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题名: 麻竹试管诱导开花及开花相关基因DlEMF2的克隆和功能分析
作者: 许红
学位类别: 博士
答辩日期: 2008-06-04
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 李德铢
关键词: 麻竹 ; 试管开花 ; 组织培养 ; 基因克隆 ; 转基因
学位专业: 植物学
中文摘要: 竹子作为重要的禾本科植物,具有特殊的经济、生态和社会价值。但是竹类集体开花和死亡的生长规律使得目前全球一半的竹类濒临灭绝。大多数竹种经历长达几十年甚至上百年漫长的营养生长周期后开花,并且开花后即死亡,自然结实率较低,使得竹子开花及相关机理的研究受到了限制,所以竹子开花现象一直成为植物学的一个难解之谜。随着植物组培开花技术的发展,通过试管诱导竹子开花已经成为可能,在此基础上建立竹子试管开花人工诱导与逆转的模式系统,不仅能为竹类分类鉴定、品种改良、杂交育种等开辟一条崭新的道路,更重要的是为竹子的开花生物学和开花机理的研究奠定了良好的实验基础。本研究一方面从麻竹试管诱导开花入手,尝试着在试管中诱导麻竹开花;另一方面,克隆控制营养生长向生殖生长转变的重要基因DlEMF2基因,并通过转基因实验初步揭示其功能和表达规律,为进一步开展竹子开花调控研究提供理论依据。 1.麻竹组织培养开花诱导研究 最近十几年,竹子试管开花现象在国内外多个实验室有过报道,这为建立竹子稳定的开花诱导和逆转体系奠定了良好的实验基础。本研究在试管中利用不同种类和浓度的植物激素,分丛芽诱导、芽增殖培养、花芽诱导三个阶段对麻竹试管开花进行了研究。建立了稳定的丛芽诱导、增殖培养体系,找到了比较合适的培养基配方,也在试管中诱导出花芽,但是受麻竹种子来源和数量限制,没有筛选到很好的开花无性系。有待获得更大量的麻竹种子后,进一步研究。 2.麻竹中开花相关基因DlEMF2的克隆和序列分析 本研究根据拟南芥EMF2基因保守区序列分析结果,设计简并引物,利用RACE技术从麻竹茎尖组织中克隆了AtEMF2的同源基因DlEMF2。序列分析表明它与拟南芥AtEMF2、水稻OsEMF2等基因结构相似,都存在三个保守区域,分别是N端核定位区、C2H2-type 锌指结构和C端acidic-W/M 区。在拟南芥中,EMF2基因属于PcG蛋白家族,通过形成EMF2蛋白复合体,结合到目标基因的启动子或基因内的特定部位,导致转录受到抑制,染色体异质后导致了基因沉默,抑制了开花基因的表达。 3.麻竹中AtEMF2的同源基因DlEMF2的功能研究 在克隆麻竹DlEMF2基因的基础上,分析了其在麻竹顶端嫩尖、茎、叶、花、根等不同组织器官的基因表达规律。实验结果表明DlEMF2在所有的组织中都有表达,在茎尖的表达量很高,这与拟南芥和水稻的EMF2基因的表达情况类似,所不同的是DlEMF2在花中的表达量非常弱,而拟南芥和水稻的EMF2基因在花中表达量同茎等部位相似,没有明显的降低。因而推测竹子DlEMF2基因在开花时间控制方面跟拟南芥和水稻会有差异。本实验中构建了DlEMF2基因的反义和过表达载体,并利用这两个载体分别转化拟南芥野生型和emf2突变体。其中反义转基因结果显示,DlEMF2基因保守区反义片段的导入,导致了拟南芥转基因植株的早花,Real Time PCR结果显示早花是由于内源AtEMF2基因表达量的明显降低引起的。麻竹的DlEMF2基因过表达使得拟南芥明显延迟了开花时间,而拟南芥自身AtEMF2基因的过表达却不能延迟开花,说明麻竹DlEMF2基因与拟南芥AtEMF2基因在作用方式上可能有所区别。用DlEMF2全长基因转化拟南芥emf2突变体发现,突变体表型可以被拯救。另外,通过基因枪轰击洋葱表皮使GFP-DlEMF2融合蛋白在表皮细胞中瞬时表达。荧光显微和微分干涉显微观察表明GFP-DlEMF2 融合蛋白存在于细胞核中, 说明DlEMF2定位于细胞核。
英文摘要: As an important plant of the grass family, bamboo has special value in economy, ecology and society. Nowadays, according to some report, 50% of the total number of bamboo species in the world are close to die out, owing to bamboo flowering. Most of bamboo species flower only once in whole life after a long vegetative stage and die after that. The long vegetative growth as well as the low natural seed production made it uneasy to understand the mechanism of bamboo flowering. By developing in vitro tissue culture, bamboo flowering in vitro became possible. It allowed us to set up a bamboo in vitro flowering and flowering-reversion system, which paved a brand new way for species identification, breeding of improved bamboo, and production of interspecific-intergeneric hybrids, especially, providing a reliable source of bamboo seed which is a basis for studying the mechanism of bamboo flowering. In this project, we focus on a tropical bamboo species, Dendrocalamus latiflorus in our study. On one hand, we tried to induce it to flower in vitro; on the other hand, we cloned an homolog gene of AtEMF2 from it, which is related to maintaining vegetative phase through repressing the flower program in Arabidopsis, and we explored its function by transgenic experiments, which offered important base for further study on bamboo flowering. Study of in vitro flowering in Dendrocalamus latiflorus In the past two decades, in vitro flowering of bamboo has been reported from many species in several laboratories of a few countries. It is allowed to set up a bamboo flowering and flowering-reversion system in vitro. In our experiment we used different kinds of cytokinins with different concentration to inducing shoots, propagating shoots and inducing flower buds step by step. Finally, we found an appropriate medium for the first two steps, and also induced flower buds in vitro at the third step. But restricted by the quantity of seeds, we did not find a good flowering line. It may need more seeds to carry on the study in the future. Cloning and sequence analysis of DlEMF2 gene in Dendrocalamus latiflorus relating to bamboo flowering Based on the conserved domain of EMF2 genes, we designed the degenerate primers and cloned the DlEMF2 gene from Dendrocalamus latiflorus. The complete cDNA of DlEMF2 was 2518 bp in length. Sequence analysis showed that it has three conserved regions, C2H2-type zinc finger motif, N-terminal basic domain and C-terminal acidic-W/M domain. In Arabidopsis, EMF2 protein complex belongs to Polycomb-group (Pc-G) proteins. In plants and animals the PcG proteins form multimeric epigenetic silencer complexes that control diverse developmental pathways. Polycomb group (PcG)–mediated gene maintains stably inherited repression of target genes to inhibit transcription which lead to gene scilencing. Functional studies on DlEMF2 gene Based on the cloning of DlEMF2 gene, we analyzed its expression among tissues including apical shoot, stem, leaf, root and inflorence by semi-quantitative RT-PCR. Its tissue-related expression pattern showed that the mRNA level of DlEMF2 was extensive in all parts of the plant and was higher in shoot, but it was much lower in inflorescence. Based on the protein sequence alignment, DlEMF2 has three conserved functional homolog regions as AtEMF2. In order to investigate the function of the DlEMF2 gene, we constructed anti-sense DlEMF2-C fusion construct, then transformed it into the Agrobacterium tumefaciens and subsequently into Arabidopsis. 59% of the transgenic plants showed earlier flowering with formation terminal flowers, others showed normal or unrelated phenotypes. Relative expression levels of endogenous AtEMF2 in these lines were tested by quantitative real-time PCR to confirm whether it has been regulated. The result confirmed that the exogenous DlEMF2 fragment was responsible for repressed endogenous EMF2 in Arabidopsis. Over expression of DlEMF2 in Arabidopsis showed late flowering in T1 generation which means there may be some differences in expression mode between DlEMF2 and AtEMF2. Rescue experiment showed that DlEMF2 can rescue the emf2-1, which means DlEMF2 has the same function as AtEMF2 in maintaining vegetative stage. A transient expression assay showed that the fusion protein of DlEMF2 and green fluorescent protein (GFP) was mainly located in the nuclei.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/398
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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麻竹试管诱导开花及开花相关基因DlEMF2的克隆和功能分析.许红[d].中国科学院昆明植物研究所,2008.20-25
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