Table_2_Mutation at Different Sites of Metal Transporter Gene OsNramp5 Affects Cd Accumulation and Related Agronomic Traits in Rice (Oryza sativa L.).docx

OsNramp5 is a key gene involved in the control of the uptake of Cd, Mn, and other metal ions by rice root cells. The functional deficiency of this gene can significantly reduce the accumulation of Cd in rice grains, but the effects of its mutation on agronomic traits such as yield and quality have not been investigated comprehensively yet. In the present study, three Huanghuazhan-based OsNramp5 mutants [LCH1 (Low Cadmium Huanghuazhan 1), LCH2 (Low Cadmium Huanghuazhan 2), and LCH3 (Low Cadmium Huanghuazhan 3)] were obtained using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. The mutation-type analysis showed that LCH1, LCH2, and LCH3 encoded defective OsNramp5 protein sequences containing only 76aa, 176aa, and 266aa, respectively. The determination of metal content and the statistics of related agronomic traits revealed that the functionally deficient OsNramp5 not only significantly reduced the accumulation of Cd in the grains of the mutants but also affected rice yield and quality. However, with the decrease of OsNramp5 mutation degree, its effects on chlorenchyma Mn accumulation, yield, and quality were also diminished. Additionally, we also found that the increase in the concentration of Mn in the soil restored the phenotype of the declined yield and quality due to the functional deficiency of OsNramp5. Our findings provide novel insights into and new materials for breeding rice varieties with low Cd accumulation and excellent agronomic traits under severe Cd pollution environment.

OsNramp5基因是调控水稻根系细胞吸收镉（Cd）、锰（Mn）及其他金属离子的关键基因。该基因功能缺失可显著降低稻米籽粒中镉的积累量，但其突变对产量、品质等农艺性状的影响尚未得到全面研究。本研究利用成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）技术，获得了3个以黄华占为遗传背景的OsNramp5突变体[LCH1（低镉黄华占1号，Low Cadmium Huanghuazhan 1）、LCH2（低镉黄华占2号，Low Cadmium Huanghuazhan 2）及LCH3（低镉黄华占3号，Low Cadmium Huanghuazhan 3）]。突变类型分析表明，LCH1、LCH2与LCH3所编码的缺陷型OsNramp5蛋白序列长度分别仅为76个氨基酸（aa）、176个氨基酸及266个氨基酸。金属含量测定与相关农艺性状统计结果显示，功能缺失型OsNramp5不仅可显著降低突变体籽粒的镉积累量，还会对水稻产量与品质造成负面影响。然而，随着OsNramp5突变程度的降低，其对叶肉组织锰积累、产量及品质的影响也随之减弱。此外，本研究还发现，土壤中锰浓度的提升可恢复由OsNramp5功能缺失所导致的产量与品质下降表型。本研究结果为镉污染严重环境下培育低镉积累且农艺性状优良的水稻品种提供了全新的研究思路与种质材料。