水稻是喜温作物，对低温敏感，特别是孕穗期低温对产量造成的损失不可弥补，因此对水稻孕穗耐冷基因的克隆与功能机理分析具有重要的意义。在前期研究中，申请人利用水稻孕穗期耐冷云南粳稻“昆明小白谷”和日本冷敏感粳稻“十和田”构建一系列包含不同孕穗期耐冷QTL的近等基因系，在此基础上本研究通过大分离群体将第四号染色体上的qCTB4-1精细定位在56.8 Kb区间内，结合核苷酸序列分析和表达分析确定候选基因CTB4a，构建互补、过表达和RNAi载体，进行遗传转化，初步验证了候选基因的功能。进一步将通过酵母双杂、Co-IP、激酶活性分析等多种分子生物学和生理生化实验技术对其作用分子机理进行解析。此外，利用水稻种质资源的基因型和表型数据对CTB4a进行单倍型和起源演化分析，明确目的基因的起源演化规律，同时还将利用分子标记辅助选择和无选择标记转基因技术创制优异耐冷遗传材料，并分析其耐冷育种利用潜力。

Rice is a thermophilic crop and sensitive to cold stress, especially at booting stage that can lead to irreparable yield reduction. So it is of great significance to isolate the genes conferring cold tolerance at booting stage in rice. In previous studies, Several NILs containing different QTLs were constructed using a cold-resistant variety from Yunnan “Kunmingxiaobaigu” and a cold-sensitive japonica variety cultivated widely in Japan “Towada”. Among these, We selected for further characterization qCTB4-1 and delimited the qCTB4-1 containing region to 56.8 kb. Nucleotide sequence analysis and expression analysis showed that one ORF was the most possible candidate gene, and designated as CTB4a. The cold resistant function of CTB4a was preliminary validated by transgenic approach. In this study, detail function and mechanism analysis will be conducted using biological, physiological or biochemical techniques, such as the yeast two-hybrid, Co-IP, kinase activity analysis, and so on. In addition, haplotype and evolution analysis will be conducted using the genotype and phenotype data of rice germplasm located in different geographical regions. Meanwhile, marker-assisted selection and marker-free transgenic technology will be used to create excellent cold-resistant genetic materials for breeding.