【目的】水稻粒形为多基因控制的复杂数量性状,同时影响稻谷产量和稻米外观及碾磨品质,挖掘粒形相关基因并解析其遗传机制,对于水稻高产和优质品种选育具有重要意义。【方法】以前期利用大粒籼稻品种特大籼(TDX)为供体亲本、小粒粳稻品种日本晴(Nipponbare,NPB)为轮回亲本获得的一个大粒近等基因系,在水稻第2染色体上初定位粒形调控基因GS2.2(grain size 2.2)的基础上,对GS2.2基因进行了精细定位和候选功能基因的克隆鉴定。【结果】利用BC₄F₂群体中2887份极小粒单株及该群体中70份基因型杂合的大粒单株自交获得的BC₄F₃群体,将GS2.2基因精细定位在2M-7-1与2M-9之间的160.6 kb的区间内。该区间编码18个基因开放阅读框(ORF1-18)。测序发现ORF18基因在大粒近等基因系与小粒日本晴等位基因间存在5个SNP差异,ORF18编码生长素调控因子蛋白OsGRF4。采用CRISPR/Cas9基因编辑技术对大粒近等基因系中ORF18进行敲除,发现ORF18敲除株系粒长变短,证实ORF18是GS2.2的功能基因。进一步对ORF18大粒近等基因系和基因编辑敲除株系进行稻瘟菌室内离体和病圃接种鉴定,发现ORF18大粒近等基因系稻瘟病抗性增强,而基因编辑敲除株系稻瘟病抗性减弱,表明ORF18正调控水稻稻瘟病抗性。【结论】本研究发现的生长素调控因子OsGRF4协同调控水稻粒形和稻瘟病抗性,为协调水稻高产和抗性育种提供了重要的基因资源。

【Objective】Rice grain shape is a complex quantitative trait controlled by multiple genes, which affects rice yield, rice appearance and milling quality simultaneously. It is of great significance to explore the genes related to grain shape and elucidate its genetic basis. Previously, a gene, namely GS2.2, which controls rice grain size, was mapped on the chromosome 2, by using a set of near isogenic lines derived from the small-grain japonica rice variety Nipponbare (NPB) as the recurrent parent, and an indica rice variety Tedaxian(TDX) as the donor parent. 【Method】In this study, fine mapping of GS2.2 and identification of candidate functional genes were carried out. 【Result】 GS2.2 was fine mapped to the 160.6 kb interval between the makers of 2M-7-1 and 2M-9, by using a BC₄F₂ population with 2887 small grain size plants and a BC₄F₃ population with 70 large grain size plants. Bioinformatics analysis showed that the 160.6 kb chromosome interval encodes 18 open reading frames (termed as ORF1-18, respectively). Sequence analysis showed that there were five SNP differences in ORF18 between the large grain near isogenic line and Nipponbare. ORF18 encoded an auxin regulatory factor protein OsGRF4. The grain length of ORF18 knockout lines, whose ORF18 was knocked out in large grain near isogenic lines by using CRISPR/CAS9 gene editing technology, became smaller. The above results confirmed that ORF18 was a functional gene of GS2.2. Further identification of ORF18 near isogenic lines (ORF18-NIL) and gene editing knockout lines by in vitro inoculation and disease nursery inoculation showed that the rice blast resistance of ORF18-NIL increased, while that of gene editing knockout lines decreased, indicating that ORF18 also positively regulates rice blast resistance. 【Conclusion】The auxin regulator OsGRF4 found in this study simultaneously regulates rice grain shape and blast resistance, which provides an important gene for breeding rice variety with high yield in coordination with enhanced resistance.