油菜根发育相关基因的克隆与功能分析

The root is a very important organ for the higher plants, and it performs essential functions during plant development, including anchoring plants, uptaking water and nutrient, and responsing to environmental factors. Shaping root development could directly affect the status of plant growth condition, and then affect the yeild. The development of Arabidopsis root has made great progress in the past twenty years. However, the root development is a complex biological process, there are still a lot of process is not clear. In this proposal, we apply a big root system (brs) mutant obtained from activation tagging approach in Brassica napus to dissection the molecular mechanism of root development..Firstly, we will colone the BRS gene by Tail-PCR, and characterize the cloned gene by over expression of the BRS gene in wild type. In addition, we will knock down the BRS gene by RNAi interference technology or knock out BRS gene by CRISPR/Cas9 to study the biological function of BRS gene. Then we will analyze the expression profile of BRS in root, stem, leaf, flowers as well as in the histological section. The subcellular localization will determine in the transient expression of BRS-GFP fusion protein in the brassica napus cotyledon cell. The relationship between the BRS gene and other genes associated with the development of the root will be detected by Realtime-PCR. The interacted proteins with the BRS will conducted with the BIFC method in Brassica napus cotyledon cell. The study has important significance for cloning our own intellectual property rights of gene and the gene used in agricultural production.

根是高等植物重要的器官，其发育状况的好坏直接影响植物体的生长状况，更影响产量。目前拟南芥根发育的研究已取得了较大的进展，但是根的发育是一个非常复杂的生物学过程，还有很多途径尚不清楚。本项目利用激活标签法获得的一个油菜大根系突变体（Big Root System, brs），通过BRS基因的克隆，结合BRS的过表达进行克隆基因的功能验证，同时应用RNAi技术和CRISPR/Cas9技术研究该基因缺失的生物学功能。对突变体根、茎中植物激素的变化进行测定，采用组织切片观察、BRS基因表达谱分析、亚细胞定位、蛋白互作研究以及BRS基因与其它根发育相关基因关系的解析，揭示BRS蛋白在油菜根发育过程中的分子机制。上述研究结果对克隆具有我国自主知识产权基因以及将该基因应用于农业生产具有重要的指导意义和参考价值。

油菜（Brassica napus L.）是世界重要的油料作物，除了具有较高的营养价值和食用价值，油菜还被用来作为生产生物柴油的原料。我国是世界上主要油菜生产国，同时也是菜籽油消费大国。然而，我国的油菜种植方式以传统方式种植为主。随着劳动力成本的提高，这种耗时耗力的种植方式严重制约我国农民油菜种植的积极性。油菜育种家一直对油菜株型进行改良，使之适合机械全程化，有利于减少劳动力成本。此外，油菜株型的改良也有利于提高油菜产量。.植物株型由地上器官组成，这些器官都是茎尖分生组织（shoot apical meristem，SAM）发育而来。因此，研究SAM发育的分子调控机制对农作物株型改良和农作物的机械化种植具有非常重要的意义。在过去的几十年间，SAM分子调控机制的研究在拟南芥和水稻取得了丰硕的成果，然而在甘蓝型油菜中的研究尚浅。我们获得了一个甘蓝型油菜多主茎突变体dt (duo tou)。dt突变体表现为茎分化异常，叶片增多，株高下降以及形成较大根系。表型和组织切片分析表明，茎尖分生组织（SAM）发育异常导致dt表型。从转录和蛋白水平上表明参与SAM活性维持、细胞分裂素生物合成和信号转导的基因差异很大，这与dt突变体中细胞分裂素水平较高有关。此外，我们通过BSA-seq方法将候选基因定位在油菜A10染色体上6.8 Mb一个区间内，并获得一个候选基因。这些结果揭示了甘蓝型油菜主茎异常分化的分子机制，为甘蓝型油菜育种和生产提供了理想的材料。