水稻氮素利用主效QTL——qCR-8的克隆与功能研究

Nitrogen is one of the major factors contributing to crop growth and yield. Characterization and identification of novel genes controlling nitrogen-use efficiency can help facilitate not only the understanding of genetic mechanism for nitrogen nutrition, but the improvement of grain yield and quality by marker-assisted selection in rice breeding program. In the study, F2, CSSL and CSSL-BC2F2 populations were constructed using Nipponbare, a japonica cultivar and Kasalath, an indica cultivar as parents with significant difference in chlorate resistance. One major QTL, qCR-8 was preliminarily mapped within 1.0 cM region between two InDel markers on chromosome 8. So far, no gene involved in the nitrogen utilization was reported isolated in the region. In comparison with Nipponare, the CSSL-qCR-8 showed significant increase in chlorophyll a content, nitrogen concentration in panicle, grain yield and nitrogen-use efficiency (NUE). CSSL-BC3F2 and CSSL-BC4F2 populations are being constructed for further fine mapping and cloning of the QTL. The transgenic lines generated by complementation test, over-expression experiment and CRISPR/Cas9 system, together with NIL-qCR-8 will be used as elite materials for expression pattern analysis of qCR-8 and metabolism and utilization determination of nitrogen, which facilitate the exploitation of molecular mechanism underlies nitrogen efficient utilization and its value in rice breeding.

氮是限制植物生长和产量形成的首要因素，发掘和深入研究水稻氮素利用相关基因对解析氮素营养的遗传机制和分子标记辅助育种具有重要的理论和实践意义。本研究利用氯酸钾抗性差异显著的粳稻日本晴和籼稻Kasalath作为亲本，构建了F2、CSSL和CSSL为基础的BC2F2遗传群体，将氮素利用主效QTL——qCR-8定位在水稻第8号染色体上两个相距约1.0cM的InDel标记之间，该区间内至今未见氮素利用相关基因克隆的报道。CSSL-qCR-8的叶绿素a含量、穗部氮浓度、单株产量和氮素利用率较对照日本晴显著增加。在此基础上，本项目拟构建CSSL-BC3F2和CSSL-BC4F2大规模回交群体进行QTL精细定位和克隆，并用遗传互补、过表达、CRISPR/Cas9转基因材料和NIL-qCR-8开展基因表达模式和氮素代谢利用研究，以了解qCR-8调控水稻氮高效利用的分子机理，并挖掘其在育种领域中的价值。

硝酸盐（NO3-）的吸收和同化效率是影响水稻产量的关键因素，氯酸盐（ClO3-）抗性是水稻硝态氮同化效率的重要指标。在本项目中，根据籼稻（9311）和粳稻（日本晴）品种之间的ClO3-抗性差异鉴定到一些主要的数量性状基因座（QTLs）。通过精细定位和图位克隆，发现其中一个主效QTL（qCR8）编码NAD(P)H依赖型硝酸还原酶（NR）OsNR2。点突变分析发现，位于OsNR2 NAD(P) 结合域的精氨酸（Arg783）和色氨酸的差别是9311（籼稻）NR活性高于日本晴（粳稻）的关键。系统进化分析表明，籼稻和粳稻OsNR2等位基因间的差异是由定向选择驱动的。籼型OsNR2基因通过与编码NO3-转运蛋白的OsNRT1.1B基因的正反馈相互作用来促进NO3-吸收。籼型OsNR2可以提高粳稻的有效分蘖、产量和氮素利用率（NUE），该促进作用可以通过聚合籼型OsNRT1.1B等位基因而进一步加强。籼型OsNR2基因为粳稻产量的可持续增长提供了新的基因资源，对未来全球粮食安全具有重要意义。