FT/TFL1基因家族调控柑橘干旱开花的分子机制

The majority of citrus ripening periods are concentrated from October to December, resulting to greater pressure on market sales, storage and processing. In the area of rainy season and dry season, lemon and grapefruit can be blossomed after the dry season, and the flowering time of kumquat and calamondin is also controlled by water stress during citrus cultivation and production process. Therefore, the drought can affect the flowering of some citrus trees and the ripening period. In order to analyze the molecular mechanism of drought regulation and flowering, the gene expression of lemons was studied by drought treatment during flower induction process, the results showed that FT/TFL1 gene family played an extremely important role in the process of drought-treated lemon to promote the process of flower formation. To analyze its physiological mechanism, this project plans to the determination of morphological and anatomical, hormonal content and other indicators during lemon flowering process; The temporal and spatial expression pattern of FT/TFL1 gene family will be also investigated by real-time PCR under different degrees of drought and hormone treatments; Meanwhile, this study attempt to identify some proteins and transcription factors which have interaction with the member of FT/TFL1 gene family and to get further functions for the study of flowering signal pathway by yeast two-hybrid assays and yeast one-hybrid assays. In addition, lemon will be grafted up/down to transgenic trifoliate orange carried fusion gene of FT:GFP driven by CaMV 35S promoter and TFL1 RNAi, respectively, and investigated its morphological and physiological changes. The results will reveal whether flowering signals are transmissible between shoot apical meristem and leaves, as well as their forms, transmission route and distribution rule, so as to enhance our knowledge about plant phase development and flowering transition. Also, the fulfill of this project will do helpful as reference for study of the interaction between rootstock and scion, and provide theoretical foundation for accelerating fruit breeding and utilization of transgenic plants to produce un-transgenic fruit.

绝大多数柑橘的成熟期集中于十月至十二月，市场销售、贮运和加工压力较大。生产中发现，雨旱季分明的地区，柠檬和葡萄柚在旱季之后可以大量开花，金柑和四季桔等花期也受控水调节，因此干旱可以影响部分柑橘的成花从而影响成熟期。为解析干旱调控成花的分子机理，课题组前期分析了干旱处理柠檬促进成花过程中的基因表达，结果显示FT/TFL1基因家族在这个过程中充当极其重要的角色。所以本项目拟对干旱调控柠檬等柑橘成花进行形态解剖，测定成花过程中的激素含量等生理指标，以探明其生理机制；利用不同程度的干旱和激素处理，调查FT/TFL1家族的时空表达；通过酵母单双杂交等分离上下游的调控基因，揭示基因与环境、基因与基因网络调控的机理；同时以超表达FT:GFP和干涉TFL1的转基因枳嫁接柠檬，研究其形态和生理变化。本项目的顺利开展不但可将柑橘成花机理的研究引向深入，还可为砧穗互作以及利用转基因材料生产非转基因果实探索新路。

绝大多数柑橘的成熟期集中于十月至十二月，市场销售、贮运和加工压力较大。生产中发现干旱可以影响部分柑橘的成花从而影响成熟期。前期研究发现FT/TFL1基因家族在这个过程中充当极其重要的角色。本项目首先对干旱调控柠檬等柑橘成花进行形态解剖，测定成花过程中的激素含量等生理指标； 通过酵母单杂交分离了与FT互作的NF-YA6。NF-YA6具有促进开花、对干旱和脱落酸超敏的功能。NF-YA6与柑橘FT启动子上的CCAAT-box元件结合。双荧光素酶试验结果表明NF-YA6可以促进FT的表达。NF-YA6还可以与柑橘FCA基因全长转录本编码的FCA1蛋白互作，但不与柑橘FCA基因短截转录本编码的FCA2蛋白互作。FCA1除了具有促进开花的功能外，还对脱落酸敏感，而FCA2对脱落酸不敏感，但FCA2可以与FCA1形成二聚体。在成年柠檬中，干旱胁迫显著地诱导NF-YA6及下游基因FT的表达，且柑橘FCA基因全长转录本FCA1的表达也被干旱胁迫诱导。脱落酸也可以诱导NF-YA6和FT的表达，但其诱导程度低于干旱胁迫。此外，FCA基因短截转录本FCA2也被干旱诱导。当干旱程度较深时，柑橘FCA基因可能通过增加FCA2的转录反馈调节开花诱导过程。因此，本研究提出了一条柑橘响应干旱和低温胁迫调控成花转变的调控途径。当水分亏缺或低温时，NF-YA6和FCA1的表达被迅速诱导，NF-YA6与FCA1形成复合体，结合在FT启动子CCAAT-box元件上促进FT的转录，FT蛋白表达后，移动至分生组织中，激活分生组织识别基因AP1和LFY的表达，使柑橘开花。当干旱程度非常严重时，柑橘FCA基因通过产生短截蛋白FCA2，FCA2与FCA1形成二聚体，减少NF-YA6-FCA1复合体的形成，反馈调节柑橘成花诱导，使柑橘不至于因过度消耗水分和代谢物质而死亡。为研究柑橘成花转变奠定了基础，为利用干旱调节柑橘花期、改良和培育童期短，花期不同的新品种提供了理论依据。