【作者】 朱杉杉；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2008， 硕士

【摘要】 多种信号均参与植物由营养生长到生殖生长的转变。在这个转变过程中,植物激素起着非常重要的调控作用。为了研究激素调节拟南芥离体花序发育的机制,首先建立了拟南芥离体花序再生系统。以拟南芥雌蕊为外植体,在含有6-BA、2,4-D的培养基上诱导愈伤组织的形成,大约25天后将愈伤组织转至含有Zeatin和IAA的培养基中诱导离体花序的产生。花序再生频率约为60%。利用半定量RT-PCR方法,对花序发育相关基因的表达进行了分析。发现愈伤组织转至分化培养基后,同源异型基因AP1、AP3、AG、LFY、TFL1、WUS、CLV1、CLV3和STM的表达先后受到诱导,而TFL2和AP2基因的表达则没有明显变化。利用pTFL1::GUS和pLFY::GUS转基因植株为材料,分析了TFL1和LFY在离体花序发育过程中表达的组织特异性。发现TFL1基因主要在具有花序轴特征的细胞,以及成熟再生花序的花序轴中表达,而LFY基因主要在即将产生花原基的区域表达,随后在成熟再生花序的花原基中表达。由于WUS和CLV3基因的表达影响拟南芥植株中花序的发育,因此对离体花序发育过程中该两个基因的表达模式进行了分析。研究结果显示,在离体花序形成早期,首先检测到WUS基因在愈伤组织内部的一群细胞中表达,称之为组织中心。随后在组织中心上方表面1～3层细胞中检测到干细胞特征决定基因CLV3的表达。随着花序原基的继续发育,两基因的表达均逐渐集中于花序分生组织中。利用可诱导的反义基因表达策略,抑制WUS基因表达,离体再生花序形成受到抑制。研究结果表明组织中心的细胞先于干细胞形成,WUS基因在此过程中起重要作用。花序的诱导需要生长素。在花序诱导过程中添加生长素极性运输抑制剂,不仅破坏了生长素的极性分布,还抑制了WUS基因的表达,最终不能产生离体花序。说明生长素的极性运输为干细胞形成及离体花序发育所必须。本研究结果表明,激素不仅诱导组织中心细胞及干细胞的形成,而且启动花序特征基因的表达,最终决定花序原基及花原基的产生,进而发育形成成熟的离体花序结构。更多还原

【Abstract】 Various signals can be involved in the transition from vegetative growth to reproductive growth in plant. Plant hormones play important roles in this process. To study the developmental mechanism of hormone-regulated regenerated inflorescence, we established a regeneration system of Arabidopsis inflorescence induced by cytokinin and auxin in vitro. In this regeneration system, pistil was used as explant to induce the formation of callus,and then the callus was transferred to another kind of media for inflorescence induction. The frequency of inflorescence regeneration was about 60%.Semi-quantitative RT-PCR analysis was performed to determine the expression patterns of genes involved in inflorescence development. The results showed that the expression of AP1, AP3, AG, TFL1, LFY, WUS and CLV3 was induced after callus was transferred to the media for inflorescence induction. In contrast, the expression of AP2 and TFL2 was constitutive during inflorescence regeneration.The expression patterns of TFL1 and LFY during inflorescence regeneration were analyzed by using pLFY::GUS and pTFL1::GUS transgenic plants. We found that TFL1 expression mainly located in the cells with inflorescence meristem identity and in the mature regenerated inflorescence. And the expression of LFY was found in the callus cells differentiating to floral primordium and in the mature regenerated inflorescence.CLV3 and WUS genes were involved in regulating inflorescence development in plants, so the expression patterns of CLV3 and WUS during the regeneration of inflorescence were determined. The results indicated that WUS was firstly expressed in a group of cells located in the inner of callus, named it organization center, and then, the expression of CLV3 was detected in the cells of layer 1-3 above the organization center. With the development of inflorescence primordium, the expression of CLV3 and WUS was focused in the inflorescence meristem. After the expression of WUS was inhibited by anti-gene strategy, the regeneration of inflorescence was reduced obviously. Thus, these results suggest that the expression of WUS, earlier than that of CLV3, is essential for the inflorescence formation.The generation of stem cell was inhibited after the polar transportation of auxin was interrupted by adding NPA, an inhibitor of auxin polar transportation, in media. And the frequency of inflorescences formation was reduced significantly after adding NPA. These results suggest that auxin polar transportation is very important for the generation of stem cell and inflorescence formation in vitro.These results indicate that inflorescence and floral meristem identity genes are induced during the regeneration of inflorescence. Further study indicates that organizing center cell and stem cell formation is necessary for the regenerated inflorescence development.更多还原