拟南芥类受体激酶SRF在受精过程中的生物学功能研究

In flowering plants, the pollen tube is emerged from a pollen grain and grows to deliver the immotile sperms to the egg, during the journey of which it communicates with several types of the female reproductive tissues and cells. Recently, the signaling mediated by receptor-like kinases and signal peptides was found to play a key role in the cell-cell communication, especially in the male-female crosstalk. From the RNA-seq data analysis in our lab, a mass of receptor-like kinases and signal peptides are found to accumulate abundantly in the male or/and female tissues, however, only some of which are well studied. In this proposal, we plan to combine the advantage of molecular biology, cell biology and live-imaging approaches to uncover the transcript activity and subcellular localization of the pollen-expressed SRF4 and SRF5 receptors. CRISPR/Cas9 technology, genetic biology and cell biology will also be well used to study the biological function of SRF4/5 in plant fertilization. Moreover, we are also looking forward to make effort to identify the corresponding signal peptide for SRF4/5 by using biochemical/cell biological methods and genetic assays. The ultimate goal of this proposal is to determine the mechanism of SRF receptors as well as their corresponding signals in male-female communication during plant reproduction.

在被子植物的有性生殖过程中，作为精细胞运输载体的花粉管需要与雌方组织或细胞间进行大量的信号与物质交流，才能保证受精作用的顺利完成。近年研究发现，植物类受体激酶和小肽信号共同介导细胞-细胞间的相互交流，并在雌、雄组织或细胞之间的相互作用中扮演重要角色。申请人所在实验室发现大量RLK受体和小肽基因在受精过程中高量或特异表达，但仅有少数基因的功能被揭示。本申请拟通过利用分子生物学、细胞生物学和活体显微成像等手段，对功能尚未被报道、花粉管特异表达的SRF4/5两个受体进行深入研究，揭示其亚细胞定位和动态表达情况，并利用基于CRISPR/Cas9基因编辑技术、细胞生物学、遗传学等学科手段，系统分析并揭示SRF4/5在有性生殖过程中的生物学功能。此外，还将综合生物化学、分子生物学和遗传学等手段，探索SRF4/5受体可能识别的信号分子，最终全面解析SRF受体所介导的信号通路在植物有性生殖过程中的功能角色

在被子植物的有性生殖过程中，雌雄双方进行密切的信号交流来保证受精成功，类受体激酶在该过程中扮演关键角色。本研究发现拟南芥类受体激酶SRF (Strubbelig Receptor Family) 4和SRF5特异地在成熟花粉中表达。为探究其潜在的生殖调控功能，本研究采用CRISPR/Cas9基因编辑技术，获得了SRF4和SRF5敲除的双突突变体srf4 srf5，发现srf4 srf5突变体能够正常完成花粉发育和授粉受精过程，且srf4 srf5雄配子的传递效率与野生型雄配子相当。以上结果暗示，可能存在其他类受体激酶与SRF4和SRF5冗余地发挥功能，SRF4和SRF5也可能调控有性生殖相关的其他过程，如抵抗逆境胁迫或介导生殖隔离。本研究结果为探究类受体激酶SRF4和SRF5在拟南芥有性生殖中的潜在重要调控功能提供了新的启示。