亚洲栽培稻与长雄野生稻种间杂种不育S44(t)位点的遗传机制研究

It is one of the accessible approaches to broaden the genetic basis in cultivated rice by utilizing the favorable genes from Oryza.longistaminata. But severe interspecific hybrid embryo abortion and pollen sterility hampered the effective utilization of the abundant genetic variations existed in O.longistaminata. Understanding the genetic mechanism of interspecific hybrid sterility is the key to exploit those rich diversity. S44(t) was a major locus controlling interspecific hybrid sterility between Oryza sativa and O.longistaminata, which was delimited to the region containing three candidate ORFs. In this study, complementation test、targeted gene mutation via the CRISPR-Cas9 system、GUS expression analysis and subcellular localization were used to elucidate gene function, moreover, gene –gene interactions were investigated by the cross. Thus, genetic component and gene number of S44(t) locus will be clear, and it will be elucidated how gene-gene interactions could result in hybrid sterility. This study will help to understand the genetic mechanism of interspecific hybrid sterility, and will provide reference to breeding of interspecific hybrid between Oryza sativa and O.longistaminata.

利用长雄野生稻中丰富的遗传变异是拓宽亚洲栽培稻遗传基础的有效途径之一，但亚洲栽培稻与长雄野生稻杂种胚败育及杂种F1花粉败育，致使长雄野生稻的育种利用十分困难。认识杂种不育基因的遗传机制是有效利用这些丰富变异的基础。S44(t)是一个控制亚洲栽培稻与长雄野生稻种间杂种不育的主效位点，定位区间内包含三个候选基因。本项目拟利用互补实验、CRISPR-Cas9基因敲除实验、GUS组化分析及亚细胞定位的方法研究候选基因的功能，并通过一系列遗传杂交分析基因间的相互作用，最终明确S44(t)位点引起杂种不育的基因类型、基因数量及基因间如何相互作用导致杂种不育。本研究有助于阐明S44(t)作用于种间杂种不育的遗传机制，并为亚洲栽培稻与长雄野生稻种间杂交育种奠定理论基础。

打破种间生殖隔离是拓宽亚洲栽培稻遗传基础的有效途径之一，也是利用水稻强大的种间杂种优势来突破产量途径的前提条件。项目组前期把控制亚洲栽培稻（RD23）与长雄野生稻（O. longistaminata）种间杂种不育位点S44(t)限定在3个ORF内，即ORF1、ORF2和ORF3。本项目在此基础上，继续深入研究S44(t)位点3个候选基因的功能。研究结果如下：利用转基因和CRISPR-CAS9的方法，明确了ORF1基因以等位抑制的方式参与杂种后代偏分离；证明了ORF2基因在杂种不育基因系统中承担“花粉杀手”的功能；来自长雄野生稻的ORF1等位基因作为“帮凶”基因，协助ORF2完成杀配子的任务；解析了ORF3基因在杂种不育系统中扮演“保护者”的角色。ORF2基因在小孢子各个发育阶段都有表达，而ORF3基因在小孢子发育后期有表达，二者均定位在细胞膜上，ORF2和ORF3基因的表达模式和亚细胞定位均与其功能相吻合。 本研究结果为进一步阐明S44(t)位点作用于亚洲栽培稻与长雄野生稻种间杂种不育的分子机制奠定基础，为创制S44(t)位点广亲和材料并培育种间杂交品种提供科学依据。在本项目的支持下，发表SCI论文3篇；项目负责人2018年度入选云南省“万人计划”青年拔尖人才称号；2020年度获“云南省中青年学术技术带头人”称号。