可提高抗倒能力和适合机械化收割从而达到增产的目的。因此，发掘和克隆调控油菜矮杆性状的新基因，阐明其调控分子机制，对进一步提高油菜单产具有重要指导意义。甘蓝型油菜矮化突变体ed1 (extremely dwarf1) 为自然突变，该突变体株高约为24cm。遗传分析表明：该突变性状受一对半显性基因控制。利用60k SNP芯片结合GWAS将该基因初步定位在C05染色体上。目前，已将目标基因精细定位在350kb的范围内（包含14个ORF），且该区间内不含有已知的株高相关基因。本申请拟在此基础上进一步精细定位，克隆矮杆基因ED1。通过转基因表型鉴定、基因表达分析、亚细胞定位及酵母双杂交等实验对ED1进行功能分析，并对其产生矮化的分子机制进行深入研究，评估其在油菜矮化杂交种育种中的应用潜力。

随着油菜杂种优势的成功利用，油菜株高也有所增高，抗倒伏则成为稳产保质的关键。适度矮化的油菜品种不仅抗倒伏性增强，而且更适合于机械化收获。因此，筛选株高适度和农艺性状优良的矮杆突变体是培育矮杆品种的有效途径之一。本项目利用图位克隆鉴定出一个控制油菜矮杆性状的ED1基因。利用遗传学和分子生物学等手段阐明了ED1基因调控油菜矮杆性状的分子作用机理。对ED1进行表达模式分析确认其为组成型表达，且在茎中表达量最高。ED1编码一个IAA7转录因子，在生长素信号途径扮演一个抑制子的角色。ed1中的点突变直接影响其被生长素降解。油菜中包含ED1等3个同源拷贝，各个拷贝对株高的影响不一。酵母互作实验表明这3个IAA7拷贝与生长素受体以及ARFs的互作强弱不同。同时，研究发现ARF8与BZR1存在互作关系，且通过Bifc实验验证。因此，ED1可以通过ARF8与BZR1的互作参与到BR信号途径来调控油菜株型。另外，本项目还图位克隆了1个控制株高的功能基因，申请3项国家发明专利，其中获批1项。借助基因编辑创制出油菜矮杆多分枝和矮杆紧凑型材料2份。本研究从种质创新，分子标记开发和基因克隆到理论突破，系统研究株型新基因的功能和作用机理，获得的突变材料可以为油菜高产株型育种提供可利用的种质资源。