马缨杜鹃响应低温解除花芽休眠关键基因RdDAM的分子调控机理

For Rhododendron delavayi，the flower bud dormancy release specifically sensitive to the low temperature, and chilling requirement is the sufficient condition in this process. Our previous reference-transcriptome analysis of four flower buds in different dormant states and other related researches definitely indicated that DAM gene is a key regulator involved the process of flower bud dormancy release response to the low temperature. It has a very important contemporary relevance to study the molecular mechanism of bud dormancy release response to the low temperature, particularly in the context of global climate change. At this before, we have obtained a method to determine the four different dormant states of flower bud in R.delavayi and cloned four RdDAM gene family members of R.delavayi. In this condition, we will firstly screen out the major gene member(s) among the DAM gene family of R.delavayi according to the their expression trends and abundance in four different states mentioned above, and then separately construct the plant RNAi-scilencing and overexpression vector for major DAM gene(s) to transform the R.delavayi. Finally, gene functional analysis will be made from phenotypes and expression profiles of relative gene clusters on DAM signal pathway in transgenic plants at natural and 4℃ treatment conditions. Eventually, we will try to discuss how the major DAM genes to regulate the flower bud dormancy release response to the low temperature in R.delavayi? This study will be a good theory cornerstone for the elucidation of molecular mechanism about DAM gene involved the process of flower bud dormancy release in woody plant with the help of the regulation network construction of DAM gene signal pathway, and also could provide new ideas for the molecular breeding related flowering time control for R.delavayi.

马缨杜鹃的花芽休眠解除特属温度敏感型，一定的低温积累是休眠解除的最主要充分条件。课题组前期有关马缨杜鹃休眠芽的有参转录组测序分析和前人研究均证实：DAM基因是响应低温解除花芽休眠的关键基因。于全球气候变化大背景下，解析低温解除花芽休眠分子机理具有重要现实意义。项目组前期已掌握有关马缨杜鹃4种重要休眠状态花芽的界定方法，并已克隆4个RdDAM家族成员基因。在此基础上，进一步分析4个RdDAM基因在4种休眠状态花芽中的表达谱，根据其表达丰度和表达趋势筛选主效基因。构建主效基因超量表达和沉默表达载体转化马缨杜鹃，通过对照株和转化株在低温处理条件下的表型及其DAM信号通路相关基因群表达响应，进行DAM基因功能鉴定及其通路相关基因调控网络的构建，最终解析DAM基因响应低温解除花芽休眠的分子作用机理。为阐明木本植物花芽休眠解除分子机制奠定理论基石，也为马缨杜鹃有关花期调控的遗传改良育种研究提供新思路。

项目以高山花卉马缨杜鹃为研究对象，从形态学、物候学及生理学角度揭示了花芽休眠的发生、发展及休眠解除过程中有关形态生理指标、环境温度及激素水平的变化规律，并据此规律制定了不同休眠状态的花芽的样品采集标准；成功克隆了休眠过程相关调控基因RdDAM家族中的4个基因成员，并采用RT-qPCR技术检测了4个成员基因在幼株各组织部位和成株中不同休眠状态花芽中的表达模式，根据其表达丰度筛选出RdDAM基因家族的主效基因成员RdDAM1；构建了四个成员基因的超量表达和沉默表达载体，并分别获得了马缨杜鹃遗传转化株15株以上；考虑到马缨杜鹃生长周期较长，苗龄尚小目前还无法进行表型统计，因此同时进行了主效基因RdDAM1的超量和沉默表达载体的拟南芥遗传转化，筛选得阳性转化株各10株以上；超量表达RdDAM1的拟南芥转化株表现出晚花且花朵发育异常（花朵萼片存留，果实发育不良等）的表型，沉默表达RdDAM1的拟南芥转化株则表现出顶端优势丧失的表型；从DAM通路下游7个开花相关基因群在超量表达或沉默表达转基因植株中的表达结果看，不同转基因株系间结果出现不一致，因此很难从中得出相关结论，这可能跟基因的异源表达有关，同时也可能跟本身调控网络的复杂性有关。下一步将延续马缨杜鹃DAM基因的本源超量表达和沉默表达研究，完成低温诱导下的表型分析及通路相关基因群的表达分析，最终完善调控马缨杜鹃花芽休眠解除的分子调控机理和调控网络。综上，通过本项目的实施，弄清了马缨杜鹃花芽的在整个休眠发展过程中的形态生理学基础，并通过基因表达分析筛选得调控花芽休眠的RdDAM主效基因，获得了马缨杜鹃相关表达载体的遗传转化株，为进一步解析DAM基因响应低温调控马缨杜鹃花芽休眠解除的分子机理奠定良好的研究基础。