一个水稻粒型新基因GLW2-1的克隆与功能分析

Rice (Oryza sativa) grain shape is a key determinant of grain yield and market values. Although many grain shape genes have been cloned in recent years, the regulation mechanism of rice grain growth and development is still unclear. It is still necessary to cloning more grain shape genes to reveal the molecular mechanism of rice grain shape. A new QTL GLW2-1, that controls grain width and length was localized in chromosome 2 by using the RIL population constructed with extra large japonica rice TD70 and small grain indica rice Kasalath. The gene was mapped to a 25 kb physical interval by map-based cloning. The results of gene prediction and functional annotation indicated that cytochrome P450 might be a candidate gene for GLW2-1. This project verifies the function of the gene by transgenic complementation and gene editing. Identify the localization pattern of the gene by subcellular localization and tissue localization. Analysis of the regulatory relationship with GW2 by protein interaction and genetic effects detection. These findings will not only aid efforts to elucidate the molecular basis of grain development, but also facilitate the genetic improvement of rice yield.

粒型是决定稻谷产量和市场价值的关键因素。近几年已经克隆了不少粒型基因，但是水稻籽粒生长发育的调控机制仍然不清晰，需要挖掘更多粒型基因以揭示水稻籽粒形成的分子机制。前期利用特大粒粳稻TD70与小粒籼稻Kasalath构建的重组自交系群体在2号染色体上定位到一个控制粒宽和粒长的新QTL位点GLW2-1。通过图位克隆方法已将其定位在25kb的物理区间，基因预测和功能注释表明，编码细胞色素P450的基因可能是GLW2-1的候选基因。本项目拟通过转基因互补、基因编辑验证该基因；通过亚细胞定位、组织定位明确该基因的定位模式；通过与粒型基因GW2的蛋白互作及遗传效应检测，分析与粒型基因间的调控关系。研究将不仅有助于阐明水稻籽粒发育的分子基础，而且有助于水稻产量的遗传改良。

粒型新基因的克隆与功能分析对研究水稻籽粒调控机制具有重要的意义。尽管已经克隆了不少控制水稻粒型的QTL，但是水稻籽粒大小的调控机制仍然不清晰。本研究在利用大粒材料TD70与小粒材料Kasalath的重组自交系群体通过传统的遗传图谱定位到一个稳定的粒型QTL位点GLW2-1。利用重测序构建的高密度bin图谱精细定位得到4个候选基因。通过候选基因进行功能注释，及序列变异分析确定GLW2-1基因；通过亲本幼穗的转录组数据分析候选基因的表达模式。通过CRISPR/Case9对候选基因进行敲除验证，验证GLW2-1基因功能，并证实该基因对粒型是负调控作用；通过扫描电镜分析基因敲除后颖壳细胞大小的变化。构建近等基因系NIL-GLW2-1并比较不同基因的效应；通过幼穗不同时期的表达谱数据，分析目标基因在幼穗发育中的表达调控机制。本研究结果有利于进一步阐述水稻籽粒调控的分子机制。