【作者】 王燕；

【导师】 李兴国； 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2015， 硕士

【摘要】 茎端分生组织(Shoot apical meristem, SAM)的离体发生是植物基因工程的先决条件,维管形成层的活动是植物次生生长和木材形成的基础。研究表明细胞分裂素在离体芽发生和形成层活动过程中均发挥了至关重要的作用,然而在木本植物中,其调控机制目前尚不清楚。本研究以84K杨为材料,建立了一套稳定的农杆菌介导的遗传转化体系。利用人工合成的生长素响应启动子DR5驱动GUS报告基因对离体芽发生过程中生长素响应信号的分布模式进行了分析。结果表明,离体芽诱导初期,生长素响应信号在愈伤组织的边缘均匀分布,并随着诱导时间的延长逐渐开始区域化分布；茎端分生组织开始形成时,DR5::GUS信号集中在茎端分生组织的上部两侧；茎端分生组织形成后,GUS信号特异分布在L1层细胞中。原位杂交结果显示,编码杨树细胞分裂素B类反应调节因子的RR12和RRl3基因的表达信号在离体芽诱导初期在愈伤组织的边缘均匀分布,随后呈现出区域化集中的分布模式,最终在除L1层细胞以外的整个茎端分生组织中均有分布。上述时空分布模式与拟南芥中的情况是一致的,表明生长素和细胞分裂素在调控杨树和拟南芥离体芽发生过程中所起的作用是相似的。利用artifical microRNA手段同时下调这两个基因的表达获得了一系列amiR-RR12,13转基因株系。选取转基因植株叶片进行离体芽诱导,结果发现与野生型相比,RR12和RR13的表达量下调后,导致离体芽发生频率和每块外植体上形成离体芽的数目均明显降低,表明RR12和RR13参与了杨树离体芽的发生过程。进一步的分析发现,RR12和RR13在维管形成层区域有较强的表达信号。下调RRl2和RR13的表达量造成杨树植株生长受到抑制。统计结果表明,amiR-RR12,13转基因植株茎第20节的直径明显小于野生型对照。细胞学分析发现,与野生型相比,amiR-RR12,13转基因植株茎的次生木质部和次生韧皮部比例降低、次生生长受到了抑制。qRT-PCR结果显示RR12和RR13在amiR-RR12,13转基因植株茎中的转录水平明显降低,而其他B类RRs未表现出明显变化,表明上述表型是特异降低RR12和RR13转录水平的结果。这些结果说明RR12和RR13通过维持维管形成层的正常活动参与了对次生生长过程的调控。本研究的结果表明,细胞分裂素通过其下游信号转导途径中的RR12和RR13介导了离体芽的发生和维管形成层的活动,并在上述过程中发挥了正向调控作用。更多还原

【Abstract】 Shoot regeneration is the prerequisite of plant genetic transformation in most plant species. The activity of vascular cambium leads to the secondary growth and wood formation. The phytohormone cytokinin plays important roles in regulating both shoot regeneration and cambium activity. However, the mechanisms underlying the regulation, especially in woody species, remain elusive.In the present study, we established a stable system for poplar genetic transformation. On the basis, we analyzed the distribution pattern of auxin response signals during shoot regeneration using DR5::GUS reporter line. The results showed that DR5::GUS signals distributed evenly at the edge of the callus at the beginning of shoot induction, and then progressively restricted into specific regions. At the initiation stage of shoot apical meristem (SAM), GUS signals were visualized at the apical and peripheral regions of the SAM. After the formation of the SAM, auxin response signals were detected at the LI cell layer. Further, the patterns of cytokinin response in shoot regeneration were examined. Sequnence analysis revealed that RR12 and RR13 of popular are homologs of the B-type cytokinin response regulater ARR1 of Arabidopsis. The results of in situ hybridization showed that the transcripts of RR12 and RR13 accumulated evenly at the edge of the callus at the beginning of shoot induction, and then restricted into specific regions. After the formation of the SAM, RR12 and RR13 were expressed throught the SAM except for the LI cell layer. These distribution patterns were in accordance with that of ARR1 in Arabidopsis thaliana during shoot regeneration process. These results indicated the similar roles of auxin and cytokinin in regulating shoot regeneration between poplar and Arabidopsis.Knock-down of RR12 and RR13 using artificial microRNA strategy resulted in the decrease of the frequency of shoot regeneration and the number of regenerated shoots on each explant. The results indicate that RR12 and RR13 are involved in the regulation of shoot regeneration. Furthermore, RR12 and RR13 were found to be preferentially expressed in the cambium zone. Decreased expression levels of RR12 and RR13 in amiR-RR12,13 lines led to the reduction of the growth rate and the diameter of the 20th internode. Cytological results showed that the formation of xylem and phloem were inhibited in amiR-RR12,13 lines, implying a reduced secondary growth in the stem. The relative transcriptional levels of RR12 and RR13 decreased significantly in the amiR-RR12,13 stem compared to those of wild-type plant, while those of other B-type RRs did not exhibit obvious changes, suggesting that the phenotype of the amiR-RR12,13 stem were resulted from decreased expression levels of RR12 and RR13.Our results suggest that cytokinin regulates shoot regeneration and cambium activity through RR12 and RR13, which act as positive regulators in these processes.更多还原