【作者】 程志娟；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2009， 博士

【摘要】 植物离体器官发生是快速无性繁殖植物的重要途径,是植物基因转化的基础,也是研究植物发育问题的主要实验系统之一。目前人们已经可以成功的诱导拟南芥离体苗的再生,在这一过程中干细胞被诱导形成。然而迄今为止,有关离体植株再生过程中是何种因素决定了干细胞的性质,仍然知之甚少。建立一个高频的拟南芥离体再生苗系统,进而深入研究其中的激素调节机制将为解释干细胞特征决定的机理提供重要信息。利用拟南芥雌蕊为外植体,在含有高浓度生长素的培养基上诱导愈伤组织的形成。然后将愈伤组织转移到含有高浓度细胞分裂素的培养基上诱导离体苗的产生。为了研究离体苗发生过程中干细胞的形成,对干细胞决定基因WUSCHEL(WUS)和CLAVATA3(CLV3)在离体苗发生过程中的表达模式进行了分析。研究结果显示,在未经诱导的成熟愈伤组织中均未检测到WUS和CLV3的表达信号。当转入诱导离体苗形成的分化培养基上,首先检测到WUS基因在愈伤组织内部的一群细胞中表达,我们称之为组织中心,随后在组织中心上方1～3层细胞中检测到干细胞特征决定基因CLV3的表达。在茎顶端分生组织形成过程中,两基因的表达均逐渐集中在茎顶端分生组织中。诱导反义WUS基因表达,干细胞和再生苗的形成受到抑制。表明组织中心的细胞先于干细胞形成,WUS在此过程中起关键作用,并决定了茎顶端分生组织的形成。在此基础上,研究了细胞分裂素和生长素在离体苗再生过程中的分布规律。利用对细胞分裂素起响应的TCS和表征细胞分裂素分布的A类反应调节因子ARR15与GFP的表达载体,证明了在离体苗再生过程中,在未经诱导的成熟愈伤组织中细胞分裂素在愈伤组织周围的数层细胞中均匀分布。然而在苗诱导分化培养过程中,细胞分裂素向着将要产生茎顶端分生组织的区域集中,并且细胞分裂素响应信号较强的区域正是WUS诱导表达的位置。利用对生长素起响应的DR5rev::GFP表达载体研究了生长素的分布。结果显示在未经诱导的成熟愈伤组织中,其分布模式与细胞分裂素相同,即在愈伤组织周围数层细胞中呈现均匀的分布。在分化培养基上,生长素在愈伤组织内亦逐渐呈现区域性分布,最终WUS基因表达区域周围生长素响应信号较强。生长素的分布模式与PIN蛋白的极性定位有关。在未经诱导的愈伤组织中,检测到PIN蛋白的微弱信号,但未呈现极性定位。然而在诱导分化后短时间内出现了PIN蛋白的极性定位信号,并且出现极性分布的区域增多。进而,研究了细胞分裂素和生长素对干细胞诱导形成的作用。当利用细胞分裂素合成抑制剂降低愈伤组织中细胞分裂素的水平,以及在细胞分裂素信号转导组分的多突变体中,生长素的分布模式均发生了改变。生长素分布模式的改变导致WUS基因不再被诱导表达,苗的诱导受到抑制。说明细胞分裂素的水平及其信号转导控制生长素的分布模式和干细胞的形成。如果使用细胞分裂素合成抑制剂,PIN1的极性定位信号消失。因此细胞分裂素对生长素分布的影响可能是通过控制PIN1的极性定位来实现的。如果在诱导过程中添加生长素极性运输抑制剂,生长素和细胞分裂素的分布不再出现区域化,维持了一种在愈伤组织周围数层细胞中近乎均匀的分布模式,并且检测不到WUS信号。这一结果在PIN1的诱导型反义转基因植株诱导苗再生过程中亦得到了证实。说明生长素的极性运输通过控制生长素的分布进而影响细胞分裂素的分布,并决定干细胞形成。综上所述提出如下实验模型:当将成熟的愈伤组织转入含有高水平细胞分裂素的苗诱导培养基后,生长素和细胞分裂素首先通过调节PIN蛋白极性定位启动生长素的极性运输。随后生长素的极性运输决定了生长素由均匀分布到区域化分布,同时影响了细胞分裂素的区域性分布。最终在细胞分裂素响应信号较强和生长素响应信号较弱的区域诱导了干细胞组织中心特征决定基因WUS的表达,进而干细胞特征决定基因CLV3被诱导表达,随后茎顶端分生组织建成。在这一过程中细胞分裂素的分布及其对WUS的诱导依赖于生长素的极性运输,而生长素的分布又受到细胞分裂素的合成及其信号转导的影响。因此细胞分裂素和生长素之间的相互作用诱导了组织中心细胞和干细胞的形成,启动了茎顶端特征基因的表达,最终决定茎顶端分生组织的出现。更多还原

【Abstract】 Plant organ regeneration plays an important role in plant reproduction and genetic engineering, and also provides an experimental system to study molecular mechanism of plant development. Shoot regeneration is an important way for plant propagation in vitro. So far, shoot regeneration of Arabidopsis was achieved successfully. Stem cells are critical for the formation of shoot apical meristem and must be induced within the callus during shoot induction. Little is understood however about the regulation of this process and how the stem-cell fate becomes determined during this process. In our current study, we showed that the fate of stem cells is determined by cytokinin and auxin within the callus after induction. Using stage-10 pistils as explants, calli were induced on the callus induced medium.Shoots were subsequently induced after each callus was transferred onto the shoot induced medium. Following induction treatment, WUSCHEL (WUS) was firstly expressed in a group of cells within callus, named it the organizing center and then, the expression of CLAVATA3(CLV3) was detected in the cells of layer 1-3 above the organization center. Finally, the transcripts of CLV3 and WUS were localized in the tissues that form the shoot meristem. By inhibition of WUS expression, the regeneration of shoot was significantly reduced. Thus, these results indicate that WUS is induced within the callus and its expression is essential for the in vitro shoot regeneration.We next visualized the behavior and interaction of cytokinin and auxin during stem-cell formation. In the non-induced callus, either cytokinin or auxin gradients were established in its surrounding edge region. However, the regional distribution of both hormones occurred following induction after which WUS was found to be induced in the region containing high levels of cytokinin and low levels of auxin. Furthermore, the polarized PIN-FORMED (PIN) proteins, which are auxin-efflux carriers, were also observed in the regions close to the edge of the callus after induction. Upon the inhibition of cytokinin biosynthesis, the auxin levels decreased and a normal distribution pattern for WUS induction was not observed. Disruption of cytokinin signaling also resulted in an abnormal distribution pattern for auxin. Following inhibition of auxin transport or inhibition of PIN1 expression, the regional distribution of auxin gradients was not detected within the callus and notably, the regional distribution of cytokinin gradients was also not observed. In addition, the pattern of cell proliferation supports the notion that cytokinin and auxin interact.Taken together, our current results suggest that a positive interaction between cytokinin and auxin controls the stem-cell fate, and we propose a model for stem-cell specification control during in vitro shoot induction in Arabidopsis by cytokinin and auxin.更多还原