绿色草莓S-RNase基因的表达调控及其介导的GSI中修饰子的鉴定

蔷薇科苹果、梨和核果类等果树配子体自交不亲和（GSI）机制的研究较深入，但同为蔷薇科的草莓属植物尚处于起步阶段；草莓属植物是基于S-RNase介导的GSI，花柱特异表达的S-RNase是GSI花柱端决定子，其特异性表达机制与蔷薇科其他物种一样尚未明晰。以自交不亲和二倍体绿色草莓为对象，以自交亲和二倍体草莓物种为参照，通过S-RNase基因启动子序列分析获得特异顺式作用元件、与启动子特异结合转录因子筛选与鉴定、与特异转录因子协同作用LncRNA筛选揭示S-RNase基因特异性表达的调控机制；从花柱、花粉酵母双杂交文库筛选出S-RNase的互作子，利用BiFC、Pull down以及在绿色草莓中基因过表达、CRISPR敲除等手段确认这些互作子与S-RNase的互作关系及其在GSI中的功能，分别鉴定出GSI反应中与S-RNase互作的花柱和花粉端的修饰子，探索草莓GSI特异性机制。

The mechanism of gametophytic self-incompatibility (GSI) in tribe Maleae and genus Prunus from family Rosaceae has been studied in depth. The self-incompatibility of genus Fragaria was clearly defined as GSI system, but only a little has been made on clarifying mechanism of this GSI. S-RNase-based GSI has been revealed in genus Fragaria SI species, and style-specific expression of S-RNase gene is the requirement as style determinant, but the mechanism of specific expression is still unclear in genus Fragaria as well as other family Rosaceae. Based on our previous study, the objective of this project is to identify the mechanism of style-specific expression of S-RNase gene and the modifiers involved in S-RNase-based GSI process of diploid Fragaria viridis (SI). By analyzing the activity of different truncated sequences of the S-RNase gene promoter, a key promoter-specific expression segment is obtained. The style-specific cis-acting elements are predicted by referring to the promoter of the self-compatible diploid strawberry species in combination with regulatory segment of the style-specific expression. And then it’s subjected to point mutation and combination mutation, and the function of the cis-acting element is analyzed according to the change in expression activity to obtain the promoter-specific cis-acting element of the S-RNase. The candidate transcription factor genes are screened from cDNA library by yeast one-hybrid system. We will identify the specific transcription factor function through overexpression, and knockout of genes in F. viridis species. LncRNAs related to the regulation of S-RNase expression are obtained by two methods including transcription factor RNA co-immunoprecipitation (RIP) and locating lncRNAs expressed by the non-coding sequence of S-RNase. The RNA pull-down assay is used to analyze the interaction between lncRNAs and S-RNase-related transcription factors. Taking S-RNase and transcription factors as reference，the expression pattern of all obtained lncRNAs are inspected by RT-qPCR, and then the correlation between the three will be further confirmed. Combining the function of transcription factors, we will obtain a synergistic regulation pattern of lncRNAs and transcription factors to S-RNase gene, and expression mechanism of S-RNase will be finalized. The S-RNase interactors are screened from the style and pollen yeast two-hybrid libraries. The assays of BiFC, Pull down, subcellular localization, and in vitro fluorescence co-localization are used to align them with the authenticity of the S-RNase interaction and the location of the occurrence. Overexpression and knockout of genes are used to verify the functional role of the interactor and further to clear which interactors are real modifiers involved in S-RNase-based GSI process. Therefore, the exploration of the strawberry-specific GSI mechanism aims to enrich the diversity of GSI in the family Rosaceae and provide clues for the evolution of genus Fragaria.