无籽沙糖桔自交不亲和相关的泛素降解途径关键酶基因功能分析

Ubiquitin and 26S proteasome system (U/26S) is a selectively proteolytic system that targets the ubiquitylation and turnover of specific proteins. The U/26S has been shown to regulate the process of self-incompatibility (SI) in model plants. The U/26S starts with the activation of ubiquitin, a 76-amino acid small peptide, by a ubiquitin activating enzyme (UBE1). The activated ubiquitin is then conjugated to a second enzyme, called ubiquitin-conjugating enzyme (UBE2). E2 performs the role as an ubiquitin deliverer to E3 enzymes (UBE3) by either directly transferring the ubiquitin onto an E3 or in many cases, collaborating with an E3 to ligate the ubiquitin onto the ε-amino group of a lysine residue in the target. SI is one important factor that can result in seedless fruit in Citrus. However, the molecular mechanism of SI in Citrus is not clear yet. In our previous studies, UBE1, UBE2 and UBE3 related to U/26S were isolated from SI Wuzishatangju mandarin. Here, the function of UBE1, UBE2 and UBE3 will be analyzed and their roles in SI reaction of 'Wuzishatangju' will be elucidated. First, UBE1, UBE2 and UBE3 will be further identified from citrus genome and the full-length cDNA of UBE1, UBE2 and UBE3 gene will be cloned from SI 'Wuzishatangju'. Second, the effects of heterologous expression proteins of UBE1, UBE2 and UBE3 on pollen growth of 'Wuzishatangju' in vitro and in vivo will be analyzed. Third, functions of UBE1, UBE2 and UBE3 genes will be analyzed by transgenic technology. Last, we will prove whether the UBE1,UBE2 and UBE3 can interact with F-box and S-RNase using yeast two-hybrid and biomolecular fluorescence complementation (BiFC). The results would be important for the understanding of U/26S in SI response of Citrus.

在模式植物中，泛素/26S蛋白酶体途径的泛素活化酶E1（UBE1）、泛素结合酶E2（UBE2）和泛素连接酶E3（UBE3）通过调控信号传导元件泛素化并降解一些特异蛋白质而参与调控植物自交不亲和反应过程。自交不亲和是导致柑桔无籽的重要原因之一。目前柑桔自交不亲和机制还不清楚。在前期研究中，我们从自交不亲和品种无籽沙糖桔中获得了UBE1、UBE2和UBE3三个与泛素降解途径密切相关的基因，本项目拟阐明UBE1、UBE2和UBE3基因的功能及其作用机理：①进一步筛选与无籽沙糖桔自交不亲和相关的UBE1、UBE2和UBE3基因；②研究UBE1、UBE2和UBE3基因表达的外源蛋白对无籽沙糖桔离体花粉萌发和活体花粉管生长的影响；③通过转基因技术分析UBE1、UBE2和UBE3基因的功能；④酵母双杂交和荧光双分子技术（BiFC）明确F-box、S-RNase与UBE1、UBE2和UBE3之间是否互作。

1、 项目的背景. 在模式植物中，泛素/26S蛋白酶体途径的泛素活化酶E1（UBE1）、泛素结合酶E2（UBE2）和泛素连接酶E3（UBE3）通过调控信号传导元件泛素化并降解一些特异蛋白质而参与调控植物自交不亲和反应过程。自交不亲和是导致柑桔无籽的重要原因之一。目前柑桔自交不亲和机制还不清楚。.2、主要研究内容.1）从自交不亲和品种无籽沙糖桔中筛选与泛素降解途径密切相关的UBE1、UBE2和UBE3基因；.2）UBE1、UBE2和UBE3基因的组织表达表达特性研究；.3）研究UBE1、UBE2和UBE3基因表达的外源蛋白对无籽沙糖桔离体花粉萌发和活体花粉管生长的影响；.4）通过转基因技术分析UBE1、UBE2和UBE3基因的功能；.5）酵母双杂交和荧光双分子技术（BiFC）明确F-box、S-RNase与UBE1、UBE2和UBE3之间是否互作。.3、重要结果和关键数据.1）从自交不亲和品种无籽沙糖桔和自交亲和品种获得了与泛素降解途径密切相关的UBE1、UBE2和UBE3基因的cDNA和DNA序列，分别命名为 CrUBE1, CrUBE2和CrUBE3。.2）CrUBE1, CrUBE2和CrUBE3基因在花粉中的表达量显著高于其他组织，且在无籽沙糖桔花粉的表达量显著高于在沙糖桔花粉的表达量。.3）成功在毕赤酵母表达CrUBE1, CrUBE2和CrUBE3基因，CrUBE1, CrUBE2和CrUBE3基因表达的外源蛋白对无籽沙糖桔离体花粉萌发和活体花粉管生长的有显著抑制作用。.4）对转WUBE1基因烟草进行了分析结果表明，转WUBE1基因的自交授粉组合花粉管在生长过程中，部分花粉管出现停止生长的现象，到达花柱基部的花粉管数量少于异交授粉和野生型自交组合。转WUBE1基因烟草的花粉生活力、发芽率、自交和异交后平均每个果荚中的种子数与野生型烟草无显著差异。这些结果表明，单独的一个WUBE1基因不能调控无籽沙糖桔自交不亲和反应，它很可能通过Ub/26S途径参与了无籽沙糖桔自交不亲和反应。.5）沙糖橘F-box基因与沙糖橘UBE2基因产生了互作，而与无沙糖橘UBE2基因未产生互作；无籽沙糖橘F-box基因与无籽沙糖橘和沙糖橘UBE3基因均产生了互作。.4、科学意义. 该结果为进一步研究柑桔自交不亲和发生机制奠定了基础。