枣树自交不亲和行为泛素降解途径关键基因鉴定及互作研究

Chinese jujube (Ziziphus jujuba) is an important fruit tree in China. Jujube have a good taste and rich nutrients, such as Vitamin C and sucrose. Although jujube is widely cultivated in China and brought great economical income to farmers, there is still lack of the theory and technology about jujube cross breeding. Because there is severely flower/fruit dropping and seedless in many jujube cultivars, it is very difficult to obtain seeds by artificial hybridization between different cultivars. Previously studies have revealed that gametophytic self-incompatibility (GSI) exists in Ziziphus jujuba and led to the seedless of many cultivars though cross-pollinated with others. In previous, we have identified the S-locus in the Z. jujuba ‘Junzao’, which was composed of pistil-specific S-RNase and pollen-specific SFB. However, there is still need to identify more critical genes in ubiquitin pathway/26S proteasome involved in GSI reaction, such as the component of SCF complex, and confirm their relationship. In this study, to investigate the function of S-RNase inhibiting growth of pollen tube in style, we first extract S-RNase from the style and co-cultured with pollens. We then construct a pollen cDNA library using the BD matchmaker library construction, and then use SFB as bait to screen the library for identify Skp using yeast two hybridization(Y2H). And then, we use the obtained Skp to test Cullin by Y2H again. We then use GST pull-down to test their interaction between Skp and SFB, Cullin, and therefore we almost find the component of SCF complex in jujube pollen. Finally, HA/c-Myc-tagged SFB and Skp were co-expressed in tobacco leaves (Nicotiana benthamiana) by agroinfiltration method, and then Co-Immunoprecipitation will be used to check the interaction between S-RNase and SFB associated with Skp. This research is aim to identify those genes involved in the ubiquitin pathway of GSI reaction in Z. jujuba, and try to characterize the molecular mechanism of GSI, which will provide the theory instruction for improving cross-breeding of Z. jujuba and maintain the sustainable development of jujube.

枣树是我国重要的经济果树。枣果种仁率低是枣树杂交育种的主要障碍，而配子体型自交不亲和（GSI）是导致枣果种仁率低的主要因子之一。枣树GSI行为中基于S-RNase泛素降解途径关键基因及其互作关系等尚未有研究。本研究在前期鉴定了枣树S位点（S-RNase和SFB）的基础上，拟通过离体试验验证柱头S-RNase对花粉管伸长的抑制效应，确定S-RNase是雌蕊中控制GSI的关键蛋白。构建花粉酵母cDNA文库，利用酵母双杂交筛选出SCF的亚基Skp-Cullin蛋白，进一步利用GST pull-down明确构成SCF复合体的Skp与SFB、Cullin的互作关系。同时在烟草中共表达SFB和Skp蛋白，利用免疫共沉淀分析枣树GSI中S-RNase与SFB、Skp的互作效应。本研究旨在深化对枣树自交不亲和分子机制的理解，为枣树杂交育种提供理论指导和实践依据。

枣果种仁率低是枣树杂交育种的主要障碍，而配子体型自交不亲和（GSI）是导致枣果种仁率低的主要因子之一。在GSI反应中，花粉SCF复合体与柱头S-RNase发生作用，导致S-RNase的泛素化降解，并最终引起SI的发生。本项目通过对枣树人工授粉观察授粉后花粉管生长状况确定了枣树中存在配子体型自交不亲和现象，对参与自交不亲和花粉SCF复合体各家族成员Skp和Cullin基因进行生物信息学和基因表达模式分析，筛选出在花粉中特异表达的基因家族成员，酵母杂交结果表明Skp基因和Cullin基因存在互作效应；对枣树自交和异交授粉后不同时间的花柱转录组测序分析，得到可能参与SI机制与钙信号途经相关、泛素降解相关的F-box基因等差异表达基因，该研究为进一步揭示枣树自交不亲和发生机制奠定了基础。