SPL成员在乙烯诱导菠萝成花不同阶段的作用机制及信号网络解析

Exogenous ethylene can significantly induce flowering transition of pineapple (Ananas comosus), and has been widely used in the production of pineapple . SPL gene family play an important role in the flowering process. However, the roles of SPLs during the process of ethylene induced pineapple flowering transition are not yet clear. The project preliminary studies showed that the expression of 5 members of AcSPL reach the peak at different stages of flower induction and development. This project is trying to identify the function by analyzing the subcellular localization and transcriptional activation activity of AcSPLs; and by obtaining the overexpressed or interfered transgenic plants of pineapple callus (isogenesis) and Arabidopsis (ectogenesis) ; Simultaneously, to analyze whether SPL14 and SPL16 react to ethylene; whether SPL4, SPL8 and key floral genes such as FT exist upstream and downstream relations; whether SPL10 regulate the key genes of floral organ such as WUS by using yeast one-hybrid and EMSA technique. Meanwhile, the yeast two-hybrid libraries are constructed to screen and identify interacting protein of AcSPLs. Building signal network of different AcSPLs at different stages of flower induction, will help build a foundation for further clarity the mechanism of AcSPLs work during pineapple respond to ethylene signal, and provide important theoretical support for the flower forcing technique improvement in pineapple production.

外源乙烯可显著诱导菠萝的成花转变，且在生产上已广泛应用。SPL基因家族在植物开花过程中扮演重要角色，但其在乙烯诱导菠萝成花转变过程中的作用机制尚不明确。前期研究表明：菠萝5个SPL成员分别在乙烯诱导成花的不同阶段表达量急剧增加。项目拟对5个候选SPL成员进行亚细胞定位和转录激活活性分析，同时构建超表达和RNAi载体转化菠萝愈伤组织（同源）和拟南芥（异源）进行功能验证，观察其成花效应；利用酵母单杂交和EMSA等实验，对早期SPL14和SPL16是否响应乙烯、中期SPL4和SPL8与FT等成花关键基因上下游关系、晚期SPL10是否调控WUS等花器官形成关键基因等进行分析，同时构建SPL不同成员的酵母双杂交文库筛选鉴定与其互作的蛋白，构建SPL不同成员在乙烯诱导菠萝成花不同阶段的信号网络，为进一步阐明SPL基因在乙烯诱导菠萝成花过程中的作用机制奠定基础，并为生产中菠萝催花技术改进提供理论支持。

菠萝生产中仍常有因催花质量不佳造成经济损失的现象，特别是近几年国内大力引进的一些菠萝新品种也存在催花难的问题，但目前解决这一问题的理论依据仍比较匮乏。前期通过乙烯催花处理后芽的表达模式分析，筛选出5个关键SPL转录因子可能在乙烯诱导菠萝成花中起着关键作用。本项目通过亚细胞定位分析明确5个AcSPLs成员均在定位于细胞内，转录激活活性分析表明5个AcSPLs成员基因均具有转录活性。转化拟南芥进行功能验证，发现5个AcSPLs成员均具有促进拟南芥提早开花的作用，其中AcSPL16的早花表型最为明显。AcSPL16同源转化菠萝发现其也具有促进菠萝提早开花的功能。AcSPL4和AcSPL16酵母双杂文库筛选得到大量互作的蛋白，酵母双杂交点对点验证发现AcSPL4与ERF72和CRF4存在互作关系，AcSPL16与PR4和ERF72存在互作关系。酵母单杂交点对点验证发现AcERF72结合到AcSPL16的启动子调控其表达。这些结果表明乙烯信号可能通过AcSPLs不同成员来调控成花。同时通过对AcSPLs成员蛋白互作网络预测可能互作的蛋白，并对ERF，MADS，BBX家族进行了分析，初步锁定可能与AcSPLs互做的关键候选蛋白。本研究通过多种手段解析了SPL基因不同成员在乙烯诱导菠萝成花过程中的作用机制和调控网络，不仅有助于阐明乙烯诱导菠萝成花的分子机制，也为菠萝成花调控技术改进及新品种选育提供理论支持。