水稻油菜素内酯缺陷型基因BRD3的功能及代谢途径研究

Plant shape and other important agronomic traits of rice are regulated by brassinosteroid (BR), and fine regulation of BR metabolic genes expression in vivo can significantly improve the yield of rice. In-depth study on the function and metabolic pathways of BR metabolism-related genes is the prerequisite for fine regulation of BR. A BR defective dominant dwarf mutant ( brd3-D ) was found from the rice T-DNA insertion mutant library. Cloning and functional verification of BRD3 gene has been performed through isolating the T-DNA flanking sequence and overexpression analysis. On the basis of the previous works, Real-Time PCR, RNA in situ hybridization, GFP-tracking and physiological and biochemical analysis will be adopted to identify the function and comprehensive expression patterns of BRD3 gene . Combined with the BR metabolism-related gene expression pattern, the role of BRD3 gene and metabolic pathways its participated will be expected to be revealed. And molecular regulatory network of BR metabolism will be further understood. These works will pave the way for investigation of the molecular regulation mechanisms of rice plant shape, fine regulation of the rice plant shape through the molecular means and maximizing the effect of ideal plant shape on rice breeding for super high yield.

油菜素内酯（BR）调控水稻株型等重要农艺性状，精细调控BR代谢相关基因在体内的表达可以显著提高水稻的产量。而深入研究BR代谢相关基因的功能与代谢途径是精细调控BR作用模式的前提。项目组从水稻T-DNA插入突变体库中发现一个BR缺陷型显性矮杆突变体（brd3-D），通过T-DNA旁邻序列及过表达分析，已完成对BRD3基因的克隆和功能验证。本项目拟在前期研究的基础上，通过Real-Time PCR、RNA原位杂交、GFP示踪等分子生物学技术及生理生化分析全面解析目的基因的功能及表达模式，同时结合BR代谢相关基因的表达谱，进一步揭示BRD3基因的功能及参与的代谢途径，完善BR代谢途径的分子调控网络。以期为探明水稻株型调控的分子机理，进而通过分子手段精细调控水稻株型，最大限度发挥理想株型在水稻超高产育种中的作用奠定基础。

油菜素内酯是植物体内广泛存在的一类甾醇类激素，在植物的生长发育过程中起着非常重要的作用。CYP734A4基因编码一个细胞色素P450单氧化酶，具有降解油菜素内酯的功能，CYP734A4基因缺失突变从未见报道，而过表达CYP734A4导致严重的油菜素内酯缺陷表型，无法获得可育的转基因植株。本项目分离了一个半显性的突变体，该突变体的T-DNA位于CYP734A4 (GenBank Accession AB488667)上游4kb左右，T-DNA插入导致CYP734A4过表达。该突变体表现为矮秆，小粒，叶片直立，对油菜素内酯钝感等表型。有趣的是，杂合突变体还表现为主穗粒数和结实率增加，这些表型在其它油菜素内酯缺失突变体中从未报道。实时定量PCR显示brd3-D 突变体的突变程度和CYP734A4的表达水平呈正相关。和CYP734A4表达量升高相一致，突变体油菜素内酯显著少于野生型植株。利用CRISPR/Cas9系统敲除brd3-D 基因可以使突变体恢复野生型表型。此外利用CYP734A4自身启动子和35S增强子转化野生型水稻可以获得和brd3-D突变体相似表型的转基因植株，进一步证实适量过表达CYP734A4基因能提高主穗粒数和结实率。过表达CYP734A4 导致BR 代谢相关基因表达显著变化，同时外施油菜素内酯可显著诱导CYP734A4 基因的表达, 这些结果证实CYP734A4 基因是BR 代谢途径的负调控因子。目前的结果表明CYP734A4具有提高水稻产量的潜能。