Abstract:

【Objective】 To accelerate the breeding process of low-Cd-accumulating rice,【Methods】 the widely applied indica pure-line cultivars, Wushansimiao and Zhongzao 35, and the popular parents of hybrids, Huazhan and Wufeng B were used as materials to create OsNramp5 knockout lines via CRISPR/Cas9. The marker-free OsNramp5 mutants together with the wild types were grown either in a Cd-contaminated field (Field A) or in a non-Cd-contaminated field (Field B) for subsequent experiments. The changes of the grain Cd content were investigated for plants grown in both Field A and Field B. The changes of the contents of other related minerals were also examined but only for those plants grown in Field A. The agronomic changes of the mutants were further assessed by examining those plants grown in Field B. 【Results】 Rice lines with mutations at OsNramp5 were successfully generated for all the four cultivars. On one hand, the grain Cd level in OsNramp5 mutants grown in Field B was reduced by 85.5% on average (<0.02 mg/kg); as a result of OsNramp5 mutation, the Cd level in plants grown in Field A, was reduced by 94.8% (<0.1 mg/kg), meanwhile, the Mn level was decreased by 52.7%; on the contrary, the Cr level was elevated by 59.5% on average for the four cultivars and an increase of 79.1% in the Pb level was also observed but only in OsNramp5 mutants of Huazhan; the levels of other mineral elements including Fe, Cu , Zn, Ca, Se and As were slightly or hardly affected. On the other hand, a slight decrease in plant height and an average yield loss of 6.9% were observed due to OsNramp5 mutation for all cultivars; the yield loss could be ascribed to a reduction in seed setting rate and seed weight and was compensated by a slight increase in effective tiller number. 【Conclusion】 Low-Cd-accumulating rice plants can be generated by OsNramp5 disruption at the cost of an acceptable yield loss and the low-Cd-accumulating rice lines produced in the present study may serve as promising cultivars to eliminate rice Cd contamination in many areas.

Key words: indica rice, cadmium, genetic editing, breeding

摘要：

【目的】 为了尝试快速培育低镉籼稻,【方法】 选取广泛应用的杂交水稻亲本华占和五丰B以及常规品种五山丝苗和中早35为材料,通过CRISPR/Cas9技术创制OsNramp5基因突变株系,在镉污染及正常土壤中种植并测定突变株系籽粒（糙米）镉含量,其他相关元素含量亦同时在镉污染土壤种植条件下测定,在非镉污染土壤种植条件下考查OsNramp5基因敲除对农艺性状的影响。【结果】 成功获得了前述品种的OsNramp5基因敲除株系;非镉污染条件下种植的4个品种OsNramp5基因敲除株系籽粒镉含量低于0.02 mg/kg,平均较野生型降低85.5%;而在镉污染土壤种植时,不同品种OsNramp5基因敲除株系籽粒镉含量低于0.1 mg/kg,平均比野生型降低94.8%;锰含量也降低52.7%,铬含量增加59.5%,铅含量在华占中增加79.1%,而在其他品种中无变化;铜、铁、锌、钙、硒和砷含量（后4种元素只在华占及衍生品系中检测）受影响较小或不受影响;OsNramp5基因敲除株系株高、结实率和千粒重较野生型小幅降低,而有效分蘖略微增加,产量平均减少6.9%。【结论】 通过OsNramp5基因敲除,可以显著降低镉积累,但在某些种植条件下,代价为小幅产量损失;通过本研究获得的低镉OsNramp5基因敲除品系在镉污染地区具有较好利用潜力。

关键词: 籼稻, 镉, 基因编辑, 育种