水稻生殖生长期耐冷基因qCTB7的克隆及耐冷作用机制研究

Low temperature is one of the most limiting factors for rice production in China and in the world. Identification of cold tolerance gene and studying the molecular mechanism of cold tolerance may play an important role in the interpretation of cold tolerance and in the practice of cold tolerance breeding in rice. Reproductive stage is the most sensitive period to low temperature in rice, and cold damage during this period would cause the most serious losses of rice yield. However, there is no report on identification of cold tolerance gene at reproductive stage by map-based cloning approach,and studying the molecular mechanism of cold tolerance in rice. In this study, cold-tolerant NILs were developed by backcrossing KMXBG, reported to be the most cold-tolerant rice variety at booting stage, as donor, with a cold-sensitive variety, Towada. Cold tolerance of a BC6F2 and other segregating populations was evaluated at several locations over two years. A stable expression QTL, qCTB7, was detected on chromosome 7. The QTL explained 21% of the phenotypic variances (spikelet fertility) and was fine mapped into a 44.5kb interval. The qCTB7 candidate has be predicted and cloned. The function of cold tolerace will be verified by complementation test, gene overexpression and silencing methods;To explain molecular mechanism of cold tolerance at reprodective stage in rice, Y2H and RNA-Seq techniques will be used to analyze interaction proteins and downstream regulated genes. Through genetic transformation and analysis of diversity, good cold tolerance rice lines and cold tolerance hyplotype will be obtaind in rice. Therefore,the results of this study will provide gene resources and breeding materials for cold tolerance breeding in rice.

低温是我国和世界水稻生产中的主要限制因子之一, 发掘水稻耐冷基因,研究耐冷分子机制，对解释水稻耐冷性，指导耐冷育种实践具有重要意义。水稻生殖生长期是对低温反应最敏感的时期，这一时期的冷害造成的水稻产量损失最为严重。但是，目前还没有水稻生殖生长期耐冷基因（影响结实率）图位克隆和耐冷作用机制研究的报道。本研究以目前世界上鉴定出来的生殖生长期最耐冷品种昆明小白谷为供体亲本、以冷敏感品种十和田为受体培育的耐冷近等基因系为材料，通过对BC6F2等分离群体进行多年多点的耐冷鉴定,在7号染色体上检测到一个稳定表达的耐冷QTL，qCTB7，对表型（结实率）的贡献率为21%，并精细定位到44.5kb的物理区间内。目前初步获得并克隆了候选基因qCTB7。通过转基因互补测验，基因超表达和沉默实验验证候选基因的功能；利用酵母双杂和RNA-Seq技术分析蛋白互作关系和下游调控基因，从而解析耐冷作用机制；通过多样性分析，获得水稻重要耐冷单倍型；通过遗传转化，获得优良耐冷水稻株系，最终为水稻耐冷育种提供基因资源和育种材料。

水稻(Oryza sativa L.)是一种起源于热带、亚热带地区，对低温极度敏感的重要粮食作物。低温不仅影响着水稻的地理分布，还影响着水稻各生育时期正常的生长发育。其中，生殖生长期冷害会影响小孢子的发育而导致花粉败育，进而直接影响产量。因此，发掘和克隆水稻生殖生长期耐冷基因，对于阐明水稻生殖生长期耐冷分子机制和开展水稻耐冷分子育种具有重要意义。在本项目中，通过连续精细定位，我们将水稻生殖生长期耐冷位点 qCTB7定位至qCTB7a和qCTB7b两个更小的区段。通过亲本序列比较、功能注释和冷诱导表达分析，分别对qCTB7a和qCTB7b区段内的预测基因进行分析，筛选出qCTB7a.3和qCTB7b.4两个候选基因作进一步分析验证。生殖生长期耐冷全基因组关联分析表明，7号染色体23.5Mb处关联区段的最高点落在qCTB7b.4的启动子区。转基因实验表明，qCTB7a.3和qCTB7b.4均调控水稻生殖生长期低温耐受性。我们将qCTB7b.4 作为重点作进一步研究。表达模式分析发现，qCTB7b.4的GUS转基因植株主要在节间、叶鞘和幼穗中被染色，且qCTB7b.4在不同种质材料中均受到不同程度的冷诱导上调表达。qCTB7b.4能特异地定位于细胞核。酵母实验发现，qCTB7b.4具有自激活活性，且它的转录激活区域位于C端169-241氨基酸位置处。qCTB7b.4可能与谷胱甘肽还原酶OsGR2和乙醛脱氢酶OsALDH2B1互作来调控生殖生长期低温耐受性。单倍型分析发现，qCTB7b.4等位基因的差异可能引起耐冷和冷敏感材料不同的低温耐受性，并且优势单倍型可能起源于中国的野生稻。