一个新的水稻每穗粒数主效QTL Gn2的图位克隆和功能分析

Rice (Oryza sativa L.) is one of the most important staple food crops in the world and is consumed by more than 50% of the world′s population. Increasing rice grain yield will be required in order to feed the increasing global population in the next decades. High-yield molecular design breeding is considered as a vital approach to enhance rice production. Grain yield in rice is determined by three main components: panicle number per plant, grain number per panicle and grain weight. So the genetic information of these genes is the foundation for high-yield molecular design breeding. In our previous study, we constructed a backcrossing introgression line population using an indica cultivar, 8006, as the recurrent parent and a japonica cultivar, Wuyunjing 8, as the donor parent. One line of the population, ZY08126, exhibited significant decreased grain number per panicle compared with the recurrent parent. To uncover the genetic basis of this variation, a segregated population was developed by selfing the F1 plants of ZY08126 and 8006. A major QTL named Gn2 regulating grain number per panicle was detected out. QTL and genetic analysis indicated that Gn2 contributed 32.3% of phenotypic variation and acted as a single Mendelian factor in this population. Gn2 has been narrowed into a 83-Kb region in chromosome 2. In this project, we are going to carry out the fine mapping of Gn2 and a complementary test will be used to confirm the candidate gene. NILs, RNAi and overexpressed transgenic lines will be developed and employed to reveal the biological function of Gn2. In addition, the allelic variation of Gn2 in various rice varieties will be analyzed by DNA sequencing. These results will provide useful information for rice molecular improvement.

水稻高产分子设计育种是提高水稻产量的一条重要途径，而产量相关基因的定位、克隆和功能等遗传信息的获取则是高产分子设计育种的基础。申请人在前期工作中，从以籼稻8006为受体、武运粳8号为供体的高世代回交导入群体中筛选出一个每穗粒数比受体亲本显著减少的株系ZY08126，以此为材料构建衍生分离群体，鉴定出一个新的水稻每穗粒数主效QTL Gn2，现已将该基因精细定位在83kb距离内。本项目拟在已有研究基础上进一步精细定位Gn2，根据生物信息学预测、cDNA测序和相关表达分析确定候选基因；通过构建互补、过表达和RNAi载体验证候选基因的功能；利用基因功能分析软件，明确基因结构和蛋白质结构；利用Real-time PCR和GUS染色等手段分析基因表达的时空特性；通过GFP-Gn2融合蛋白进行亚细胞定位；通过组织切片解析Gn2调控的形态学和生理学基础。本研究为揭示水稻每穗粒数调控的分子机制提供重要信息。

本项目围绕水稻重要产量影响因子之一每穗粒数展开深入研究，取得的主要结果如下：.1、以日本晴为供体、广陆矮4号为受体，构建了一套染色体片段代换系，以其中85个单片段代换系为材料，进行水稻每穗粒数QTL的鉴定，共检测到27个每穗粒数QTL，对其进行重叠片段分析，有8个在2～4个单片段代换系中同时检测到，通过代换作图对其中15个QTL定位到更小的区间范围内，本研究中鉴定和定位的每穗粒数相关QTL为水稻每穗粒数基因的克隆、调控网络的解析以及分子育种的开展奠定基础；.2、以籼稻8006为受体亲本、粳稻武运粳8号为供体亲本构建了一套回交导入系群体，以其中一个株系ZY08126（主穗粒数约为190粒）为研究对象，将其与8006（主穗粒数约为245粒）杂交并自交，构建衍生的F2和F3 群体用于QTL定位和克隆，最终将目标基因Gn2限定在标记C15和S2-255之间约83kb的物理距离内；.3、从中花11迟抽穗品系中发现了一个突变体，株高显著降低，穗长变短，颖花数显著降低，籽粒明显变小，将该突变体命名为gns4。将突变体与9311杂交后自交获得F2群体用于基因定位和克隆，最终将目标基因定位在166.7Kb范围内，通过序列和表达分析克隆了目标基因，该基因编码一个细胞色素P450蛋白CYP724B1，该蛋白是BR生物合成中的一个关键酶。干扰和过表达分析表明，GNS4不仅控制着株高，还调控每穗粒数和籽粒大小，且GNS4过表达之后，表现出粒重增加以及单株产量提高的表型特征，具有潜在的育种价值。利用qRT-PCR技术对GNS4的表达情况进行了分析，发现GNS4在叶鞘中表达量最低，在穗中表达量最高。同时对BR生物合成和信号转导相关基因进行表达量分析，结果发现，4个BR生物合成相关基因和9个BR信号转导相关基因在突变体中的表达量均显著降低，但是，13个BR相关基因均未出现反馈调节作用。