【作者】 李丹丹；

【导师】 傅小鹏；

【作者基本信息】 华中农业大学 ， 园林植物学， 2017， 硕士

【摘要】 对于观赏性植物来说,花不仅是植物繁衍后代的重要器官,更是决定其观赏价值和经济价值的关键因素,因而花发育研究一直是观赏植物研究的重点。石竹（Dianthus chinensis）作为观赏植物,因其花朵繁盛,此起彼伏,观赏期较长等优点被广泛的应用于园林绿化和人们日常生活的美化、装饰中。从早期的ABCE模型中可知,SEP类基因在各个花器官中都发挥着重要作用,并且在植物进化过程中出现功能冗余、新功能化和亚功能化。但在石竹的发育过程中是否出现了新的功能还有待探究。本研究从石竹中克隆到5个SEP类基因,并分别对它们的氨基酸结构和蛋白特性进行了分析。同时对石竹不同时期的花芽和不同的花器官的表达情况进行了探究。在此基础上对DcSEP1、DcSEP3-1、DcSEP3-2、Dc MADS7、DcSEP4基因功能进行了初探。主要研究结果如下:1.从石竹中克隆出DcSEP1、DcSEP3-1、DcSEP3-2、Dc MADS6、Dc MADS7、DcSEP4六个基因,氨基酸比对发现它们都具有完整的M、I、K、C域,并且在不保守的C域内都具有E类基因特有的SEPI和SEPII基序。2.实时荧光定量分析表明在不同的花芽分化时期中,石竹DcSEP3-2、Dc MADS7和DcSEP4在花原基形成期就有所表达,而DcSEP1、DcSEP3-1在萼片原基形成期开始表达,说明DcSEP3-2、Dc MADS7和DcSEP4可能参与了花原基的形成,DcSEP1、DcSEP3-1发挥功能的时间相对较晚。另外在对不同花器官的表达中发现只有DcSEP4在营养器官茎和叶中表达,DcSEP3-2、DcSEP4在四轮花器官中都有所表达,DcSEP1在花瓣和心皮中的表达量比较高,DcSEP3-1除了在花瓣和心皮上表达,在萼片中的表达也比较高,但Dc MADS7特异性的在萼片和花瓣中表达,说明它们可能参与调控不同花器官的功能。3.在ABCE模型的基础上,运用酵母双杂交和双分子荧光互补对石竹E类蛋白之间的相互作用进行了分析。结果表明:DcSEP3-1、DcSEP3-2和DcSEP4之间具有强相互作用,两两之间可以形成同源或异源二聚体;DcSEP4和Dc MADS7之间存在弱相互作用;DcSEP1和任何基因之间都不存在相互作用。且双分子荧光互补试验表明SEP亚家族基因全部在细胞核中发挥作用。4.构建了石竹E类基因的超量表达载体和干涉载体,并进行了石竹和拟南芥的遗传转化,得到了石竹DcSEP1、DcSEP3-1、DcSEP3-2、Dc MADS7、DcSEP4过量表达转化拟南芥T₁代转基因株系,DcSEP1、DcSEP3-2过量表达转化石竹T₀代转基因株系,DcSEP3-1、DcSEP3-2、DcSEP4缺失突变转化石竹T₀代阳性苗。通过观察转基因植株的表型发现,35s:DcSEP3-1、35s:DcSEP3-2和35s:Dc MADS7超表拟南芥中都出现了早开花的现象,此外35s:DcSEP3-2和35s:DcSEP4超表拟南芥还出现了强突变的表型,表现为植株异常矮小,莲座叶2-4枚,顶端花序提前终止,花器官异常不结实等现象,但只观察了T₁代转基因拟南芥表型,这两个基因的功能还有待进一步试验验证。更多还原

【Abstract】 For ornamental plants,flowers are not only important organs of the multiplication of plant progeny,but also the key factors that determine its ornamental and economic value,and thus studies on flower development has been the focus of ornamental plant.Dianthus chinensis are widely used in landscaping and the beautification and decoration of people’s daily as ornamental plant for its advantages,such as flowers flourish,the longer view and admire,etc.From the early ABCE model,SEP genes plays an important role in flower organs,and functions redundancy,neofunctionalization and subfunctionalization during the evolution of plants,but whether the emergence of neofunctionalization in the development of the carnation remains to be explored.In this study,five SEP genes were cloned,and their amino acid structure and protein characteristics were analyzed respectively.At the same time,the expression of flower buds and different flower organs were studied in different periods.O n the basis of this,the preliminary function of DcSEP1,DcSEP3-1,DcSEP3-2,Dc MADS7,DcSEP4were got.The main results are as follows:1.DcSEP1,DcSEP3-1,DcSEP3-2,Dc MADS7,DcSEP4 genes were cloned from the single Dianthus chinensis,amino acid pairs have found that they all have complete M,I,K,and C domains,and possesses SEPI and SEPII motifs for unique class E genes in non-conserved C domains by analyzing the amino acid sequence.2.Real-time quantitative analysis showed that DcSEP3-2,Dc MADS7 and DcSEP4 were expressed during the flower bud differentiation period,while DcSEP1and DcSEP3-1 were expressed at the time of septal primordium formation,Indicating that DcSEP3-2,Dc MADS7 and DcSEP4 may be involved in the formation of flo wer primordia,DcSEP1 and DcSEP3-1 may specifically determine the formation of floral organs.In addition,only DcSEP4 has a relatively high expression in vegetative organs stems and leaves,the expression of DcSEP3-2 and DcSEP4 were detected in four whorl of floral organs.The expression of DcSEP1 in petals and carpels was relatively high,and we also found expression of DcSEP3-1 in petals and carpels,but Dc MADS7specific expression in sepals and petals,suggesting that they may be involved in regulating the function of different floral organs.3.Based on the ABCE model,the interaction between the E proteins of Dianthus chinensis was analyzed by yeast two hybridization and bimolecular fluorescence complementation.The results showed that DcSEP3-1,DcSEP3-2 and DcSEP4 had strong interactions,which could form a homologous or heterogenous dimer.DcSEP4and Dc MADS7 have weak interactions,and DcSEP1 does not interact with any E class gene.The bimolecular fluorescence complementation assay showed that the SEP subfamily genes were localized in the nucleus.4.The overexpression vector and interference vector of E gene were constructed,and the genetic transformation of Dianthus chinensis and Arabidopsis thaliana was carried out.DcSEP1,DcSEP3-1,DcSEP3-2,Dc MADS7,DcSEP4 overexpression was transformed into Arabidopsis thaliana T₁ transgenic lines,DcSEP1 and DcSEP3-2overexpression was transformed into transformed T₀transgenic lines,and DcSEP3-1,DcSEP3-2 and DcSEP4 deletion mutations were transformed into the T₀ generation.By observing the phenotype of transgenic plants,we found that Arabidopsis thaliana appeared early flowering in 35s:DcSEP3-1,35s:DcSEP3-2 and 35s:Dc MADS7 in the transformation of Dianthus chinensis.In addition,conversion of 35s:DcSEP3-2and 35s:DcSEP4 in Arabidopsis also appeared strong phenotypic mutations,characterized by the performance of abnormal plant,rosette leaves 2-4,the top of the inflorescence early termination,flower organ abnormalities.更多还原