【作者】 张杰道；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 核基质结合区(Matrix Attachment Regions,MARs)是基因组上通常富含A/T的能够将DNA或染色质附着到核基质上的DNA序列。通过与结合蛋白的互作,MARs在维持和/或修饰DNA或染色质结构及调控相关基因方面表达中发挥重要作用。TM2是我们从烟草基因组中分离得到的一个MAR序列,在体外与核基质具有很强的结合能力,并且在水稻、烟草等转基因植株中对侧翼基因表达表现出很强的增强作用。为了进一步阐明TM2的表达调控机理,本研究对TM2在不同基因表达系统中调控侧翼基因表达的功能特点进行了详细分析,以探讨TM2可能的作用机制及其在植物基因工程中的应用。主要试验结果如下:1.在烟草转基因植株和细胞悬浮系中,双端TM2序列均能显著提高侧翼表达盒中报告基因gusA的表达水平。细胞类型对转基因表达有一定影响,但不影响TM2的调控功能。MAR序列的串联重复能够增强其表达调控作用,但增强效应与串联拷贝数不成比例。2.烟草TM2序列具有双向表达调控能力,且作用没有明显的序列方向性。无论在表达盒上游和下游,TM2均能提高侧翼基因的表达水平,表现为双向调控特性。尽管上游MAR表现出比下游MAR更强的调控作用,但双端TM2对于提高侧翼基因的表达无疑都是必要的。TM2在重组位点上正向插入和反向插入对其侧翼基因表达水平的增强作用没有显著影响,说明TM2的功能不受插入方向的影响。3.烟草TM2能够改变侧翼基因的表达水平,但不改变其启动子的表达模式。启动子缺失分析表明,TM2对侧翼基因的表达增强作用要求启动子具有调控基础表达的必需元件。无论对于组成型CaMV 35S启动子还是光合组织特异的PNZIP启动子,TM2只改变启动子的调控水平,不改变启动子调控的基因表达模式。4.TM2序列上的拓扑异构酶Ⅱ结合位点、DNA解旋结合位点和T-box均是功能元件。烟草TM2位点缺失分析表明,三种元件的缺失都会引起TM2侧翼基因表达水平的降低,是TM2 MAR的主要功能元件,但位点之间存在一定的功能冗余。5.烟草TM2序列能够显著提高侧翼启动子区对核酸酶的敏感性。微球菌核酸酶敏感试验表明,TM2显著降低了转基因植株中CaMV 35S启动子的特异扩增水平,说明TM2能够使侧翼DNA结构松散,具有局部染色质开放能力。几个功能元件对于提高核酸酶敏感性起关键作用。6.烟草TM2序列的功能不严格依赖核基质的存在,但需要在浓缩的DNA结构中才能发挥作用。在植物瞬时表达中,TM2没有明显的表达增强作用,但在酵母核外质粒表达系统中可以通过提高转录效率来增强侧翼基因的表达,几个功能元件在核外表达增强过程中同样发挥重要作用。7.通过酵母单杂交分离到几个TM2序列的潜在结合蛋白,凝胶阻滞分析表明这些蛋白能够与TM2片段进行体外结合。多种侯选靶位点暗示TM2可能有多个核基质结合位点,也间接说明TM2作用机理的复杂性。8.利用TM2序列和其他表达调控序列,构建了植物高效超表达载体和干涉表达载体,能够用于植物高效转化及转基因表达调控。利用In-Fusion Smart技术,可以将植物高效表达载体与cDNA池重组,用于大规模基因表达筛选。这些载体的构建可为植物基因功能分析提供有效的研究工具。更多还原

【Abstract】 Matrix Attachment Regions (MARs) are the DNA sequences with rich A/T nucleotides that may be involved in anchoring DNA/chromatin to the nuclear matrix. By the interaction with the binding proteins, MARs play an important role in the maintance and modification of the DNA/chromatin structure and the regulation of the gene expression.TM2 is a matrix attachment region isolated from the genomic DNA of tobacco, which can strongly bind to the nuclear matrix and significantly enhance the transgene expression in transgenic plants of rice and tobacco. To gain insights into the regulatory mechanism of TM2 by which transcription enhancement of transgene occurs, we give the detailed analysis of the expression variation of flanking transgenes drived by different promoters in different expression systems. We present the main results as follows:1.The tandem repeats of TM2 sequence can enhance the transgene expression activation mediated from the flanking MAR in transformants, although this effect does not correspond with the copy number of the MARs. The average level of transgene expression in tobacco calli is markedly lower than that in plants, however, this difference from the cell development does not influnce the function of TM2.2 . The TM2 bidirectionally regulated the flanking transgene expression without dependence on the sequence orientation on the integrating site. The 5′-TM2 and 3′-TM2 can both enhance the transgene expression, although the increasing distance of the MAR relative to the promoter might decrease the activation to a certain extent. This characterization indicates TM2 enhance the transgene expression in a bidirectional manner. The effect of location suggests both the 5′-TM2 and 3′-TM2 flanking the transgene in the construct are desirable for maximal enhancement of transgene expression. In addition, when the individual TM2 was constructed in the opposite direction relative to the transgene, either in the upstream or in the downstream of the cassette, the GUS assay does not show a significant difference between the constructs. These results indicate there is no direction effect of the TM2 sequence on transcription enhancement of the target gene expression.3.The TM2 can change the expression lelvel of the flanking transgene rather than the expression pattern mediated from the promoter. The effect of TM2 on GUS expression in transgenic cells that harbored the CaMV 35S or PNZIP minipromoters suggests that transcription enhancement from the tobacco MAR requires the basic expression of reporter gene. Without the upstream enhancer elements of minipromoter the MAR present to be invalid.4.The effect of the site-specific deletion of two unwinding sites, one topoisomerase II binding site and one T-box element in TM2 indicated that they are all functional elements. Deletion of any kind of elements always results in the decreasement of the flanking transgene expression. These four sites perform the vast majority of the enhancement mediated from TM2, although there is some functional redundancy in their contribution.5.According to the micrococcal nuclease accessibility analysis, the CaMV 35S promoter adjacent to the TM2 was degraded more rapidly than the control without MAR. By contrast, the time-course digestion of the promoter linked to mutant MAR was similar with that of the control. Considering the increasing accessibility to micrococcal nuclease would result in the decrease of PCR products for the regions of interest, the difference revealed that TM2 plays a role in nucleosome remodeling of the promoter region. The deletion of the four sites determined the effect of TM2 on the micrococcal nuclease accessibility.6.The transcription activation of flanking gene expression from the tobacco TM2 does not simply depend on the nuclear matrix but the condensed DNA structure. The MAR does not present any regulation ability in agrobacterium-mediated tobacco transient expression of the transgene expression. However, it can enhance the transcription efficiency so as to increase the his3 gene expression on plasmid expression vector out of the yeast nuclear. The above elements in TM2 also play important role in out-of-nuclear expression enhancement.7.Several potential genes encoding TM2 sequence-binding proteins were screened from the cDNA pool by yeast one-hybrid method. These proteins show high affinity with the TM2 fragment by electrophoresis mobility shift assay. These potential multiple targets implicate the functional complexity of the TM2 MAR. 8.With TM2 and other regulation sequences, we construct the plant overexpression and RNAi vectors. These constructs can be used in high-efficient transformation and regulating gene expressions. The recombination between the high-efficient plant expression vector and the cDNA pool by the In-Fusion Smart method bring an important perspective to large-scale screening of genes. These constructs will provide efficient tools for function study of the plant genes.更多还原