水稻雄性发育相关基因TP1的克隆及其影响花药和花粉发育的机制

Male-sterile germplasms have been widely used in hybrid rice breeding. Therefore, it is particularly important to study the male sterility of rice to increase the yield of hybrid rice. Although the predecessors have done a lot of research in this area, our understanding of the mechanism of rice pollen development is still insufficient. We previously isolated a new rice male-sterile mutant（namely tp1）, which had abnormal tapetum degradation and microspore development. Using map-based cloning method, we mapped the TP1 gene to a 73-kb interval. PCR products sequencing revealed a 1-bp substitution in the third exon of a gene encoding the acyl-CoA thioesterase (ACOT) in the mutant. This mutation induced a stop codon, which caused the protein encoded by the gene was greatly truncated. Using the CRISPR/Cas9 system of genome editing, we confirmed that the TP1 gene is required for male development in rice. qPCR results showed a strong expression of TP1 gene in anthers and young panicles, further implying that this gene has important functions in rice male development. It was reported that the ACOT family is involved in the bio-metabolism of lipid, however, the function of this TP1 gene has not been identified in rice. In this work, we intend to accurately identify the detailed abnormalities of the degradation of tapetal cell layer and pollen development in the tp1 mutant by applying cytological observations on their fine structures. We also intend to understand the possible lipid metabolism pathways of anthers that TP1 may participate in, via in vitro enzyme activity and Gas Chromatography Mass Spectrometry (GC-MS) analysis. We will use live cell imaging system to characterize the protein distribution of TP1. We also aim to explore the genetic relationship between this gene and other genes involved in the same process in rice by expression analysis, multiple mutation analysis and other methods. Overall, our research will reveal the role of the TP1 gene in rice male development, and may explore new genetic resources for expanding male-sterile germplasms of rice.

雄性不育种质资源早已在水稻杂交种子的生产中被广泛应用，因此研究水稻的雄性不育对于提高杂交水稻的产量就显得尤为重要。我们前期鉴定了一个新的水稻雄性不育突变体tp1，该突变体的绒毡层和小孢子花粉壁发育异常。应用图位克隆和基因编辑技术，我们克隆了该突变体的因果基因。该基因编码一个酰基辅酶A硫酯酶(ACOT)家族成员，并在幼穗和花药中优势表达，进一步暗示该基因在水稻雄性发育过程中有重要功能。据报道，ACOT家族蛋白可能参与生物中脂肪的代谢，但TP1在水稻中的功能是未知的。因此，本项目拟通过应用超微细胞学观察，明确突变体中绒粘层降解和花粉发育的具体缺陷；利用体外酶活鉴定和GC-MS了解TP1可能参与的花药脂肪类物质代谢通路；利用活体细胞成像技术探究该蛋白的分布特征；并通过基因表达、突变分析等方法探讨该基因与其它育性基因的遗传关系；以揭示该基因在水稻雄性发育中的作用，拓展水稻雄性不育系的基因资源。

雄性不育系的应用对杂交水稻育种至关重要。然而，水稻雄性发育的分子机制仍存在许多未知。在本研究中，我们分离到一个粳稻背景下雄性不育突变体tp1，与此同时也鉴定并得到一个籼稻背景下的等位突变体osgelp34-1。细胞学分析表明osgelp34-1突变体表现出花药壁层降解延迟和花粉发育异常，从而导致皱缩和无活力花粉粒的产生。基因克隆和CRISPR/Cas9分析证实TP1/OsGELP34的点突变是导致雄性不育表型的原因。. TP1/OsGELP34基因在生殖组织中高表达，编码一个假定的脂肪酶。OsGELP34蛋白定位于内质网( ER )，在陆生植物中保守。综上所述，我们的研究结果表明OsGELP34在水稻雄性生殖中起着至关重要的作用，在水稻杂交育种中具有潜在的应用价值。在项目执行期内，以第一标注发表研究论文3篇，培养硕士研究生2人，博士生研究生1人。