microRNA参与调控油松胚珠雌配子体游离核时期发育的机制研究

The development of the female gametophyte (FG) is one of the key processes of alternation of generations for gametophyte and sporophyte in plant, which decides the fate of the seeds in the future. It is well demonstrated that the free nuclear period of FG is crucial for the development of ovule. However, studies of molecular mechanism of ovule and female gametophyte FG development were mainly focused on angiosperms, such as Arabidopsis thaliana, investigation on gymnosperms remains to be further elucidated. In consideration of the insufficiency of previous investigations, this project mainly focuses on the regulation mechanism of ovules growth as their female gametophyte in free nucleus stage in female sterility (Clone 28), the Chinese pine ovule in seed orchard in Xingcheng Liaoning Province. First, miRNA libraries and degradome libraries were constructed from different periods of free nuclei division of FG from female sterility (Clone 28) ovule and the normal Chinese pine ovule to select and identify the special miRNAs and target genes, related to free nuclei division. Then we will analyze the relationship between the degradome and the transcriptom obtained from previous works, as well as the negative regulation of miRNAs on their target genes. In addition, Laser capture microdissection (LCM) will be employed to isolate the FG from the surrounding diploid tissues, and the spatial and temporal specificity of miRNAs and target gene's expression will be determined by means of single-cell sequencing coupled with total RNA extraction and linear amplification. Finally, we will further explore the key miRNAs and determine their biological function. Surely, the results of this investigation will lay foundation for the better understanding of the regulatory mechanisms of miRNAs involved in the functions of ovules growth and the abortion of clone 28, thus facilitate the theory of xylophyta developmental biology.

植物雌配子体的发育过程是植物细胞配子体和孢子体世代交替的关键过程之一，其中雌配子体游离核时期是胚珠发育关键环节。然而，目前已有胚珠发育的报道主要集中在拟南芥等被子植物上，在裸子植物中尚缺乏深入研究。针对前人工作的不足，本项目以辽宁兴城油松种子园中28号雌性不育系和可育系油松胚珠为材料，着重开展如下研究：(1) 构建雌配子体游离核分裂不同时期miRNA和降解组数据库，筛选并鉴定相关miRNA及其靶基因；(2) 分析降解组与前期获得的转录组信息的关系，探究miRNA对靶基因的负调控作用；(3) 利用激光捕获显微切割技术获得雌配子体，通过微量RNA提取和线性扩增以及单细胞测序技术，确定miRNA及其靶基因表达时空特异性；(4) 挖掘关键调控miRNA并进行生物学功能验证。本研究将为阐释油松胚珠发育的miRNA调控机制及28号雌性不育系败育的分子机理，丰富木本植物发育生物学理论的研究奠定基础。

雌性不育是植物界普遍存在的现象，会导致结实率下降，影响种质资源的繁殖和利用。因此，加强对植物雌性不育的研究对良种繁育和经济效益提高具有重要的意义，但目前的报道仅局限在拟南芥等被子植物，裸子植物中还未进行系统研究。本项目以油松28号不育系以及15号可育系游离核分裂前、中、后时期的胚珠为材料，着重开展如下研究：(1)构建雌配子体游离核分裂不同时期miRNA数据库，筛选并鉴定相关miRNA及其靶基因，探究miRNA对其靶基因的负调控作用；（2）运用基于生物信息学的多组学联合分析方法，辅以形态学、解剖学和分子生物学等研究手段，基于二代及三代转录组测序对差异基因进行功能分析进而对油松雌性不育系胚珠败育的分子调控机制展开研究；（3）检测相关基因在油松胚珠组织中的表达规律并锁定关键基因进行生物学功能验证、互作基因探寻及组织定位，深入揭示油松胚珠雌性不育的分子调控机制。以上研究在转录水平阐释了油松雌性不育的分子调控机制，并为裸子植物胚珠发育的深入研究提供了新的思路。