叶锈菌侵染后小麦F-box家族基因表达模式及其互作靶蛋白的筛选

In the ubiquitin-mediated protein degradation pathway, F-box protein is a key component of ubiquitin-protein ligase (E3), which performs very crucial role of conferring specificity to the SCF complex for recognizing appropriate targets in many plant biological processes, such as growth and development, hormone signal transduction, resistance to the pathogens and abiotic stresses. At present, little is known about the F-box genes and their biological function in wheat.In our previous work, several F-box genes had been cloned.The project firstly looks for F-box genes expressed specifically in wheat leaves or stems by quantitative real time PCR, secondly, we further looks for the genes whose expression are induced by infection of wheat leaf rust pathogens by the same method mentioned above, finally, the project hopes to find out the downstream candiate proteins interacting with the selected F-box gene which is closely related with disease resistance by Yeast two-hybrid, and identifys the nature of the candidate protein. Aboving results will make clear the relationship of expression of F-box genes and wheat disease resistance/susceptibility,and obtain some information for the candidate proteins interacting with the F-box protein, thereby, lay the foudations for deeply understand- ing the biochemical network of F-box protein family in wheat disease resistance /susceptibility.

F-box蛋白是泛素介导蛋白质降解途经中泛素-蛋白连接酶E3的关键组份，因其专化性识别待降解目标蛋白而在植物生长发育、激素信号传导和抗病、抗逆境胁迫中发挥重要作用。目前小麦上关于F-box基因克隆及其功能研究的报道较少。本项目在前期获得小麦F-box基因的基础上，利用实时定量分析技术，首先找到在小麦叶片或茎秆中专化性表达的F-box基因；其次，找到受叶锈菌侵染后诱导表达的F-box基因；最后选择1条与叶锈菌侵染密切相关的F-box基因，利用酵母双杂交技术筛选与其互作的靶蛋白，并对靶蛋白进行鉴定。本项目可明确F-box基因的表达与小麦抗/感叶锈病性的关系，并获得与之互作靶蛋白的相关信息，从而为深入探讨F-box蛋白家族在小麦抗/感病过程中的生化网络奠定基础。

为了解小麦中F-box家族基因的存在状况及功能，本项目对其开展了系统研究。通过数据库检索，获得38条小麦F-box家族基因，主要归为三类：只强烈显示F-box结构域的序列15条，兼具F-box和LRR结构域的序列17条，兼具F-box和Kelch结构域的序列6条；根据这些基因序列设计了48对基因专化性引物，对TcLr15和中国春（CS）两个小麦材料苗期和成株期的不同组织部位cDNA进行扩增，共获得F-box家族基因片段20条，其中F-box/LRR重复蛋白基因 9条，只包含F-box结构域的基因 7条，F-box/Kelch基因4条；选取7条基因（TaSKP2A-1 、TaSKP2A-2、TaFBL14-1、TaFBL14-2、TaFB6、TaFK-2和TaFK-3）进行叶锈菌侵染和组织特性表达分析，5条基因可能参与小麦对叶锈菌的抗性防御，1条基因可能参与小麦对叶锈菌的感病过程，另两条基因对叶锈菌侵染无响应，7条基因均呈现品种和组织专化性表达。对TaSKP2A-1 和TaSKP2A-2在TcLr15和CS中受IAA、ABA和MeJA三种激素诱导后的表达模式进行了分析，TaSKP2A-1在CS中对三种激素的诱导均非常敏感，TaSKP2A-2在TcLr15中对三种激素的诱导比较敏感；构建了一个受叶锈菌侵染的CS小麦酵母双杂交文库，文库滴度为9.9×108 cfu/mL，插入片段分布范围为250bp～2500bp之间；构建了3个基因酵母诱饵载体（pGBKT7-TaSKP2A-1、pGBKT7-TaFK-2和pGBKT7-TaFK-3）。完成了pGBKT7-TaSKP2A-1的酵母文库筛选和候选靶蛋白验证。与TaSKP2A-1有相互作用的候选蛋白共8个，分别为小麦蛋白激酶、信号锚、铁蛋白、AZM、SKP1/ASK1、核酮糖-1,5-二磷酸羧化酶/加氧酶、Kunitz-type蛋白酶抑制剂、磷酸核酮糖激酶PRK。进一步验证SKP1/ASK1与TaSKP2A-1具有相互作用。TaSKP1/ASK1为泛素降解途径中泛素蛋白连接酶E3的重要组分，与F-box蛋白TaSKP2A-1一起组成E3的基本骨架，暗示着所获得的TaSKP2A-1可能在泛素介导的蛋白质降解途径中发挥作用。项目研究结果为深入解析小麦中F-box的功能和代谢网络奠定了基础。