【作者】 卢龙涛；

【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， 2009， 硕士

【摘要】 目前通过植物转基因技术进行作物品种的改良已经成为作物品质改良的重要手段。我们都希望外源转入的优良基因能在作物中得到高效和稳定的表达,但是外源的基因在转入植物中一代或者几代后会出现不再表达、表达变弱的现象。这种现象的发生很大程度上是由于转基因沉默现象导致的。由于转基因沉默严重阻碍转基因商品化的进程,因此,如何提高转基因后代的遗传稳定性、减少转基因沉默的发生是一个亟待解决的问题。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。以往的研究者发现核基质结合序列在减少转基因沉默方面具有重要作用。TM6是本实验室从烟草核基质中分离得到的一个具有一般MARs结构特征的DNA序列,核基质体外结合实验证明TM6与烟草核基质具有很强的体外结合能力。为了研究TM6在植物体内的功能,主要是在其提高转基因后代遗传稳定性中的作用,我们构建不同的植物表达载体并转化了双子叶植物烟草和拟南芥,对转基因植株中TM6连接的外源基因的表达情况进行了分析。为了进一步研究TM6的作用机理,本实验还分析了TM6对其邻近启动子区域DNA甲基化的影响,主要实验结果如下:1.在双子叶植物烟草中,双TM6能够明显的降低杂交后代的转基因沉默比例,提高转基因后代的遗传稳定性。这个实验结果为TM6在提高外源基因在转基因植物中的遗传稳定性方面的应用提供了实验依据。2.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DNaseI处理后的转TM6的转基因烟草细胞核内,TM6显著降低了CaMV35S启动子区域的特异扩增水平,初步说明TM6能够减低其邻近启动子区域DNA甲基化水平,从而降低基因沉默的发生,提高基因表达。3.对转基因烟草中CaMV35S和NOS启动子区域的DNA甲基化程度进行检测,分析得知:在CaMV35S启动子区域,没有TM6的转基因烟草植株的DNA甲基化水平较高大约在13%～37%,而有TM6的转基因烟草植株中几乎没有DNA甲基化的发生;在NOS启动子区域,没有TM6的转基因烟草植株的DNA甲基化水平较高大约在24%～51%,而有TM6的转基因烟草植株的DNA甲基化水平在9%～14%左右。该实验进一步验证了TM6能够显著降低其邻近启动子区域DNA甲基化水平。4.双子叶植物烟草中分离的TM6同样可以在拟南芥中增强外源基因的表达,并且在不同类型的启动子(组成型35S启动子、冷诱导型COR启动子和光合组织特异型启动子PNZIP启动子)的驱动下都能有增强表达的作用。说明TM6是一个能广泛提高外源目的基因表达的核基质结合序列,并且这种对外源基因表达的提高并不改变启动子的作用类型。更多还原

【Abstract】 Gene transfer technology is becoming a fundamental tool to enhance agronomic performance or improve quality traits in a wide variety of crop species. But gene transfer technology are sometimes severely handicapped by transgene silencing and instability of gene expression. Transgene expression is frequently lost after one or a few sexual generations. So it is important to adopt efficient strategies to overcome gene silencing and increase transgene expression level for optimizing transgenic technology.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. They have been seen as fundamental tools to reduce or eliminate some forms of transgene silencing because of their capacity to increase transgene expression levels or reduce the variance between transgenic events when flanking a transgene cassette in both plants and animals.TM6 is a matrix attachment region isolated from the genomic DNA of tobacco, which can strongly bind to the tobacco nuclear matrix in vitro. In this study, we investigated the effect of TM6 on stability of transgene expression in the progeny of the primary stably transformed tobacco and the function of TM6 in Arabidopsis. We also investigated the effect of TM6 on the DNA methylation status of promoter regions. The main results were as follows: 1. TM6 can decrease transgene silence of backcross progeny in tobacco, and increase stability of transgene expression in the progeny of the primary tansformed tobacco. This results make it possible for the application of TM6 in transgene plants to increase stability of transgene expression.2. According to the DNaseI accessibility analysis, the CaMV35S promoter adjacent to the TM6 is degraded more rapidly than the control without MAR. Considering the increasing accessibility to DNaseI would result in the decrease of PCR products for the regions of interest, the difference reveals that TM6 plays a role in decreasing DNA methylation of the promoter regions. This maybe one mechanism of TM6 decrease transgene silence and increase gene expression.3. we also detect DNA methylation status of the CaMV35S region and NOS region in transformed tobacco with and without TM6. In our results, there is almost no DNA methylation of CaMV35S regions in transformed tobacco with TM6 but there is 13%～37% DNA methylation of CaMV35S regions in transformed tobacco without TM6. In NOS region, frequence of DNA methylation is 24%～51% in transformed tobacco with TM6 compared 9%～14% those without TM6. This results indicate that TM6 can decrease the DNA methylation in both CaMV35S regions and NOS regions.4. Although TM6 is isolated from tobacco, it can also enhance transgene expression in arabidopsis. We use different type promoters (CaMV35S, PNZIP, COR) to test the influence of TM6 in transgenic arabidopsis, and find that TM6 can enhance gene expression in the transgenic Arabidopsis with three promoters. This indicated TM6 can widely enhance gene expression in different kinds of plants, not change the gene expression pattern.更多还原