【作者】 张颜；

【导师】 任华中；

【作者基本信息】 中国农业大学 ， 蔬菜学， 2014， 博士

【摘要】 黄瓜(Cucumis Sativus L.)属单性花植物,花的性别分化直接影响着增产潜力。前人已就黄瓜雌花发育的调控机制开展了较多研究,而雄花发育分子机制则一直被忽视,尚难全面了解黄瓜花芽性别发育的分子机制。赤霉素(GA)是重要的植物激素之一,可促进黄瓜雄花形成,但其作用机制尚不清楚。本文以雌雄同株型黄瓜为材料,通过时空表达、亚细胞定位和功能分析,研究了GA信号转导因子CsGAIP(负调节因子)和CsGAMYB1(正调节因子)对黄瓜雄花形成的调控机制,旨在丰富黄瓜花芽性别发育的理论基础。取得的主要结果如下：1、黄瓜CsGAIP属于DELIA家族的同源基因,其N端含有DELLA和、/HYNP两个保守域；时空表达分析表明,CsGAIP主要在黄瓜的茎和雄花芽处表达,而在雄花发育过程中,CsGAIP的表达量主要集中在初期的花序分生组织、花分生组织、维管束和两性期的萼片原基、花瓣原基、雄蕊原基、雌蕊原基以及后期的花粉母细胞、花粉囊壁、花粉粒中,且在两性期的表达量最高；亚细胞定位分析显示,CsGAIP蛋白定位于细胞核；通过在拟南芥中的功能验证,发现CsGAIP的异位表达可部分恢复双突变体rga-24/gai-t6的植株高度、雄蕊发育以及植物育性表型,同时可通过下调花官属性基因AP3和PI的表达抑制野生型拟南芥的雄蕊发育。2、黄瓜CsGAMYB1属于GAMYB家族的同源基因,其序列中含有一个R2R3重复序列以及3个GAMYB家族特有的保守元件Box1、Box2、Box3;时空表达分析表明,CsGAMYB1主要在黄瓜的雄花芽处表达,且外源GA3处理可上调其表达,而在花发育过程中,CsGAMYB1的表达主要分布在初期的花序分生组织、花分生组织和两性期的萼片原基、花瓣原基、雄蕊原基、雌蕊原基以及雄花的花粉母细胞、花粉囊壁、花粉粒中,而在雌花的子房中有微弱的表达；亚细胞定位分析显示,CsGAMYB1蛋白定位于细胞核；通过在拟南芥中的功能验证,发现CsGAMYB1能部分恢复双突变体myb33myb65中雄蕊发育异常和不育的表型,但其过量表达可导致野生型拟南芥的雄性不育,推测其作用机制中存在一种剂量依赖效应；此外,通过黄瓜遗传转化体系,获得了CsGAMYB1干扰转基因植株,与对照相比,干扰植株中的雄花节与雌花节之间的比例显著下降,但乙烯合成基因F、M和内源乙烯含量没有明显变化,表明CsGAMYB1可调节黄瓜的性别表达,而乙烯没有参与此调控过程。更多还原

【Abstract】 Cucumber (Cucumis sativus L.) is a typical monoecious vegetable with individual male and female flowers, and the sex determination directly affects the product yield. Extensive studies have been performed previously on the mechanism of cucumber female flower development, while the molecular regulation of male flower development has been neglected, which greatly hinders the understanding of sex expression in cucumber. Gibberellin (GA) is an important plant hormone that can promote male flower formation in cucumber, but the regulatory mechanism remains elusive. In this study, two GA signaling factors, CsGAIP (a repressor of GA action) and CsGAMYB1(a positive regulator for GA signaling pathway), were identified from the monoecious cucumber. Through expression, subcellular localization and functional analyses, we researched the regulator mechanism of CsGAIP and CsGAMYB1in male flower formation of cucumber. The main results are as follows:CsGAIP belongs to DELLA family in cucumber, and it contains DELLA and VHYNP domains in the N-terminal region. CsGAIP is predominantly expressed in stems and male flower buds. During male flower development, CsGAIP RNA was found in the inflorescence meristem, floral meristem, vascular strands of the early stage, developing sepals, petals, stamens, carpels of the hermaphrodite stage and microsporocytes, anther wall, pollen grains of the later stages, while the expression in the hermaphrodite stage is the highest. Further, CsGAIP protein is localized in nucleus. Ectopic expression of CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6mutant, as well as inhibit stamen development of wide-type Arabidopsis through transcriptional repression of floral homeotic genes APETALA3(AP3) and PISTILLATA (PI). CsGAMYB1belongs to GAMYB family in cucumber, and it contains an R2R3repeat domain and three conserved motifs Box1, Box2and Box3, which are typical structures in GAMYB family. CsGAMYB1is predominantly expressed in male flower buds of cucumber, where its expression is upregulated by GA3treatment. During flower development, CsGAMYB1RNA was found in the inflorescence meristem, floral meristem of the early stage and developing sepals, petals, stamens, carpels of the hermaphrodite stage. For male flowers, CsGAMYB1is predominately expressed in the microsporocytes, anther wall and pollen grains. For female flowers, the expression of CsGAMYB1is detected in the developing ovary, but the signal is weaker. Further, CsGAMYB1protein is localized in nucleus. CsGAMYB1can partially rescue stamen development and fertility phenotypes of Arabidopsis myb33myb65double mutant. However, overexpression of CsGAMYB1in wild-type Arabidopsis resulted in male sterility with dose-dependent manner. Besides, we generated the CsGAMYB1-RNAi transgenic plants in cucumber and found that knockdown of CsGAMYB1decreases the ratio of nodes with male and female flowers, but no effect on expression of F and M genes and ethylene production, suggesting that CsGAMYB1can regulate sex expression of cucumber, and ethylene is not involved in this process.更多还原