水稻高位分蘖调控基因TUP1的克隆与功能研究

Rice (Oryza sativa) is one of the most important food crops in the world and also is the model plant in monocotyledonous crops. Tillering is the key agronomic trait determining grain yield per plant in rice. Tillers usually develop from axillary meristems (AM) or axillary buds which are initiated in the axils of leaves on the basal unelongated nodes. The axillary buds on the upper elongated nodes generally become arrest. The molecular mechanism underlying this phenomenon remains elusive. Previously, we screened and identified a rice mutant, named tillering on upper nodes 1 (tup1) that keeps tillering on the upper elongated nodes. The candidate gene TUP1 was cloned through map-based cloning. TUP1, which is located on chromosome 4, is a new functional gene. The gene function was confirmed by using RNAi and gene editing technology. In this project, we will analyze the function and the spatiotemporal expression pattern of TUP1 during the development of AM by using cytological analysis. We intend to investigate the changes of auxin distribution pattern in tup1. In addition, we will compare expression patterns and levels of tillering-related genes in tup1 and wild type. Moreover, the TUP1-involving pathway will be explored by transcriptome analysis and qRT-PCR. Furthermore, we will use multiple methods to screen the regulators or interactors of TUP1, and the candidates will be further characterized in detail. This project aims to elucidate the regulatory mechanism of TUP1 during the development of AM. The achievements would provide new cues for the study of the molecular mechanism underlying AM development in monocotyledon crops.

水稻是重要的粮食作物，也是单子叶作物研究的模式物种。分蘖是决定水稻单株产量的要素之一，由叶腋处的侧生分生组织（腋芽）发育而成。一般仅基部未伸长节上的腋芽可发育成分蘖，高位伸长节上的腋芽不能发育为分蘖，调控这一现象的分子机理还不明确。前期我们筛选到一个在高位伸长节上产生分蘖的突变体tup1，通过图位克隆定位到突变基因。TUP1定位于4号染色体，是一个新的功能基因。在野生型中对TUP1进行RNA干涉或基因编辑敲除均导致突变体类似表型，从而确认了TUP1抑制高位分蘖的功能。本项目拟在此基础上，详细分析TUP1在叶腋分生组织不同发育阶段的作用及时空表达模式，比较生长素在突变体中的分布变化，分析相关分蘖调控基因受到的调控影响及TUP1介导的调控途径，筛选TUP1互作蛋白，最终揭示TUP1在水稻分蘖发生过程中的作用机制。研究结果将为深入理解单子叶作物叶腋分生组织发育调控的分子机理提供新的线索。

水稻是重要的粮食作物，也是单子叶作物研究的模式物种。分蘖是决定水稻单株产量的要素之一，由叶腋处的侧生分生组织（腋芽）发育而成。一般而言，仅基部未伸长节上的腋芽可发育成分蘖，高位伸长节上的腋芽不能发育为分蘖，调控这一现象的分子机理还不明确。前期我们筛选到一个在高位伸长节上产生分蘖的突变体tup1，通过图位克隆定位到突变基因TUP1，RNA干涉以及基因编辑敲除均导致突变体类似表型，确认了TUP1抑制高位分蘖的功能。本项目在前期的基础上构建了TUP1全长互补株系，互补株系可以恢复tup1-/-腋芽伸长的表型。对tup1-/-茎节长度与腋芽生长的关系进行了分析。结果显示tup1-/-在第II、IV、V节更容易产生高位分蘖。利用proTUP1-GUS进行GUS染色分析，TUP1比较特异的茎端分生组织、腋芽、伸长腋芽、茎节基部、成熟的叶脉中表达。在水稻原生质体中对TUP1进行亚细胞定位分析发现，TUP1定位在细胞核中。利用第II节腋芽组织构建的酵母双杂cDNA文库对TUP1的互作蛋白进行筛选，从中筛选到与TUP1互作的蛋白P10，并进行初步酵母双杂验证。酵母双杂结果初步揭示TUP1与P10在体内存在互作。构建了P10cc Crispr/Cas9转基因株系，则表现出高位分蘖的表型，暗示着TUP1与P10结合共同抑制高位分蘖。RNA-seq进行GO分析显示，这些差异表达的基因富集于生物合成相关途径。在第II茎节分蘖芽中对细胞分裂素相关基因进行了QRT-PCR分析，结果显示细胞分裂素合成基因IPT3、细胞分裂素激活酶基因LOG、信号转导基因OsRR1细胞分裂素信号基因显著上调，暗示着在腋芽分生组织伸长的过程中，TUP1-P10通过抑制细胞分裂素合成以及信号抑制腋芽伸长。研究结果将为深入理解单子叶作物叶腋分生组织发育调控的分子机理提供新的线索。