水稻籼粳杂种不育Sa复合体的分子机理研究

Reproduction isolation caused hybrid male sterility between rice subspecies indica and japonica limits utilization of the strong heterosis of interspecific hybrids to further increase the yield in rice. Hybrid male sterility is controlled by nuclear genes and dozens of related loci have been mapped genetically, but only four genes have been cloned so far, including the complex locus Sa whose molecular mechanism will be further studied in this project. We aim to study the molecular functions of SaF+, SaF-, SaM+, and SaM- proteins encoded by the indica and japonica alleles of the two Sa genes, SaF and SaM, as well as their interactors to reveal the molecular mechanism of hybrid male sterility controlled by Sa locus. We have achieved some primary data on this project and plan to obtain following results by the funding support: 1) Providing direct molecular evidences of intercellular transport of SaF+ and SaM+ for "two-gene three-factor" model using immunohistochemistry. 2) Identifying the important region for interaction and subcellular localization with yeast two-hybrid and BiFC assays. 3) Establishing an interactom of SaF and SaM with yeast two-hybrid screening. 4) Knock-down or knock-out some interactors of SaF or SaM by RNAi or CRISPR-Cas9 system, to understand the biological function of these factors and mechanism underlying Sa-mediated hybrid male sterility.

由籼粳稻亚种间生殖隔离机制引起的杂种不育是扩大杂交组合，充分利用亚种间杂交优势，提高杂交稻产量的最大限制因素。水稻杂种不育主要受核基因控制，涉及几十个相关遗传位点，但目前实现分子克隆的只有4个，包括本研究涉及的Sa复合基因座。深入研究籼粳稻2个Sa基因SaF和SaM编码的四种蛋白产物SaF+、SaF-、SaM+和SaM-及其互作因子的分子功能，将有助于揭示籼粳杂种不育的发生机制。近几年我们在Sa研究中积累了一定新基础，试图通过本项目资助达成如下成果：1）通过免疫组化技术为Sa"双基因三因子"模型的SaF+和SaM+胞间转移提供直接分子证据。2）用酵母双杂交和双分子荧光互补技术确定影响SaF、SaM互作和亚细胞定位的重要区段。3）利用酵母双杂交文库筛选建立SaF和SaM互作蛋白的网络。4）借助基因沉默或敲除确定与SaF和SaM互作且具有生物学功能的因子，探索Sa基因控制杂种不育的分子机制。

水稻杂种不育是远缘杂种优势利用，提高杂交稻产量的最大限制因素，也是研究生殖隔离分子机制的重要模型。在国家基金面上项目（31471564）的资助下，本项目四年来深入研究了由Sa介导的籼粳杂种不育分子机理，完成了预期的研究内容，并取得了如下研究成果：1）构建了表达SaF+和SaM+标签融合蛋白的稳定转基因材料，并利用免疫组化技术验证Sa“双基因三因子”模型，但实验结果并不支持原有模型的假设；2）用酵母双杂交技术确定了SaF、SaM互作和亚细胞定位的重要区段并筛选获得了若干与SaF和SaM互作蛋白。利用双分子荧光互补技术对部分的互作蛋白进行再确认；3）利用体外纯化的融合蛋白，验证SaM+具有SUMO E3连接酶的生物学功能；4）借助RNAi基因沉默或CRISPR/Cas9敲除SaF和SaM获得了Sa位点亲和的新型育种材料；5）开发了研究蛋白与核酸互作的新技术方法，有利于后续研究工作的开展。本研究对其他类型的杂种不育具有重要的借鉴作用，所创制的杂种亲和材料，有望克服远缘杂种不育，实现强杂种优势利用。本项目执行期共发表论文6篇，申请专利3项，获得省部级奖励3项。培养硕士毕业生2名，博士毕业生2名，博士后1名，圆满完成了既定目标。