水稻低镉基因lcd的图位克隆及功能验证

Rice (Oryza sativa L.), as the most important food crop in China, is known to be strong cadmium (Cd) accumulator. Identification of rice cultivars with low Cd-accumulating abilities is a novel approach towards minimizing our dietary Cd intake, but little is known about the genetic and molecular mechanisms underlying Cd translocation and accumulation in rice plants. In this study, a low cadmium (lcd) mutant was isolated from EMS-generated rice mutant library (Oryza sativa L. indica, cv. 9311). Phenotypic and physiological characterization of the wild-type (WT) and mutant under Cd stress were investigated under solution culture. For the positional cloning of lcd gene, two F2 populations derived from rice inter-subspecific crosses, the lcd mutant/ japonica Nipponbare and the lcd mutant/ japonica Taizhong65 were constructed to isolate the lcd candidate genes by map-based cloning. The functions of the candidate genes were further validated by construction of complementary vector, RNAi vector, Promoter-GUS fusion vector and lcd-EGFP fusion vector of the candidate genes. These results will preliminarily illustrate the genetic and molecular mechanisms underlying Cd translocation and accumulation in rice plants, and provide a theoretical foundation for breeding of low Cd accumulation rice varieties and improving food safety in Cd-contaminated soils.

水稻是我国最主要的粮食作物，也是一种对重金属镉 (Cd) 富集相对较高的作物，降低水稻对镉的吸收和累积是提高我国稻米质量安全的关键之一，然而目前对水稻镉积累的遗传机理和相关分子作用机制还是缺乏深入了解。本项目拟以籼稻9311 EMS诱变筛选到的镉低积累突变体lcd (low cadmium) 为材料，通过苗期水培镉处理，对lcd突变体镉低积累特性形成的生理生态特征进行分析研究；在此基础上，以lcd突变体和粳稻日本晴、台中65杂交构建的F2群体为材料，采用图位克隆技术分离lcd候选基因；最后，克隆候选基因并构建候选基因的互补表达载体、沉默表达载体、Promoter-GUS和lcd-EGFP融合表达载体，对候选基因进行功能验证，初步阐明水稻镉低积累的遗传机理和分子作用机制，为今后进一步通过分子标记辅助育种等手段来培育低镉水稻新品种、提高我国粮食质量安全提供理论依据。

镉是影响我国稻米质量安全的首要污染物，开展水稻镉积累的遗传机理和相关分子作用机制研究，不仅有助于加强对作物重金属镉积累分子机理的理解，同时还可为今后进一步通过分子标记辅助育种等手段来培育籽粒镉低积累水稻品种提供分子手段。本研究中，以EMS诱变获得的镉低积累突变体lcd1和野生型9311杂交构建的F2群体为材料，lcd1糙米镉含量在不同生态环境下相比野生型均下降约97%，且根系对镉的吸收速率降低，根系可溶性组分中贮存的镉含量增加。遗传分析和基因定位结果表明，lcd1的低镉表型受隐性单基因控制，并最终将该基因定位于七号染色体8883713-8890474bp间，对应于OsNRAMP5（自然抗性相关巨噬细胞蛋白）的第七外显子。lcd1中OsNRAMP5编码框ATG下游2688核苷酸处C突变成T，导致根系对镉的吸收速率大大降低，进而导致糙米中镉含量大幅度降低。组织特异性和亚细胞定位结果显示OsNRAMP5基因主要在水稻根系成熟区和分生区中表达，在茎杆和叶片中表达量较低，说明OsNRAMP5基因主要在根系对镉的吸收中起着重要作用。GFP融合蛋白瞬时表达和Western Blot结果表明，lcd1突变体中OsNRAMP5-GFP融合蛋白和OsNRAMP5蛋白不同剪切体的表达量相比野生型均明显降低，说明该蛋白表达功能发生了一定程度的缺失，这可能是导致突变体镉吸收能力显著下降的主要原因。此外，lcd1中OsHMA3基因表达量相比野生型显著上调，将大量吸收的镉区隔化在根系可溶性组分，从而降低镉向地上部分的迁移。本研究结果初步明确了水稻镉低积累的遗传机理和分子作用机制，为镉低积累水稻品种选育提供理论基础。在项目资助下，发表国内外论文6篇，授权国家专利1项，参加国内学术会议交流2人次，培养硕士生1名。