水稻新型不育系武香S育性转换分子机理与应用基础研究

The status of two-line hybrid rice is becoming increasingly important for our country's rice production, but the molecular mechanism of its fertility alteration for photoperiod-thermo-sensitive genic male sterile line is still not very clear in two-line hybrid rice. To address this important issue, in this project, we propose to use new two-line sterile line Wuxiang S as experimental materials, and apply epigenetic analysis techniques combined with the cytogenetic methods, molecular genetics methods and other research methods. Specifically, the molecular mechanism of fertility alteration of Wuxiang S is studied, including the nuclear chromatin histone modification study at different developmental stages, genome-wide DNA methylation studies, transcriptome, miRNA research and proteomics research and so on. By investigating the phenotypic difference between the former and the post of fertility alterations, we can comprehensively and systematically reveal the occurrence of molecular events in the fertility alteration process involved in the two-line sterile line Wuxiang S, leading to the construction of regulatory networks of fertility alterations and the discovery of the key regulatory nodes and important related genes. The proposed research tasks are expected to provide experimental evidence for elucidating the molecular mechanism of fertility alteration in rice two-line sterile line. Meanwhile, we also aim to investigate a variety of ways for the utilization of two - line sterile line Wuxiang S; to explore the value-in-use of quality germplasm; to explore effective ways for applying two-line hybrid rice sterile lines as a series of multi-purpose applications in genetics and breeding; and to provide experimental evidence and theoretical foundation for the applications of "a series of multi-purpose" hypothesis. All these proposed research activities are expected to advance and provide guidelines for practical productions of the two-line hybrid rice.

两系杂交稻在我国水稻生产中的地位越来越重要，但对其光温敏核不育育性转换的分子机理仍然不十分清楚。为此，我们以新型两系不育系武香S为材料，拟利用表观遗传学的分析技术并结合有关细胞、分子遗传学等研究方法，对其进行育性转换的分子机理研究，包括开展不同发育时期染色质核小体组蛋白修饰研究、全基因组DNA甲基化研究、转录组学、miRNA研究以及蛋白质组学研究等， 寻找育性转换前和育性转换后的各种表现差异，从而较为全面系统的揭示水稻两系不育系武香S在育性转换过程中所涉及以及发生的分子事件，构建育性转换的调控网络，寻找关键的调控节点和重要的相关基因，为阐明水稻两系不育系育性转换的分子机理提供实验依据；同时，尝试两系不育系的多种利用途径，发掘优质种质资源的利用价值，探索杂交水稻两系不育系一系多用在遗传育种应用中的有效途径，为"一系多用"设想提供实验依据以及应用理论基础，有利于指导两系杂交稻的生产应用。

以两系不育系武香S为材料，对其育性转换的分子机理进行了较系统地研究，结果表明：（1）在武香S miRNA 表达谱分析中，共预测到373个新的miRNA分子，提出了一个依赖miRNAs 参与育性转换可能的调节途径；（2）对染色质核小体组蛋白修饰研究表明，在育性转换期H3K9ac和H3K4me2均主要富集于转录起始位点处。获得16,141个育性转换显著差异表达基因，以及开放染色质区域差异关键转录因子 688个；发现转录因子ERF141、 MADS7、MADS50和MYB它们构成一个非常复杂的调控网络，在花药角质层和外壁形成起关键作用，提出ERF141和MADS50 等转录因子作为UbL40 mRNA 的上游调控因子，对育性的转换进行调控的假设；（3）iTRAQ 定量蛋白质组学和转录组学的比较分析，发现在武香S育性转换期前后分别有65个和55个差异表达蛋白而在转录组也有相应的差异表达基因，它们形成cor-DEPs-DEGs 在武香S 育性转变过程中有重要的作用，共同构成蛋白-蛋白相互作用调控网络；（4）在lncRNA 表达谱和circRNA表达谱研究中，共鉴定出3948个lncRNA分子，选出622个差异表达lncRNA，其中97个lncRNA作为miRNA的合成前体参与转录后调控，89lncRNA能以内源竞争性伪靶标的方式通过lncRNA- miRNA-mRNA网络调控miRNA靶基因的表达，来调控武香S育性的转换；得到9994个候选circRNA分子，获得186个差异表达circRNA，预测了circRNA-miRNA-mRNA网络互作关系，获得15个差异表达circRNA参与ceRNA网络调控，从而调控花粉发育；（5）在与花粉育性相关的水稻PLL 基因家族和PPR 基因家族的研究中，鉴定了12个PLL基因，构建人工miRNA表达载体对OsPLL3和OsPLL4两个基因进行沉默，结果表明其沉默引发了花粉部分不育的现象；鉴定出491个PPR基因，预测49个miRNA和54个对应的PPR基因靶标，结合分析了武香S 的RNA-Seq结果，说明PPR基因与相应miRNA共同参与到水稻的花粉发育以及育性转换；（6）武香S已经提供给全国多家育种单位进行配组，本组育成武香S 四个新组合：武两优17、武两优16、武两优42、武两优73。.共发表SCI论文6篇，获得S新品种权一项。