【作者】 于明明；

【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， 2008， 硕士

【摘要】 细胞分裂素调节植物生长和发育的许多过程,如促进细胞分裂、影响开花时间、延缓叶的衰老以及控制顶端优势等。人们对拟南芥(Arabidopsis thaliana)花分生组织特征基因和花器官特征基因的功能以及光、温度等环境因素对花发育的调控作用已有较深入的研究。在拟南芥中,转基因表达pAP1:IPT4的研究结果显示,与野生型植株相比,转基因植株花序中的细胞分裂素含量显著提高,表现出花排列密集、花原基和花器官数目增多、顶端优势降低等表型(李兴国等,未发表资料)。为进一步阐明细胞分裂素对花发育的调控作用,我们主要利用原位杂交和GUS染色技术,对pAP1:IPT4转基因植株中花发育相关基因的表达模式进行了较详细的分析。原位杂交和GUS染色分析结果显示,在pAP1:IPT4转基因植株中,花分生组织特征决定基因LEAFY(LFY)的表达部位与野生型相比没有明显变化,但表达量显著提高。原位杂交结果同时显示,在pAP1:IPT4转基因植株中,花器官特征决定基因APETALA1(AP1)、PISTILLATA(PI)、AGAMOUS(AG)的表达量均有不同程度的提高。在转基因植株处于时期4的花中,AP1不仅在萼片原基中表达,在花中心的分生组织中亦有较多的mRNA积累,这与其在野生型植株中的表达模式有所不同。PI在转基因植株的花序分生组织和时期2的花中均有表达,随着花的进一步发育,PI的转录水平较野生型明显提高。AG在转基因植株的花原基中有较强的表达信号。当花发育至时期3和4时,AG在萼片原基内方分生组织中的mRNA水平比野生型明显增加,在萼片原基中也检测到AG的表达。我们推测转基因植株的表型可能与上述基因的表达变化具有一定的关系。对pAP1:IPT4转基因植株进行的全基因组表达谱分析发现,LATERAL ORGAN BOUNDARIES DOMAIN(LBD)基因家族成员LBD3的表达量受到上调。为了进一步探究LBD3的功能,我们构建了35S:LBD3和35S:anti-LBD3表达载体并转化野生型拟南芥。结果显示,35S:LBD3转基因植株主花序轴的顶端优势显著下降,而35S:anti-LBD3转基因植株未见明显的表型变化。RT-PCR的结果表明,在35S:LBD3转基因植株中,MORE AXILLARY GROWTH4(MAX4)表达量明显提高,MAX2的表达量则略有降低。MAX途径的下游基因BRANCHED1(BRC1)的表达量也有所上调。MAX途径通过抑制生长素的极性运输来抑制侧枝生长。LBD3与生长素极性运输以及侧枝发育相关基因的作用关系有待进一步研究。更多还原

【Abstract】 Plant hormone cytokinin participates in regulation of diverse plant growth and development processes including cell division, flowering, leaf senescence and maintenance of apical dominance. The functions of Arabidopsis thaliana floral meristem identity genes and floral organ identity genes have been well studied, and some environmental factors such as light and temperature were implicated in floral development modulation. In Arabidopsis, cytokinin level in pAP1:IPT4 transgenic plants is elevated compared with wild type plants, resulted in abnormal phenotypes including compacted flower arrangement, increased floral primordium and floral organs,reduced apical dominance and so on (Li et al., unpublished data). To investigate the roles of cytokinin regulation in flower development, we examined the expression patterns of genes controlling flower and floral organ development in pAP1:IPT4 transgenic plants of Arabidopsis.In situ hybridization and GUS staining analyses indicated that floral meristem identity gene LFY was up-regulated in pAP1:IPT4 transgenic plants, whereas its expression domain was not affected compared with that in wild type plants. Analyses of in situ hybridization revealed that the up-regulation of floral organ identity genes AP1, PI and AG occurs in pAP1:IPT4 transgenic plants. AP1 mRNA was first detectable in flower primordia. As flower development proceeds, AP1 trancripts accumulated in sepal primordium, as well the central meristem interior to sepal primordial in pAP1:IPT4 transgenic plants, suggested that the AP1 expression pattern was affected by transgenic expresssion of IPT4. PI was expressed in the floral meristem and stage 2 flower of pAP1:IPT4 transgenic plants, and the PI mRNA level increased along with subsequently flower development. AG signal was detectable in the floral primordium of pAP1:IPT4 transgenic plants and stronger in floral meristem than that of wild type in stage 3 and 4 flowers. Additionally, In pAP1:IPT4 plants, AG signal was discovered in sepal primordium, which differed from that of wild type plants. Thus, it suggested that changed expression patterns of these genes partially contribute to the phenotype of pAP1:IPT4 plants.Genome-wide expression analyses by gene chip revealed that the expression of LBD3, a member of the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family in Arabidopsis, was up-regulated in pAP1:IPT4 plants. To further study the function of LBD3, 35S:LBD3 and 35S:anti-LBD3 construct was introgressed into wild type plants of Arabidopsis, respectively. Overexpression of LBD3 resulted in reduced apical dominance, while 35S:anti-LBD3 transgenic plants had no detectable phenotypic altreration. Previous studies have uncovered that MAX pathway represses lateral bud growth by suppression of polar auxin transport, and BRANCHED1(BRC1) functions downstream of MAX pathway. The results of RT-PCR analyses indicated that the expression of MAX4 was evidently up-regulated and MAX2 down-regulated in LBD3-overexpressing plants. In addition, the abundance of BRC1 mRNA was elevated in 35S:LBD3 plants. These data raised the possible relationships between LBD3 and polar auxin transport as well as genes controlling lateral bud growth.更多还原