水稻粒形基因GS9的克隆及其功能研究

Rice grain size affects not only rice yield but also rice quality. It is an effective way to breed rice cultivars with high yield and good quality by selecting rice with favorable grain size. In our previous work, a major QTL GS9 (Grain size 9), which can specifically regulate rice grain size, was identified from the chromosome segment substitution lines derived from a cross between a japonica cultivar Nipponbare (receptor) and an indica cultivar Qingluzhan 11 (donor). Study of the near-isogenic lines of gs9 in the Nipponbare background showed that gs9 allele from the donor could specifically increase the grain length, decrease the grain width and reduce the grain chalkiness, but without any negative effects on milled rice percentage and other qualitative or agronomic traits of rice, indicating that gs9 has the potential application value in future rice breeding. Meanwhile, our preliminary data indicated that plant hormones may participate in the regulation of grain size by GS9. . In this project, we are going to clone and analyze the sequences of the candidate genes in the target region based on the previous fine mapping result. Then, complementary test will be carried out to confirm the target gene of GS9. Further experiments including RNA-sequencing, protein-protein interaction assays, genetic interaction test and plant hormone regulation analysis will be performed to dissect the genetic effect and biological function of GS9. Also, the molecular mechanism and the regulation network program of GS9 will be revealed. In addition, the potential application of gs9 in breeding will be evaluated by introducing gs9 allele into the commercial rice varieties. Finally, the natural variation of GS9 will be identified in different rice germplasms. The results from this project will provide novel genes or gene resource for molecular breeding of elite rice cultivars by modulating grain size.

水稻粒形不但影响水稻产量，也与稻米品质密切相关。因此，选育优良粒形稻米品种是进行高产优质育种的一个有效途径。申请人从以粳稻日本晴为受体、籼稻清芦占11为供体的染色体片段代换系中鉴定了一个调控水稻粒形的主效QTL GS9。利用近等基因系证明来自清芦占11的等位基因gs9可特异性增加粒长、减少粒宽和降低垩白，对整精米率等其他加工品质和产量性状均无明显影响，具较好的应用潜力；同时发现植物激素可能参与GS9对水稻的粒形调控。本项目拟在前期精细定位GS9的基础上，对目标区域内候选基因进行克隆和测序分析，通过互补试验验证GS9的候选基因，并进一步从转录组学、蛋白互作、遗传互作、激素调控等角度系统分析其遗传效应和生物学功能，探明GS9调控水稻粒形形成的分子机制。同时，在主栽水稻品种中评价gs9的育种价值，鉴定不同种质资源中GS9的自然等位变异，为水稻粒形的分子改良提供优异的基因资源。

水稻是我国重要的粮食作物之一，高产与优质一直是水稻品种改良的最主要目标。水稻粒形不但影响水稻产量，也与稻米品质密切相关。因此，选育优良粒形稻米品种是进行高产优质育种的一个有效途径。前期研究中我们在以粳稻日本晴为受体、籼稻清芦占11为供体的染色体片段代换系中鉴定到一个调控水稻粒形的主效QTL GS9，并对GS9进行了精细定位。在此基础上，本项目重点围绕GS9的基因克隆、生物学功能解析及调控稻米粒形的分子机制开展系统研究，并取得了预期的研究成果；相关结果发表论文3篇，其中包括Nature Communications在内的SCI论文2篇；申请专利2项，其中有关GS9基因的专利获得授权；培养博硕士生5人，其中2名已毕业；项目主持人李钱峰入选江苏省双创团队（2017）、江苏省青蓝工程优秀青年骨干教师（2019）、江苏省六大人才高峰（2019）等。. 首先，利用图位克隆法顺利克隆到粒形调控基因GS9，通过测序明确其第二外显子上一段7Kb外源DNA片段插入是导致GS9基因突变的原因，后续功能互补实验确认GS9是籽粒细长突变体的目标基因。GS9突变导致籽粒变得细长，而其过表达则使籽粒变得短圆。GS9编码一个功能未知的蛋白，其通过影响细胞分裂调控籽粒粒形。利用酵母双杂筛库，鉴定到与GS9互作的OFP家族蛋白OFP8和OFP14。已知OFP8本身受到植物激素BR信号通路中GSK2激酶的调控，后续转录激活实验等证明OFP8和OFP14均可通过互作抑制GS9的转录活性。因此，初步建立了一个GSK2-OFPs-GS9协同调控籽粒粒形的分子模式，且GS9与BR信号通路之间存在着复杂的影响与调控。同时，通过创建gs9与gs3、GW5的聚合材料，明确gs9调控水稻粒形独立于gs3和GW5，代表一种籽粒粒形调控的新机制。鉴于gs9等位基因具有特异性增加粒长的表型，且可显著提高稻米的外观品质，因此，我们利用CRISPR/Cas9技术在武运粳8号、武运粳27号、2611等推广粳稻品种中敲除GS9，均获得籽粒细长、外观品质提高的优质材料，进一步表明gs9具有很好的育种利用价值。