水稻籽粒钙含量QTL的克隆及基因功能研究

Mineral elements such as calcium, iron, zinc are essential for human’s wellbeing. However, it is estimated that over two-thirds of the world’s population lack one or more essential mineral elements. Currently, mineral malnutrition, better known as “hidden hunger”, is considered to be one of the most serious global challenges to humankind. Rice is one of the most important staple crops, providing more than one fifth of daily calories for half of the world’s human population as well as being a major source of mineral nutrients. Biofortification of rice varieties with mineral nutrients through breeding is considered to be a cost-effective strategy to alleviate micronutrient malnutrition such as calcium deficiency. Such strategy is based on the identification of QTLs/genes that control the accumulation of mineral nutrients in rice grains. We have previously identified 134 QTLs that control the variations in the concentrations of 16 mineral elements in unmilled rice grain by using two synthetic rice mapping populations. Among the identified four QTLs that control calcium accumulation in rice grain, one QTL was identified in both mapping populations on multiple years. This QTL explains up to 57.1% of variation in grain calcium concentration. In this project, we aim to identify the gene responsible for this QTL, characterize the function of the causal gene, and elucidate the potential working mechanisms and regulatory networks. This project will shade light on the molecular mechanisms for the regulation of calcium translocation and accumulation in rice grain, and provide genetic materials and basic knowledge for breeding rice varieties with high calcium in grains using strategies like molecular marker-assisted selection.

钙、铁、锌等矿质元素是人体必需的营养元素。然而，全世界有超过三分之二的人口存在一种或多种必需矿质营养元素摄入不足的问题，由此造成的“隐性饥饿”已成为影响人类健康的全球性问题。水稻作为重要粮食作物之一，不仅为世界人口提供超过五分之一的能量，同时也是矿质营养元素的主要来源。挖掘水稻籽粒矿质元素积累相关基因，培育籽粒富含矿质营养元素的生物强化水稻品种将有助于缓解缺钙等矿质营养元素缺乏的问题。在前期工作中，我们利用两个水稻定位群体定位到了134个控制16种元素在籽粒中积累的QTL，包括4个控制籽粒钙含量的QTL。本项目将对其中一个贡献率最高达57.1%的主效QTL进行精细定位，克隆相关基因，阐明该基因的功能、作用机制及其控制水稻籽粒钙含量的分子机理和调控网络。本项目将对理解水稻中钙的迁移及其在籽粒中积累的分子机理具有重要意义，并为进一步通过分子标记辅助育种等手段培育籽粒高钙水稻品种提供理论指导。

钙、铁、锌等矿质元素是人体必需的营养元素。然而，全世界有超过三分之二的人口存在一种或多种必需矿质营养元素摄入不足，由此造成的“隐性饥饿”已成为影响人类健康的全球性问题。稻米是人体摄入矿质营养元素的主要来源。挖掘水稻籽粒矿质元素积累相关基因，培育籽粒富含矿质营养元素的生物强化水稻品种将有助于缓解缺钙等矿质营养元素缺乏的问题。本项目在前期定位到4个控制籽粒钙含量的QTL位点的基础上，对其中一个贡献率最高达57.1%的主效QTL位点qGCa-3进行精细定位和基因克隆。通过对水稻重组自交系群体进行全基因组重测序，构建了高密度遗传连锁图谱进行QTL分析，并构建大规模高世代回交分离群体进行精细定位，最终将控制籽粒钙含量的主效QTL位点qGCa-3的因果基因确定为GCSC1。两个亲本间籽粒钙含量的差异主要由GCSC1基因启动子序列差异造成其表达量变化引起。GCSC1编码一个含有HMA保守结构域的功能未知蛋白，定位在叶绿体被膜上。该基因主要在叶片中表达，具有明显昼夜节律变化，但不受缺钙诱导。微量热泳动技术分析表明，GCSC1蛋白可以结合Ca2+，其Kd值为8.33 μM，是一个亲和力较强的Ca2+结合蛋白。在酵母异源表达显示GCSC1具有Ca2+转运能力，但两亲本间的氨基酸序列差异对Ca2+转运活性没有影响。通过非损伤微测技术检测纯化的叶绿体Ca2+流向，发现GCSC1基因敲除突变体的叶绿体Ca2+外排活性显著低于野生型，表明GCSC1是定位在叶绿体上的钙离子外排蛋白。敲除GCSC1基因造成钙从老组织向新生组织的再分配增加，同时孕穗期钙从叶片向籽粒转运的也增加。转录组分析表明gcsc1突变体叶片中木质素合成和甲酯化果胶降解等代谢途径中的相关基因表达发生了变化，造成细胞壁结合的钙离子减少，从而向籽粒转运增加。本项目通过QTL定位成功克隆了控制水稻籽粒钙含量基因GCSC1，阐明了GCSC1作为叶绿体Ca2+外排蛋白调控Ca2+从叶片向籽粒的再分配，对解析水稻中钙的迁移及其在籽粒中积累的分子机理具有重要意义，并为进一步通过分子标记辅助育种和基因编辑技术培育和创制籽粒高钙水稻品种提供有用新基因。