TIFY 转录因子参与水稻耐寒过程的机理研究

Low temperature is one of the major environmental factors that influence rice (Oryza sativa L.) growth, development and production. Rice seedlings are particularly sensitive to chilling in early Spring, improvement of chilling tolerance in rice may significantly increase rice production. To identify the genes involved in the increased chilling tolerance found in cultivars from growth regions with low yearly-temperatures, we chose one kind of strong cold-resistance rice, named Gonggaogui (O. sativa ssp. japonica, refered as Ggg) as materials from GuiZhou province. In our previous study, we constructed the cold-resistant gene library through Suppression Subtractive Hybridization (SSH). From the SSH library, we isolated a GCT1 (Gui Chilling-Tolerance 1) gene which maybe involve in the pathway of cold resistance of Ggg. Expression of OsGCT1 was up-regulated by cold temperature, and genomic DNA sequence comparisons between the Ggg and 93-11 (O.sativa ssp. indica) showed that one single-nucleotide mutation results in a change in an encoded amino acid. Here, in order to explore the function of OsGCT1, we will construct GCT1 -overexpression (OE) and RNA interference (Ri) transgenic rice lines in Ggg and 93-11, and investigate their chilling tolerance. Meanwhile, we will analyze the effects of GCT1 on rice yield and quality, to evaluate the potential value of this gene in proving rice chilling-tolerance. In addition, we will check the expression pattern of GCT1 and its protein localization under normal and cold conditions. By means of biochemical approach and transcriptome profiling analyses, we will perform some functional analysis and detect the interaction proteins of GCT1. At last, we try to investigate the role of GCT1 in rice adaptation to chilling temperature. Our studies may reveal a novel pathway that controls cold adaptation in rice and will help to broaden the possibilities for genetically engineering cold-tolerant rice cultivars.

环境低温会严重影响水稻的产量和品质。研究水稻响应低温胁迫的分子机制，对于全面了解水稻的抗寒基础及培育抗寒新品种具有重要意义。为了克隆更多的水稻耐寒基因，我们选择了贵州当地一种耐寒的粳稻品种“公告鬼”(Ggg)作为研究对象，构建了Ggg低温胁迫响应SSH文库，从文库中筛选克隆了一个TIFY类型的转录因子，命名为GCT1 (Gui Chilling- Tolerance 1)。研究发现该基因在粳稻Ggg中受到低温诱导表达，并且GCT1的蛋白序列在粳稻Ggg和籼稻93-11之间存在着一个氨基酸差异。本项目拟通过获得过量表达GCT1和低量表达GCT1的转基因植株，以各项耐寒生理指标和相关基因表达为依据，确定转基因植株的耐寒性，以此对GCT1的功能进行验证；并利用生理生化手段和转录组测序分析技术，探讨GCT1可能的调控水稻低温应答的作用机制，为水稻的抗寒育种提供一定的理论基础。

环境低温会严重影响水稻(Oryza sativa L.)的产量和品质。研究水稻响应低温胁迫的分子机制，对于全面了解水稻冷适应的分子基础及培育抗寒新品种具有重要意义。项目组前期从水稻低温胁迫响应文筛中克隆了一个TIFY类型的转录因子，命名为GCT1。利用CRISPR/Cas9基因组编辑系统，对GCT1进行编辑。通过分析野生型、敲除突变、过表达植株在冷处理条件下的植株表型、生理生化指标以及基因表达，发现GCT1确实在水稻的冷适应过程中起重要作用。冷处理使gct1突变体的细胞膜受到极大破坏，离子渗漏率随冷处理时间的延长而迅速升高；叶片萎蔫失水严重，叶绿素严重降解，并且植株体内的可溶性糖含量积累明显减少，有害物质MDA积累增加。RNA-seq和荧光定量PCR分析发现，冷处理后，gct1体内大量冷响应基因的表达与野生型相比，明显下调。这些基因主要涉及细胞活性氧平衡调控、非生物胁迫响应、细胞膜磷脂代谢以及细胞壁生物合成途径。该结果暗示着GCT1参与调控水稻的抗寒性可能和冷胁迫条件下，细胞中活性氧平衡的保持、细胞膜磷脂合成代谢以及细胞壁组分重组有关。从野生型和突变体的差异基因中筛选到一个与真核细胞膜鞘磷脂代谢相关的长链基激酶（Long Chain Base Kinase 1）OsLCBK1。研究结果表明 LCBK1和GCT1一样，正向调控植物的抗寒抗冻性。进一步利用酵母双杂交技术筛选GCT1的互作蛋白，发现GCT1可能和家族基因中的TIFY11b相互作用，共同调控水稻的冷响应过程。本研究探讨了GCT1在水稻冷适应过程中的生物学功能，对于进一步解析水稻的冷适应过程及培育抗寒新品种具有重要意义。