ARF3非细胞自主性调控拟南芥雌性生殖细胞命运决定的机理研究

植物体内存在高度精密的调控机制，准确地“指定”极少数的体细胞特化为生殖细胞。大孢子母细胞MMC的形成代表着体细胞向雌性生殖细胞的转变，标志着雌性生殖细胞的起始，其形成的分子机理还不清楚。我们在前期的研究工作表明tasiR-ARF通过靶向调控生长素响应因子ARF3在胚珠原基体细胞中的极性分布，介导了生殖细胞MMC的特化。本项目将深入揭示ARF3调控MMC特化的分子机制：①通过基因融合表达、northern 杂交及原位杂交技术检测tasiR-ARF在胚珠中的表达模式；②驱动ARF3在胚珠原基不同细胞层内的异位表达，揭示ARF3非细胞自主性调控MMC特化的机理；③通过比较转录组、ChIP-测序技术及生物学实验鉴定ARF3下游调控基因，构建ARF3调控网络。本项目将挖掘更多的参与生殖细胞命运决定的基因，为解答有性生殖过程中生殖细胞命运决定、体细胞与生殖细胞的互作等关键科学问题提供重要的参考依据。

The specification of the MMC is of critical importance as it is the first committed cell of the female germline lineage. In light of this, how signaling among somatic cells, or between somatic cells and the potential MMC, occurs to ensure the specification of only one MMC is of much interest. In the previous, we found the polar distribution of ARF3 in ovule primodium mediated by tasiR-ARF controlled the MMC specification. This project intends to reveal molecular mechanism of ARF3 regulates reproductive cell fate determination in Arabidopsis. We will ①apply fusion expression system，small RNA northern blot and small RNA in situ hybridization to study the expression pattern of tasiR-ARF in ovule; ②adopt ectopic expression of ARF3 driven by different promoter to evaluate the non-cell-autonomous regulation of ARF3 on MMC specification; ③use RNA sequencing and chromatin immunoprecipitation- sequencing technology to screen ARF3 downstream genes and construct ARF3 dependent-signaling networks. Our finding will excavate more gene which are involved in MMC formation and shed light on the study of germ cell fate decision, gametes initiation and somatic cell-germ cell interactions.