植物RNA结合蛋白LSM调控种子成熟的分子机制研究

The postembryonic growth and development, as well as the crop yields of the higher plants, all depend on the embryogenesis. The storage reserves accumulated in the late embryo during seed maturation are the basis of the plant postembryonic development and the main source of the human food. Therefore, the study of plant seed maturation has important theoretical significance and application value. Based on the preliminary findings, RNA-binding protein LSM plays important roles in the seed oil and seed storage protein accumulation. And the seed maturation of the lsm mutant showed severe defects and the lsm seeds are wrinkled. These findings suggested that LSM plays important roles on the embryogenesis during seed maturation stage. The present project intends to analysis the function of LSM in the seed maturation, the molecular and genetic mechanisms of the LSM in regulating the seed maturation, by comprehensively using the technology of the cell biology, molecular biology, genetics, biochemistry and seed biology, on the basis of the preliminary findings; and to screen and identify the interactional proteins and target genes of LSM through the methods of yeast two-hybrid, affinity purification, BiFC, RNA-Seq and so on; to elucidating the molecular network of the RNA-binding protein LSM-mediated post-transcriptional regulation of gene expression in the regulation of seed maturation. The final results obtained in the proposed research will clarify a novel mechanism regulating the plant seed maturation and provide the theoretical basis and genetic resource for molecular breeding of crops.

高等植物胚后的生长发育以及作物的产量依赖于胚胎发生，晚期胚胎发生即种子成熟过程中所积累的贮藏物质是植物胚后生长发育的基础和人类粮食的主要来源，因而研究植物的种子成熟具有重要理论意义和生产应用价值。本课题组在前期研究结果中发现，RNA结合蛋白LSM不仅参与种子油脂和贮藏蛋白的积累，并且突变体的种子皱缩，其种子成熟过程受到了明显抑制，表明LSM在种子成熟时期参与了植物胚胎发生的调控。本项目拟在前期研究的基础上综合运用细胞生物学、分子生物学、遗传学、生物化学和种子生物学等技术深入分析LSM在植物种子成熟中的功能，解析LSM调控种子成熟的分子遗传机制，并利用酵母双杂交、亲和纯化、BiFC、RNA-Seq等方法筛选和鉴定LSM的相互作用蛋白与靶基因，以期深入阐明LSM调控种子成熟的分子调控网络。项目的实施将阐明一种新的植物种子成熟调控机制，同时也能为农作物的分子设计育种提供理论依据和基因资源。

Pumilio蛋白是真核生物中一类高度保守的具有Pumilio同源结构域的RNA结合蛋白，目前有关植物Pumilio蛋白的功能和作用机理还不清楚。本项目以拟南芥为研究对象，对植物Pumilio蛋白基因APUM24（即LSM）在种子发育中的功能及其在调控种子成熟过程中作用机制进行了研究。研究结果表明APUM24在种子成熟过程中高表达， APUM24定位于细胞核中；APUM24的突变体apum24-2呈现出种子变小及皱缩等表型，APUM24过表达能产生较大的种子；apum24-2突变体种子成熟受阻，油脂含量明显降低，油脂合成相关基因的表达量明显下调，但是WRI1的表达量不受影响；遗传学关系分析发现，apum24-2wri1-4双突变体中产生与apum24-2类似的种子表型；APUM24过表达不能恢复wri1-4种子皱缩的表型，但是WRI1过表达可以恢复apum24-2种子皱缩的表型；此外，在apum24突变体中WRI1的转录后剪接方式不受影响，但是WRI1的蛋白水平明显降低，蛋白稳定性实验结果显示apum24-2突变体中WRI1的蛋白稳定性受26S蛋白酶体降解所调控，表明APUM24可能通过WRI1调控种子的成熟； CULLIN3的E3连接酶适配器BTB / POZMATH（BPM）基因在apum24-2突变体中明显上调，在APUM 24过表达植物中明显下调。RNA Immunoprecipitation（RIP）实验结果表明APUM24可以与BPM基因的mRNA的3’UTR结合调控BPM基因mRNA的稳定性；在apum24-2突变体中miroRNA敲减BPM（6xamiBPM）和CUL3（2xamiCUL3）的表达可以部分恢复apum24-2突变体的皱缩的种子表型、油脂合成相关基因的表达、种子贮藏蛋白和油脂含量；APUM24蛋白中PUF结构域的核心氨基酸突变后的过表达不能恢复apum24-2突变体的皱缩种子表型，也不能产生比WT大的种子，表明PUF结构域对APUM24调控种子成熟是必需的；APUM24蛋白通过与BPM基因mRNA的3'UTR中预测的保守的pumilio响应元件结合，调控BPM的表达，进而影响WRI1的蛋白水平从而调控种子的成熟。本项目初步阐明了APUM24在拟南芥种子成熟中的关键作用和机制，对提高作物种子品质和产量具有重要意义。