B类MADS-box基因调控紫花地丁两型花发育的功能研究

The dimorphic plant produce both open, potentially out-crossing chasmogamous (CH) flowers and closed, self-pollinating cleistogamous (CL) flowers. CH flowers have bright-coloured petals and special odour which can attract insects. In CL flowers, reduced corolla, androecium and anther sacs are quite common. Usually, petals and partial stamens of CL flowers are poorly developed or even absent. The mixed breeding system not only is a result of adaptive evolution to environment , but also provides a good material for the research of floral organ development.Many ecologists and biologists have conducted research on the mixed breeding system, but the underlying molecular mechanisms remain largely unknown. Viola yedoensis is a species with the chasmogamous-cleistogamous mixed breeding system. Our preliminary work indicates photoperiod is the most crucial factor that effects the development of dimorphic flower. CH flowers are formed in response to short daylight, CL flowers in response to long daylight, with intermediate CL flowers produced during transitional periods.The genetic study of flower devlopment indicates that the B-class genes are PISTILLATA(PI) and APETALA3(AP3), which code for MADS-box transcription factors are not only involved in the specification of stamen and petal identity but also in control of organ maturation and morphology during later developmental stages through the maintenance of expression. So we suspect that the expression of B-class MADS box genes may be regulated by photoperiod and influence the development of the CH and CL flowers. In this project, we will clone and isolate the homeotic sequence of B-class MADS box genes in Viola yedoensis. Under the different daylight，we will compare the expression quantity of B-class MADS box genes in CH flowers, intermediate CL flowers, and CL flowers, and examine the expression pattern in different floral organ. Meanwhile,we will study the protein-protein interactions, and reveal the phenotype changing through gene silencing of B-class MADS box genes. Through the experiments above, our study will explore the molecular mechanism of formation of CH and CL flowers, and understand the significance of evolutionary biology about formation of the dimorphic flower plant.

两型自花受精植物中，开放花鲜艳的花瓣与特殊气味吸引昆虫可能远交，闭锁花部分雄蕊与花瓣或发育不充分或完全缺失，自交。这种两型花混合繁育系统被认为既是对环境的适应性进化，又为研究花器官的发育提供了难得的素材，但目前对其发育机制还缺乏了解。紫花地丁是一种典型的两型自花受精植物，光周期是影响其形成两型花的关键生态因子，短日照、中日照与长日照分别对应的是开放花、过渡闭锁花与完全闭锁花。花发育的遗传学研究表明B类MADS-box基因主要决定花瓣与雄蕊的身份，同时调控这些器官的发育过程和形态。因此，我们推测光周期有可能通过调控B类MADS-box基因的表达来影响紫花地丁两型花的发育。本项目将分离紫花地丁中B类MADS-box基因的同源序列，研究不同光周期下这些基因在花发育过程中的表达模式，揭示它们蛋白间的互作样式及基因特异沉默后的表型变化，探讨两型花形成的分子机制，理解两型花植物形成的进化生物学意义。

自然界中，一些植物具有开放花和闭锁花的混合繁育特征，这种两型花的混合繁育特征被认为既是对环境的适应性进化，又为研究花器官的发育提供了难得的素材，然而，这种潜在的分子机制目前仍不清楚。紫花地丁是一种典型的两型闭花受精植物，光周期是影响其形成两型花的关键生态因子，短日照下，开的是开放花，随着光周期的延长，会形成过渡闭锁花与完全闭锁花。长日照下，花瓣与雄蕊的数量与大小均低于或小于开放花的，并且在大于14-h光周期下，会形成没有花瓣且只有2枚可育雄蕊的完全闭锁花。ABC模型表明B类MADS-box基因会在很大程度上影响雄蕊与花瓣的发育。因此，我们的工作聚焦在这些基因的特征上来理解这种特异的花型转变。本研究中分离到3个基因，分别命名为VpTM6-1，VpTM6-2和VpPI。这3个基因在不同的花型中其表达存在明显的差异，尤其是在花瓣与雄蕊中，而且在开放花中表达量最高，过渡闭锁花中有明显的降低，在完全闭锁花中的表达量最低。当花诱导与花器官发生后，VpTM6-1，VpTM6-2和VpPI基因表达的差异与光周期的变化密切相关。因此，我们认为在过渡闭锁花与完全闭锁花中，花瓣与雄蕊发育的抑制可能与长日照调控的B类MADS-box基因的下调有关，从而诱导了完全闭锁花的形成。在本氏烟草外源体系中，通过VIGS实验方法研究了紫花地丁B类MADS-box基因的功能。研究表明，以紫花地丁的TRV2::VpPI（ORF）与TRV2::VpPI（3’UTR）为探针，能敲除本氏烟草花芽中的NbGLO基因，使得本氏烟草的花发育发生异常，花瓣皱缩，雄蕊部分或完全转变为雌蕊。以TRV2::VpTM6-1（ORF）（VpTM6-1和VpTM6-2序列同源性很高，因此只做了一个探针）为探针，敲除了本氏烟草中的NbTM6基因，使得花粉发育出现异常。因此，紫花地丁中VpPI与VpTM6基因的功能分别与本氏烟草中NbGLO和NbTM6的功能是相似的，既调控花瓣与雄蕊的身份，又调控花瓣与雄蕊的发育。本研究将有助于我们理解两型花植物形成的适应性进化机制，并为两型花发育的进一步研究提供理论依据。

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