组蛋白乙酰转移酶BrcuHAC1调控菜心（菜薹）抽薹时间的分子机理研究

Histone acetylation is an important post-translational modification correlated with gene activation. In Arabidopsis, there are five histone acetyltransferases AtHACs homologous to animal p300/CBP proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation and apoptosis. The functions of p300/CBP in animals are well characterized, whereas little is known about the roles of HACs in developmental control in plants. In Arabidopsis, AtHAC1 affects flowering time by epigenetic modification of factors upstream of FLC. We previously cloned BrcuHAC1 and functional domain( HAT )from Chinese flowering cabbage( Brassica rapa syn. campestris L. ssp. chinensis var.utilis Tsen et Lee). In order to study the function of BrcuHAC1, overexpression and RNA interference of BrcuHAC1 will be studied to effects of the objective gene to bolting time in Chinese flowering cabbage.The difference of phenotype and physiological indexes will be draw a comparison between transgenic plants and its control after BrcuHAC1 overexpressed and RNA interfered in Chinese flowering cabbage. The temporal and spatial expression pattern of BrcuHAC1 will be analyzed with used of a BrcuHAC1 promoter driving GUS and real-time PCR and RNA transcriptional levels of FLC and members of the autonomous pathway will be analyzed in transgenic plants. Chromatin immunoprecipitation assay will be revealed the histone modification of FLC and GST-pull down will be researched the effects on histone acetyltransferases activity of BrcuHAC1 to FLC and proteins of the autonomous pathway in vitro. Based on these results of this project, we will not only clarify the function of histone acetyltransferases BrcuHAC1 controlling bolting time in Chinese flowering cabbage, but the new histone code of epigenetic inheritance to regulate plant development.

组蛋白乙酰化修饰是一类重要的翻译后修饰，通常与基因转录激活相关。动物中p300/CBP是很重要的一类组蛋白乙酰转移酶，目前动物中的功能研究比较清楚，但在植物中p300/CBP的同源蛋白HACs的功能还知之甚少。拟南芥AtHAC1可通过表观调控FLC上游因子影响FLC染色质重塑，从而抑制FLC表达后促进植物开花。由此可否用我们已从菜心（菜薹）中克隆的BrcuHAC1研究对FLC表达的影响来调控菜心的抽薹时间？本项目拟通过菜心中超表达和RNAi敲除BrcuHAC1，初步确定目标基因对菜心抽薹发育的影响；通过目标基因在转基因材料中时空表达特性及对FLC、自主通路基因转录水平影响分析，FLC的ChIP检测，目标蛋白对FLC、自主通路蛋白体外乙酰转移酶活性影响检测，阐明BrcuHAC1的生物学功能，找出目标蛋白直接调控的信号（基因）蛋白，为芸薹属作物组蛋白修饰调控抽薹时间分子机理研究提供理论依据。

组蛋白乙酰化修饰是一类重要的翻译后修饰，通常与基因转录激活有关。从菜心早、晚熟品种分别克隆了组蛋白乙酰转移酶基因，命名为BrcuHAC1，分析其功能结构域。结果表明：目的基因的全长6061 bp，包括CDS 区5067 bp，编码1688个氨基酸；对应的gDNA全长为7376bp，含有15个外显子和14个内含子。BrcuHAC1蛋白属于组蛋白乙酰转移酶家族成员，具有6个高度保守域，分别是2个ZZ型锌指结构域、2个TAZ型锌指结构域、PHD结构域和负责乙酰转移酶活性的HAT结构域（975~1504）。在正常生长条件下BrcuHAC1沉默植株表现出叶片卷曲、抽薹开花时间晚、育性差等发育缺陷，而超量表达植株则表现为抽薹时间提早，叶色浅绿等特征。与对照植株比较，BrcuHAC1沉默植株的株高、薹粗、可溶性糖含量、MDA含量等抽薹指标均为下降，相关抗氧化酶（SOD、POD、CAT）活性无明显变化，而超量表达植株除了株高、薹粗上升外，其它指标与对照植株无显著性差异。组织特异性分析结果表明，BrcuHAC1在菜心（菜薹）的根、茎、花、叶中均有表达，其中花中的表达量最高，叶次之，根最低，表明BrcuHAC1可能与花的发育有关。在对照植株中，BrcuHAC1的表达量随苗期至完全抽薹开花期呈现上升趋势；超量表达的T1代株系中，表达量最高为16.15，最低为1.56，表明BrcuHAC1基因已在转录水平上得到表达。BrcuHAC1沉默后，开花抑制因子FLC的表达量升高，而自主通路基因表达量均为不同程度下降，该结果说明BrcuHAC1通过调节FLC的表达来调控菜心（菜薹）的抽薹时间，然而组蛋白乙酰化修饰与基因的转录激活有关，故推测FLC不是BrcuHAC1作用的直接靶位点。FLC的ChIP检测表明，沉默植株中FLC染色质的组蛋白未发生任何修饰，且自主途径相关基因的表达量未发生任何变化，表明BrcuHAC1可能通过表观调控FLC基因的上游因子来影响菜心的抽薹时间。体外检测BrcuHAC1对自主途径蛋白乙酰转移酶活性的影响，结果表明自主开花通路的FPA蛋白被BrcuHAC1乙酰化修饰，表明BrcuHAC1可能通过翻译后修饰FLC的上游因子FPA的组蛋白乙酰化来实现菜心抽薹时间调控的。该研究为芸薹属作物组蛋白修饰调控抽薹时间分子机理研究提供理论依据。