一个水稻分蘖调控基因的克隆及其调控机制研究

Rice tillering, one of the most important agronomic traits that may influence panicle numbers and further affect grain yield, is controlled by multiple factors such as endogenous physiology and exogenous environmental factors. Identified new genes control rice tillering and revealing its molecular mechanism will be an important direction for rice ideal plant breeding. In our previous study, a rice mutant line with reduced plant height and delayed tillering was found. In the early vegetative growth, the contents of soluble protein, free amino acid, and the activities of glutamine synthase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH) in the mutant ts2 were significantly lower than those in the wild type plants, thus indicating that the nitrogen metabolic intensity in the mutant ts2 is significantly weaker than the wild type. And the expression levels of genes related to the synthesis and signal transduction of strigolactones and cytokinin were increased in the mutant plants. At this time, the growth of axillary buds was inhabited. Subsequently, the activities of GOGAT, GS and GDH were recovered to the normal level. And the genes related to the synthesis and signal transduction of hormones were down-regulated in the mutant plants. Meanwhile, the inhibition effect of tiller growth was relieved. In this project, we will try to determine the biological function and regulatory network of TS2 by using wild type and ts2 mutant plants. The method of genetic complement and CRISPR/Cas 9 will be used in this study to verify the biological function of candidate genes. RNA-Seq, histochemical staining, RT-PCR and many other methods will be used to analysis the gene function and regulation network of TS2. Meanwhile, the relationship between TS2 and hormones will be studied in this project, and the regulatory mechanism to nitrogen assimilation will be involved. With these approaches, we hope that the biological function of TS2 and its regulation network to rice tiller growth could be found. For ts2 mutant, the molecular mechanism of transformation between tiller inhibition and growth will be discussed in this project.

分蘖是影响水稻产量的重要农艺性状之一，鉴定克隆影响水稻分蘖的遗传因子，对高产育种具有重要的指导意义。申请人获得了1株株高降低、分蘖发生时间推迟的突变体ts2，该突变体在播种后36天之前氮同化能力减弱、独脚金内酯及细胞分裂素合成和信号转导相关基因表达量升高，腋芽生长受到抑制，植株维持单茎状态；随后，突变体氮代谢指标恢复正常，激素合成及信号转导相关基因表达量下调，分蘖抑制效应解除。在此基础上，本项目拟利用野生型植株和ts2突变体材料，通过CRISPR/Cas9、遗传互补等方法对TS2候选基因进行功能验证，利用组织化学染色、RT-PCR、转录组测序分析等技术分析TS2的生物学功能及其对腋芽生长的调控网络。同时，本项目拟从激素调控和氮素吸收利用角度研究TS2对水稻分蘖调控的生理生化基础，揭示ts2突变体分蘖抑制与发生之间进行转换的分子机制，为TS2应用在水稻高产育种中奠定理论基础。

水稻分蘖是影响水稻产量的重要农艺性状之一，挖掘鉴定影响水稻分蘖的调控因子，对于高产育种具有重要的知道意义。本项目通过化学诱变获得了水稻突变体ts2，该突变体分蘖芽生长被抑制、株高降低、主穗粒数减少。生理生化分析表明，突变体植株碳氮同化能力减弱。本项目利用ts2突变体和五山丝苗构建了定位群体，通过分离群体分析法和隐性群体分析法，将TS2定位在水稻的第8号染色体上182.4 kb范围内。随后，本项目通过测序对候选基因的基因组序列进行了检测，筛选出突变体和野生型之间具有序列差异的ORF，并利用CRISR/Cas9技术对候选基因的生物学功能进行了验证，结果表明TS2编码ABC转运蛋白OsABCG18。分析转录组数据发现，野生型和基因敲除植株地上部差异表达基因主要在激素信号转导、脂肪酸代谢、氨基酸代谢、糖代谢、病原菌防卫反应等通路富集，而在根部，差异表达基因主要富集在糖代谢、氨基酸代谢、类苯基丙烷合成和病原菌防卫反应等途径。代谢组数据表明，野生型和基因敲除植株之间差异代谢物主要富集在氨基酸代谢、萜类化合物合成、玉米素合成、ABC转运子、脂肪酸代谢等途径。此外，对氮素转运、同化基因表达分析发现，TS2敲除植株根部氮素吸收、同化相关基因表达水平显著上调，而地上部植株编码谷氨酰胺合成酶基因表达量显著下调，但氮素再利用相关基因显著上调，后续对野生型和基因敲除植株氮素利用相关生理生化、基因调控网络进行分析将为揭示TS2的生物学功能奠定基础。