水稻PLA3介导miRNA调控种子萌发的分子机制研究

miRNAs are involved in the regulation of seed germination. In plants it is generally accepted that miRNAs mediate the cleavage of target gene mRNA, thereby regulating gene expression at the mRNA level and inhibiting seed germination. The research group found that the PLA3 gene affects the germination of rice seeds, and the PLA3 knockout mutant pla3-5 of the Nipponbare background was obtained using the CRISPR/Cas9 technique. The mutant has an eukaryotic type, and it has been confirmed through experiments that PLA3 and miRNA have Correlation. This project intends to use mutant pla3-5, PLA3 overexpressing plants and wild-type rice seeds as materials, using the Ribo-Seq new technology established by the research group for translation studies, combining Small RNA-seq, RNA-seq and iTRAQ proteomics technology studies how PLA3 mediates the germination of miRNA-regulated seeds. This project is expected to establish the Ribo-Seq technology system in rice, and at the same time enrich the molecular mechanism of miRNAs regulating seed germination. The results of this study are also expected to provide a theoretical basis for the effective regulation of "sprouting" in rice cultivation and seed production.

miRNA参与种子萌发过程的调控。在植物中普遍认为miRNA是通过介导靶基因mRNA的切割，从而在mRNA水平调节基因表达，抑制种子萌发。课题组发现PLA3基因影响水稻种子的萌发，且利用CRISPR/Cas9技术获得了日本晴背景的PLA3基因敲除的突变体pla3-5，该突变体具有易萌发表型，同时通过实验证实PLA3与miRNA具有相关性。本项目拟以突变体pla3-5、PLA3超表达植株和野生型水稻种子为材料，利用课题组建立的、可用于翻译组研究的Ribo-Seq新技术，结合Small RNA-seq、RNA-seq及iTRAQ蛋白质组学技术，研究PLA3以何种方式介导miRNA调控种子的萌发。本项目有望在水稻中率先建立Ribo-Seq技术体系，同时可丰富miRNAs调控种子萌发的分子机制。本研究成果也有望针对水稻栽培和制种中“穗发芽”的有效调控提供一定理论依据。

种子萌发是植物个体发育中最关键的时期，种子萌发一直是人们重点研究的问题。课题组以PLA3相关突变体为材料开展研究，结果表明与野生型相比，pla3-5突变体植株矮小，容易在母本上萌发；pla3-5突变体的胚在种子中所占比例较大；pla3-5纯合突变体大部分生长一周后就死亡，仅极少部分能够存活，且高度不育；pla3-5突变体比野生型更容易萌发。PLA3亚细胞定位结果显示PLA3定位于内质网。通过通过转录组测序、Small RNA及iTRAQ蛋白质组学分析，从中筛选出了影响水稻种子萌发关键的miRNAs及其具有调控关系靶基因，并对其进行表达验证，结果显示主要有2类小RNA分别为miRNA156和miRNA166，表达趋势与转录组测序数据一致。同时采用酵母双杂交实验，我们也筛选出可能与PLA3互作的基因，并将进一步验证。本项目还建立一种植物中miRNA检测新方法。综上所述，本项目通过挖掘PLA3调控种子萌发途径中的关键因子，建立PLA3与miRNAs在调控种子萌发的联系，初步解析PLA3调控水稻种子萌发的作用机制，对指导农业生产实践具有一定的理论价值和现实意义。同时，课题组建立的植物中miRNA检测新方法，可助推miRNAs的相关研究。