稳定表达的耐冷QTL qCT-3-2的克隆和耐冷机制分析

Rice is one of the most important food crops. However, cold stress frequently happens, and causes huge losses to the global rice production, especially when the cold stress appears at the reproductive stage. So, mining cold tolerance genes of rice at the reproductive stage and researching on mechanism of cold stress have important significance to ensure the food safety. So far, more than 60 cold resistant QTLs were identified at the reproductive stage, but few were fine mapped, cloned or utilized in breeding, and the cold tolerance mechanism of rice at the reproductive stage is not clear. Previously, a cold resistant QTL qCT-3-2 with high practical application value in breeding was identified by GWAS and was further fine mapped in the interval of 192.9kb by us. It can resistant stably to the low temperature at seedling and reproductive stages. Based on the existing research results and near-isogenic lines, the applicant intends to clone qCT-3-2 and analyzing the tolerance mechanism of qCT-3-2 through the strategy of combining QTL mapping and whole genome expression profile. Our research results will provide value information for breeding rice varieties with cold tolerance.

水稻是最重要的粮食作物之一。然而冷害频繁发生，对全球稻米生产造成巨大损失，特别是发生于生殖期的障碍性冷害。因此识别耐冷基因和研究水稻对冷胁迫的机制对选育耐冷品种具有重要现实意义。迄今已有超过60个生殖期耐冷QTL被定位，但被精细定位、克隆和应用于育种实践的QTL寥寥无几，人们对水稻生殖期的耐冷机制也没有清楚的认识。先前申请人通过GWAS和物理作图相结合的方法在水稻第3染色体上将一个稳定表达的耐冷QTL qCT-3-2精细定位到192.9kb的物理区间，该QTL的有利等位基因来自一个不耐冷的籼稻品种黄华占，其不但在不同环境下的孕穗期稳定表达，而且对苗期低温也具抗性，表现出较高的育种利用价值。基于已有的研究结果和近等基因系，申请人拟利用QTL精细定位、表达谱分析、生物信息学分析等多种手段相结合的策略，克隆qCT-3-2,并对其耐冷分子机制进行研究，以期为水稻耐冷育种提供有利用价值的信息。

通过构建次级分离群体，qCT-3-2被定位到分子标记RM6297与ZCT-7之间85kb的物理区间且与分子标记ZCT-7紧密连锁。生物信息学分析，受ABA介导的no apical meristem protein家族基因LOC_Os03g04070被列为优先候选基因并被克隆。转基因株系表型鉴定分析表明，与野生型相比，过表达株系可一定程度上提高水稻苗期、孕穗期的耐冷性。瞬时表达研究显示该基因转录蛋白表达于细胞核。分子机制分析表明，OsNAC22通过介导ABA信号途径，赋予水稻非生物胁迫耐性。研究结果对进一步理解水稻复杂的抗逆机制，通过MAS手段从苗期筛选有利耐冷基因的品种提供了有效信息。