水稻光周期不敏感抽穗期基因Ghd5的克隆和功能分析

Rice grain yield is closely related to heading date.In our previous studies, one QTL affecting heading date, plant height and spikelets per panicle was mapped on chromosome 5 using a recombinant inbred population derived from the cross between Zhenshan 97 and Teqing. Here we name the QTL Ghd5. Comparison of heading dates of two Ghd5-NIL(near isogenic line) homozygotes under short day and long day conditions indicated that Ghd5 was insensitive to photoperiod. Ghd5 responsed to exogenous GA3 in field condition. In this project, we will plant a large near-isogenic F2 population of 5000 individuals and select about 800 extreme recessive plants with late heading date, tall plant height and big panicle to fine map Ghd5. Candiadates of Ghd5 are determined by gene annotation in the target region and comparative sequencing between two parents. In order to confirm the identity of Ghd5, Zhenshan 97 alleles of the candidate genes are introduced into NIL-Teqing by Agrobacterum-mediated transformation. We will discover its spatial and temperal expression patterns, identify its up-stream and down-stream genes and unveil the mechanism of its response to GA3 using several biological methods. Our targets are to establish the gene regulatory network of heading date and paincle differentiation regulated by Ghd5 and comprehensively understand the flowering pathways Ghd5 involved. In addition, we will make association analyses to identify the favorable alleles of Ghd5 for rice genetic improvement from 363 accessions with a wide genetic variation. Cloning and characterization of Ghd5 will make great contributions to elucidating their relationship between heading and panicle differentiation.

水稻抽穗期和谷物产量密切相关。前期，我们利用珍汕97/特青衍生的RIL群体在第5染色体定位到一个同时控制抽穗期，株高和每穗颖花数QTL，我们将它命名为Ghd5。Ghd5对光不敏感，Ghd5对赤霉素GA3产生响应。本申请将在此基础上，利用近等基因系F2大群体的极端隐性单株，采用图位克隆的方法将Ghd5精细定位到20 kb；生物信息学分析结合亲本比较测序，确定候选基因。同时利用转基因完成功能互补验证，确证分离该基因。利用分子生物学技术查明该基因的时空表达特点，鉴定上下游调控基因，揭示Ghd5响应赤霉素的调控机理。最终解析抽穗期和穗大小形成的分子机理，丰富我们对激素调控的水稻开花途径的认识。另外，我们将对国内外363份材料的Ghd5进行核酸多样性和关联分析，确定适宜于生产的优良等位基因，服务于水稻遗传改良。Ghd5的分离和机理分析，将对水稻抽穗和穗分化相互关系的研究具有重要意义。

水稻抽穗期和谷物产量密切相关。我们在以珍汕 97 作为轮回亲本, 特青为供体亲本的近等基因系群体中，发现一个主效QTLGhd5同时调控每穗颖花数、株高和抽穗期，Ghd5对光周期不敏感。我们利用近等基因系F2大群体的极端隐性单株，将Ghd5精细定位到20 kb，区间内包含之前克隆的Hd1；我们比较测序两亲本的Hd1的等位基因，发现特青等位基因在CCT结构域存在一个4 bp的缺失，造成移码，导致功能破坏。因此Ghd5很可能就是Hd1。我们获得了珍汕97背景携带特青Hd1等位型的材料的Hd1超表达，互补表达和基因敲除植株，发现开花期，株高和每穗颖花数性状都出现了期望的变化，表明Hd1就是控制这3个性状的基因。转录分析表明，在珍汕97背景下，Hd1总是上调Ehd1和Hd3a，从而总是表现促进开花。.但是，为什么Hd1在珍汕97背景下总是表现促进开花，与之前报道的长日抑制，短日促进开花不一致呢？我们之前发现Ghd7，Ghd8和Hd1之间存在遗传互作。珍汕97没有Ghd7和Ghd8功能等位基因，而明恢63则没有Hd1和Ghd8功能等位基因。Ghd7很可能是决定Hd1可变功能的因子。于是，我们分析明恢63背景的Hd1近等基因系，这个材料的Hd1表现出短日促进开花，长日抑制开花。为了提供更充分的证据，我们分别构建了珍汕97背景和明恢63背景的Ghd7和Hd1二位点纯合的4种基因型，进行长短日处理，结果表明的确是Ghd7的状态决定了在长日条件下的Hd1功能反转。Y2H和BiFC等多种分析手段证明Ghd7和Hd1蛋白互作。激素喷施试验表明Ghd5/Hd1响应赤霉素和细胞分裂素，延迟开花。我们对国内外328份栽培稻材料和41份野生稻材料的Ghd5/Hd1进行测序，发现Ghd5/Hd1核酸多样性丰富，有29个等位基因类型。另外，我们还克隆了与Hd1可能存在互作的Ghd7.1，发现野生稻中有功能的等位基因和随后产生的无功能新突变等位基因在栽培稻中共存。无功能等位基因使水稻种植区继续往高纬度地区扩张做出重要贡献。.本课题的研究表明Ghd7的状态决定Ghd5/Hd1的可变功能！合理利用这2个基因对水稻遗传改良具有重要意义。