【作者】 冀芦沙；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 植物基因工程， 2009， 博士

【摘要】 核基质结合序列(Matrix Attachment Region,MAR)是真核生物基因组上一段富含A/T,且能够将DNA或染色质附着到核基质上的一段DNA序列。当染色体周围富集大量置换蛋白时,这些置换蛋白能特异识别并结合到MAR序列区域并替换掉H1组蛋白,使MAR区域的染色体形成一种易于调节因子及修饰因子接近的开放结构,因而MAR序列的出现使染色质上核小体处于松散状态,从而影响邻近基因的表达。根据研究推断,MAR在维持和/或修饰DNA或染色质结构及调控相关基因表达中发挥重要的作用。TM6是本实验室从烟草核基质中分离得到的一段具有MAR结构特征的DNA序列。为了验证TM6在植物体内的功能,我们构建不同的植物表达载体转化烟草,对转基因烟草中外源基因的表达情况进行了分析检测。为了进一步研究TM6的作用机理,本实验分析了TM6周围的染色体区域活跃程度及TM6的结合蛋白与TM6相互作用关系,主要实验结果如下:1.在双子叶植物烟草中,双端TM6序列均能显著提高表达盒中报告基因gusA的表达。虽然细胞类型对转基因表达有一定的影响,但并不影响TM6的调控功能。本实验将P35S,PPNZIP,PCOR及Pmini35S特异型启动子引入MAR研究,结果显示,TM6的作用依赖于启动子的类型,但TM6并不能改变启动子的作用模式。本实验结果为以后在转基因植物特定发育时期或特定器官中提高外源基因的表达提供了实验依据。2.在转化烟草的过程中发现,连有TM6的载体其转化效率高于不连TM6的载体。通过对转基因烟草中卡那抗性基因的表达分析发现,抗性基因的表达量的高低与转化频率存在着直接的关系,因此推测TM6通过增加抗性基因的表达来提高了转化效率,同时这种转化效率的提高使抗性芽发生提前,数量增多,并且抗性苗的生长势也明显高于对照。3.对TM6的缺失分析表明,TM6II(551-1193bp)区域是TM6的核心功能区,占TM6功能的79.1%。TM6II序列上的拓扑异构酶Ⅱ结合位点、MRS区域及AT?box区域在调控基因表达上起到了协同作用。推测TM6通过序列上的拓扑异构酶Ⅱ结合位点与植物体内的拓扑异构酶Ⅱ相结合,从而促进T?DNA插入位点附近的DNA解旋,进而一些外源的调控元件识别并结合到TM6上的AT富含区及ARS区,同时加快外源基因的复制和转录。4.烟草TM6序列能够显著提高侧翼启动子区对核酸酶的敏感性。在微球菌核酸酶(MNase)处理后的转TM6的转基因烟草细胞核内,TM6显著降低了35S和NOS启动子区域的特异扩增水平,说明TM6能够使邻近启动子区域DNA结构松散,便于外源转录因子的结合从而开启外源基因表达。5.从烟草cDNA文库中分离到两个TM6序列的潜在结合蛋白NtMBP1和NtHMGB,凝胶阻滞(EMSA)分析表明这些蛋白能够与TM6片段体外结合。在竞争性EMSA实验中,蛋白NtDBP1和NtHMGB分别特异结合到TM6II的两个不同区域TM6II-I(761-870 bp)和TM6II?II(934-1013 bp)。同时多种侯选靶位点竞争结合实验验证:NtHMGB能特异结合到TM6II上MRS区域,而NtDBP1蛋白在TM6II上可能有多个潜在的核基质结合位点,也间接说明TM6作用机理的复杂性。6.根据以上的实验结论,对TM6作用模型进行推测:TM6周围的染色体区域与外源染色质调控蛋白(拓扑异构酶II等)识别并结合后,解开TM6染色体区域的高级结构从而使染色体敏感性增强。同时TM6结合蛋白(NtMBP1和NtHMGB等)将TM6II上特定位点的DNA双链释放,便于外源调控因子(RNA聚合酶和甲基化酶等)的结合从而增加外源基因表达。更多还原

【Abstract】 Matrix Attachment Regions (MARs) are the DNA sequences with A/T rich nucleotides that may be involved in anchoring DNA/chromatin to the nuclear matrix. The displacement proteins specifically bind to MAR regions in chromatin to facilitate the mediated displacement of H1 histones, and the enrichment of those proteins might open the chromatin structure of TM6 regions. MAR plays an important role in the maintance and modification of the DNA/ chromatin structure and the regulation of the gene expression.TM6 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 tobacco. To gain insight into the regulatory mechanism of TM6 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. TM6 is a novel element to increase the transgene expression levels in both dicotyledons (tobacco) and monocotyledons (arabidopsis). The difference of the cell development in plants and calli does not influnce the function of TM6. Four different promoters (P35S, PPNZIP, PCOR and Pmini35S) have been used to test the influence of MAR on transgenic plants, and the results show that TM6 does not change the gene expression pattern. The result, that MAR increase gene expression in desired tissues with tissue?specific promoters, is a new way to enhance the transgene expression.2. Assays of transgenic plants show that the expression level of nptⅡ, the quantitative GUS activity and the transformation efficiency are quite consistent, TM6 transgenic plants have higher transformation efficiency than those without TM6. We presume that TM6 increases the transgene efficiency through the increase expression levels of nptⅡ, and the TM6 transgenic plants show the stronger seed germination.3. Deletion analysis shows that TM6II (551-1193 bp) plays 79.1% role of the whole TM6 sequence. The effect of the site?specific deletion of one topoisomerase II binding site, one AT?box and one MRS element in TM6II indicate that they perform the vast majority of the enhancement mediated from TM6. There is some functional redundancy in the contribution to open the TM6 chromatin structure and recruit transcription factors, increasing the genes transcription.4.According to the micrococcal nuclease accessibility analysis, the CaMV 35S promoter adjacent to the TM6 is degraded more rapidly than the control without MAR. Considering the increasing accessibility to micrococcal nuclease would result in the decrease of PCR products for the regions of interest, the difference reveals that TM6 plays a role in nucleosome remodeling of the promoter region. The deletion of the four sites determines the effect of TM6 on the micrococcal nuclease accessibility.5. Two potential genes encoding TM6 sequence?binding proteins, NtMBP1 and NtHMGB, are screened from the cDNA pool by yeast one?hybrid method. The two proteins show high affinity with two specific fragments, TM6II?I (761 to 870 bp) and TM6II?II (934 to 1013 bp), by electrophoresis mobility shift assay (EMSA). NtHMGB can specifically bind to MRS element of TM6, and NtMBP1 might associate with other elements on TM6. These potential multiple targets implicate the functional complexity of the TM6.6. Proposed models for the chromatin regulation of MARs on gene expression. We persume that some DNA duplex?destabilized enzymes and chromatin regulated factors, such as the helicases and topoisomerases, bind to specific elements in TM6 loosening the TM6 chromatin. The NtMBP1 and NtHMGB proteins act as the architectural factors to displace the H1 histones to decrease compactness of the chromatin fiber at the TM6 regions. Meanwhile the transcription factors (such as FACTs) bind to the opening chromatin regions to increase the transcription.更多还原