【作者】 杨海峰；

【导师】 卢孟柱； 万贤崇；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2007， 博士

【摘要】 基因组学技术研究植物发育某一过程往往获得许多参与该过程的基因，如何采取快速、有效的方法来鉴定这些基因的功能是解析该生物学过程分子机制的基础。拟南芥在人工诱导条件下由形成层活动产生相当数量的木质部，在一定程度上可以模拟树木木材形成。本研究利用该系统分析相关候选基因的拟南芥突变体来研究其参与树木次生维管系统发育的功能。根据毛白杨维管系统再生过程中差异表达基因信息，从国际拟南芥突变体库NASC、RIKEN、CSHL筛选了89个基因的151个拟南芥突变系，利用次生诱导体系来研究相应基因对维管系统的影响。在诱导条件下，这些突变体表现出不同的形态和结构的变化，共有22个表型和结构发生变化。20个突变系发芽率或存活率较低，发芽率由10％—60％，其中2个突变系存活率分别为0％和50％。5个突变系在茎或下胚轴的结构上发生变化，其中一个突变系arris-stem(as)发生如下表型变化：从真叶出现至开花结实的整个生长期，生长速度要明显比野生型拟南芥缓慢；叶缘有明显锯齿，莲座叶呈螺旋状排列；茎的基部有较多侧枝，侧枝间距明显缩短，茎有明显扭曲；突变体茎侧面有1至数条棱形突起，内部存在一至数个排列紧密的细胞团，由多层细胞呈环形排列，细胞壁明显加厚，内部存在管状分子，推测为维管组织。根据突变体库提供的资料，as突变体T-DNA插入位点位于基因组中At2g44110基因。我们从基因补偿、基因表达分析及基因超量表达等方面分析该基因功能。通过PCR由拟南芥中分离该基因和启动子，构建基因补偿表达载体、基因表达模式分析表达载体和基因超量表达载体并导入as突变体或野生型拟南芥。研究结果表明，通过基因补偿，突变植株表型基本恢复；基因表达分析研究表明，该基因在花和幼叶中沿维管束表达，在幼嫩的根茎中呈束状表达，在幼嫩组织中表达量高，在成熟叶片和成熟茎中不表达。超量表达试验也获得了转化植株，尚未进行表型鉴定。根据基因补偿研究结果，证明At2g44110为造成突变体表型变化的决定基因，很可能参与调控维管组织的发育式样。表达分析研究表明，该基因在茎的初生生长初期就已经表达。由上述研究结果，综合推断At2g44110基因在茎的初生生长初期就已经参与了维管组织的发育，调控维管组织的发育式样，而在经充分次生诱导后，该基因的突变能充分表现出来而造成as突变体的表型变化。as基因在拟南芥维管系统发育中具有重要的作用，其同源基因在毛白杨维管系统再生过程中也呈差异表达，说明拟南芥和木本植物可能具有一定相似的维管系统发育调控模式。同时本研究作为一个范例，为应用拟南芥次生诱导研究体系来研究参与木材形成的基因的功能，进而为揭示次生维管系统发育的分子调控机制奠定基础。更多还原

【Abstract】 Wood formation is a comprehensive process which possesses thousands of genes underprecise control. To understand the molecular mechanism of wood formation, a quick andefficient method is necessary to study the function of these genes. Arabidopsis can produceconsiderable amounts of secondary xylem under certain condition, which can mimic theprocess of wood formation. In this study, the induced Arabidopsis secondary growth system areapplied to verify the function of genes previously obtained may involved in the development ofsecond vascular system in poplar.According to information of differentially expressed genes during regeneration ofsecondary vascular system in poplar, we obtained 151 Arabidopsis mutant lines comprising 89Arabidopsis genes form mutants center of NASC, RIKEN, CSHL. These were raised underinducing condition to test if any morphology and structure changes. Twenty two Arabidopsismutants had various changes in morphology and anatomical structures. Twenty mutants hadlow germination rate from 10% to 60%, of which 2 had survival rates at 0% and 50%. Fivemutants occurred structure changes in the hypocotyls or stem. One of these lines, namedarris-stem (as), showed some unique changes: slower growth rate in comparison with the wildtype from germination to florescence; serrated margin of leaf blades, spiral rosette; morebranch in the bottom part of the stem, shorter nodes, twisted stem and branch. There were oneor several arrises along the stem. Across sections of the arrises showed one or several compactcells lumps which were round, made up of several layers of cells, looked like vascular-bundle.The tissue was more fluorescent under UV light than the surrounding cells, which suggestedthere was the thickened cell wall. Tracheary elements were observed inside the arrises undermicroscope, suggesting they are similar to vascular bundles.According to information from Arabidopsis Mutants Center(http://signal.salk.edu/),T-DNA insert site located in the gene At2g44110. The At2g44110 gene functions wereinvestigated with three approaches, i. e., gene complementary; gene express profile; geneover-expression. The results showed that the phenotype of as mutants came to the normal aftergene complement. The expression profiles showed that the as gene was mainly expressed inthe vascular bundles of the inflorescence, young leaves, and also presented in bundle in young root and stem. The express occurred only in young tissues, and no express was found in oldleave and stem. The transformants intended to over-expression of the gene was obtained butnot characterized yet.Based on the complementary study on the as mutants, we suggest T-DNA insertion of theAt2g44110 cause the as phenotype. In addition, its expression pattern indicated that this geneexpressed in the primary growth stage of the stem. These results indicate it may predetermineon the potential vascular development pattern and also fimction in the later development of thevascular systems.This study showed that as may play an important role in the vascular system developmentin the Arabidopsis, and this gene have been found differentially expressed in the secondaryvascular system in poplar (Populus tomentosa.). Taking together we suggest that Arabidopsisand poplar may be similar in the vascular system development and regulation mechanisms.This study may serves as an example to study functions of genes involved in wood formation,using the induced secondary vascular system in Arabidopsis, which could provide the basicdata to elucidate the molecular mechanisms of the development of the secondary vascularsystem.更多还原