油菜油体发生及其早期调控机制研究

Improving oil content is the main goal of rape breeding and uncovering the detailed developmental process of oil body formation can provide important theoretical basis for creating new cultivars of rape with high oil content. Through years of breeding, we have created near isogenic lines in Brassica napus containing different QTLs with board range of oil production ratio from 36% to 60%. In this project, based on these new varieties of rapeseed we will explore the occurrence and accumulation of the oil bodies and lipid by cell biological methods. Via transcription profile analysis and comparative embryology and other means, we plan to trace the gene expression related to oil body formation and to select and test differential genes associated the formation of oil body and its dynamic process, thus it may provide a theoretical guidance for further research on high-oil rapeseed breeding. In addition, it can make great contributions to solve the problem of oil shortage in our country.

提高含油量是当前油菜主要育种目标之一，而揭示油菜油体发生过程则可能为培育高含油量的油菜品系提供理论基础。本申请拟以课题组构建的甘蓝型油菜含油量从36%至60%的近等基因系材料来研究不同含油量油菜胚珠发育及种子成熟过程中油体发生及其早期形成的调控机制。通过显微操作、细胞染色、荧光显微镜和透射电镜观察及磁共振扫描等细胞生物学及微量物质检测方法明确油体及脂类物质的时空分布规律；利用比较胚胎学和单细胞转录组学等多种技术手段，对高油和低油油菜种子发育过程中的油体蛋白调控基因以及其他基因的动态变化进行追踪，对油体发生相关的差异基因进行筛选和验证。本研究将会为揭示油菜种子含油量形成机制、为进一步通过分子设计育种提高油菜种子含油量提供重要理论指导。

研究油菜油体发育过程可为培育高含油量的油菜品系提供理论基础。显微操作、细胞染色、荧光显微镜和透射电镜观察结果发现发现油体出现在油菜胚胎发育早期，在授粉9 ~ 11 d后（球形胚时期）的胚体和胚柄中均存在直径小于0.5 μm的油体，还发现油菜DH系低油Q318和高油Q181材料中油体个数在球形胚和心形胚时期并无差异，但在晚期的鱼雷形胚细胞中油体个数差异明显。转录组分析表明晚期胚胎较早期胚胎下调表达基因明显多于上调表达基因，低油材料318不同时期胚胎中下调基因均高于高油Q181各时期胚胎中差异表达基因数。GO功能与KEGG路径富集分析，鱼雷胚（YLP）和心形胚（XXP）差异基因在高油、低油及混合比较过程中均富集于脂质代谢过程。差异基因验证结果表明C2011与C789早期表达量是晚期表达量的4倍多；C930早期表达量是晚期的1.5倍；C357、C453与C1083晚期表达量高于早期；C414与C219晚期表达量是早期的2倍。另外，除BnCLO3的表达量在整个胚胎发育阶段无明显变化外，其他油体蛋白基因Oleosins、Steroleosins和BnCLO1的表达量在心形胚时期就明显增多，并持续增长；脂肪酸合成转录因子BnLEC1、BnL1L、BnWRI1和BnFUS3在胚胎发育阶段，基因表达规律均呈先上升再下降的趋势。基因功能验证结果表明在油菜及萝卜早期胚胎发育过程中均发现LEA基因的强烈差异表达情况，初步揭示BnLEA18基因在油菜角果及胚珠数量上可能参与调控，BnLEA3在光合效率增强及降低活性氧等生理过程发挥重要作用。