AcERF3转录因子在乙烯诱导菠萝成花中的功能及其作用机理研究

The use of ethephon is critical to induce flower for pineapple (Ananas comosus) production. Economic losses due to bad flower-induction happened often. Therefore, it is of great significance to clarify the mechanism of pineapple respond to ethylene signal. The ERFs transcription factors are key regulators in ethylene-signaling pathway, forty-two Unigene sequences that share high homology with ERF protein were obtained from the floral transcriptome database of pinapple. Through a preliminary analysis, we presume that AcERF3 might play an important role in floral induction of pineapple by responding to ethylene signals. In this study, taking pineapple plants ‘Comte de Paris’ cultivar (a leading variety) as research material, the present proposal aimed to identify its function by analyzing the expression pattern of the AcERF3, its subcellular localization and transcriptional activation activity; to analyze the function of AcERF3 by obtaining the overexpressed or interfered transgenic plants; simultaneously, to screen its DNA binding sites from whole genome by using ChIP-seq analysis; to screen using yeast two-hybrid, to confirm whether the genes of downstream relevant to flowering-regulating pathway using association analysis with RNA-Seq. Taken together, the study will disclose the mechanism of ethylene induced flower in pineapple, which will provide theoretical basis and technical support to obtain transformation resistance plants by genetic engineering methods.

应用乙烯催花是当前菠萝生产中最关键的栽培技术之一，但因催花不当造成经济损失的情况仍时有发生，因此阐明乙烯调控菠萝成花的内在机理就具有十分重要的意义。ERFs是乙烯信号转导途径中的关键转录因子，前期在菠萝花芽转录组中发现了42个ERFs转录因子成员，通过初步表达分析推测AcERF3可能在响应乙烯信号诱导菠萝成花中起着重要作用。项目拟以主栽菠萝品种‘巴厘’为试材，通过分析AcERF3的时空表达特征、亚细胞定位及转录激活活性，初步阐明AcERF3的表达特性；通过在菠萝中过量或干涉AcERF3转录因子表达，探析其在菠萝成花中的功能；同时利用ChIP-seq技术从全基因组水平分析AcERF3转录因子与DNA的结合位点，通过与转录组关联分析，揭示其下游调控的靶基因是否与菠萝成花相关，以期从转录水平阐明菠萝成花的分子调控机制，为通过基因工程手段定向改良菠萝的成花性状提供理论依据和技术支持。

利用乙烯及其衍生物对菠萝进行人工诱导开花已成为菠萝生产中普遍使用的栽培技术，但目前乙烯诱导菠萝成花的具体机理尚不清楚。本项目以易受乙烯诱花的‘巴厘’菠萝为材料，分析了AcERF3的转录调控特性，并通过转基因拟南芥（异源）和菠萝（同源）对其功能进行了鉴定；同时采用ChIP-seq技术结合转录组数据对AcERF3下游的靶标基因进行了筛选，对乙烯诱花处理后AcERF3与靶标基因之间的协同表达关系进行了综合分析。取得主要结果如下：AcERF3在乙烯利处理后1天时的顶端分生组织中具有一个极显著的表达高峰，且具有促进拟南芥早花的功能；利用ChIP-seq分析筛选得到了关键的靶基因AcSPL16，并发现AcSPL16表达特性和功能与AcERF3的相似，可能在乙烯诱导菠萝成花转变的过程起着重要作用，推测AcERF3在响应乙烯信号后与AcSPL16启动子结合进而调控AcSPL16的表达来诱导菠萝成花。本项目解析了菠萝AcERF3转录因子在乙烯诱导菠萝开花中的具体作用机制，为深入揭示乙烯诱导菠萝成花的分子机理提供重要的基础。