油菜素内酯信号通路关键基因在甜菜块根发育过程中的功能研究

For a long time, the sugar beet industry is the pillar industry at many poverty-stricken areas in Inner Mongolia. The biological problem of this project will be focused on the mechanism of yield and quality formation for sugar beet. Based on the previous study of us, this study will take insight into the role of BR in regulatory mechanism of taproot growth and sucrose accumulation. Three candidate genes which are the crucial genes of BR signaling, including CPD (constitutive photomorphogenesis and dwarfism), BZR1/2 (brassinosteroid resistant 1/2) and XTH (xyloglucan endotransglucosylase/hydrolase) , are the gene of rate-limiting enzyme for BR, transcription factor gene for BR signal transduction pathway, and the target of BZR1/2, respectively. Gene silencing line, overexpression line and the line of transcriptional regulation analysis will be constructed as the research materials. Using bioinformation, phytophysiology, cytobiology and molecular biology, we identify the relationships between the spatial and temporal expression of these three genes, and anatomical structure, physiological metabolism, weight and sucrose content of taproot. Thereby we understand the regulatory function of BR signaling for taproot development. Meanwhile the response of CPD to cultivation factors will be identified, and analyze the regulatory pathway when cultivation factors influence the yield and quality of taproot. The study will reveal the regulatory mechanism of BR in taproot development. Besides, this study will provide the theoretical basis of the molecular breeding and chemistry regulation for increasing yield and sucrose content.

长期以来，甜菜制糖业是内蒙古特别是一些贫困地区的支柱性产业。本项目围绕如何提高甜菜产量和含糖的问题，在已有研究基础上，研究油菜素内酯（BR）在甜菜块根膨大和糖分积累过程中的调控机理。应用生物信息学、植物生理学、细胞生物学和分子生物学研究手段，以油菜素内酯信号通路中的3个关键基因（BR合成限速酶基因CPD、BR信号通路中转录因子基因BZR1/2和BR信号下游靶基因-木葡聚糖内糖基转移酶/水解酶基因XTH）为候选基因，以基因沉默株系、超表达株系和转录调控分析株系为材料，研究油菜素内酯信号通路关键基因在甜菜块根发育过程中的时空表达特性及其与块根形态解剖结构、生理代谢和根重、含糖的关系。同时研究CPD对主要栽培因子的应答反应，解析栽培因子对产质量影响的可能调控机理。从而阐明油菜素内酯对调控甜菜块根发育及其产质量形成的作用机理，为进一步挖掘甜菜产质量，开展分子育种和化学调控提供理论基础和实践依据。

甜菜是以榨取块根糖分为生产目的经济作物。本研究应用分子生物学及植物基因工程等技术手段，深入研究了油菜素内酯（BRs）信号通路中的3个关键候选基因（BR合成限速酶基因BvCPD、BR信号通路中转录因子BvBZR1和下游靶基因木葡聚糖内糖基转移酶/水解酶基因BvXTH），对甜菜块根发育的调控机制。研究表明（1）BR可通过扩大甜菜块根维管束环间薄壁细胞面积，显著增加各维管束环的环距，进而促进甜菜块根的增粗。（2）在甜菜块根发育过程中,BvCPD表达量与BR含量呈正相关，均在块根及糖分增长期达到最大值；该基因编码的蛋白定位于细胞核和细胞膜上；通过同源或异源过表达及沉默BvCPD基因证实了该基因能够显著增加植株的株高、叶面积、地上部鲜重、根长、根体积和根重，沉默BvCPD使甜菜叶柄加长、叶片变窄卷曲；通过酵母单杂交实验证实 BvbZIP1与BvAREB1能够结合在BvCPD启动子区域，且该基因的表达受水分、温度、光照、氮素等因素的调控。（3）BvBZR1表达量随BR增加而显著上调；该基因的亚细胞定位于细胞核上；过表达BvBZR1甜菜的株高、叶片长、叶片重、叶柄重、块根直径和长度都显著大于野生型，该基因能够增加甜菜块根各维管束环间薄壁细胞的大小和层数，从而促进块根直径的增大；BvBZR1通过上调块根SPS的表达量，下调INV的表达量增加块根蔗糖含量；BvBZR1可通过调控与细胞壁形成相关的BvXTH和BvCESA下游靶基因实现促进根发育。（4）BvXTH23、 BvXTH17基因的亚细胞定位于细胞膜上；经拟南芥异源过表达和突变体互补实验证明BvXTH可增强BvXTH酶活性，调控根尖伸长区细胞的生长。（5）初步建立了BR信号通路相关基因在甜菜块根膨大及糖分积累中的调控模型。上述研究成果填补了甜菜块根生长发育调控的分子生物学研究领域空白，通过遗传转化创制了甜菜高产新资源，为利用BR调控甜菜产质量形成提供了理论依据和技术措施，对提高甜菜生产水平具有重要的理论意义和应用价值。