Study on the higher order structure of DNA that enhances the integration of transgene into the host genome

研究增强转基因整合到宿主基因组中的 DNA 高阶结构

• 批准号: 11460156
• 负责人: MORIKAWA Hiromichi
• 金额: $ 7.1万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460156/
• 关键词: Matrix attachment region (MAR); scaffold attachment region (SAR); tobacco; gene delivery; green fluorescent protein (GFP); integration; higher order structure of DNA; trangene; MAR (matrix attachment region); SAR (nuclear scaffold attachment regi

A 507-bp sequence (designated TJ1) that has been clone from a transgene locus of tobacco cell, and found to be a nuclear matrix / scaffold attachment region (MAR/SAR) was flanked to the 5' and 3' side of the expression cassette, in four different orientations, of the green fluorescent protein (GFP), a non-selective marker, gene. In each of these expression cassette, the GFP gene was under the control of cauliflower mosaic virus 35 S promoter and nopaline synthase polyadenlyation signal. These constructs were introduced separately into tobacco cells in order to investigate the effect of the MAR on the integration frequency of the transgene into the host genome. The presence of the MAR sequences in the cassette appeared to increase the transformant yield more than 2 times as compared with the control construct lacking the MAR sequences. Since there is no selection pressure to obtain those transformants in the present study, it is conceivable that MAR-based enhancement of the gene expression is not involved in the present result of the transformant yield. Therefore, the observed transformant yield should purely reflect the frequency of the integration of the transgene into the host genome. It is thus concluded that TJ1 MAR increases the integration frequency more than 2 times over the control. No significant effect of the orientations of the MAR sequence in the transformant yield was observed. Our findings provide direct evidence for the first time that the MAR cloned from the transgene locus stimulates the integration of transgene into the eukaryotic genome

从烟草细胞转基因位点克隆了一个507bp的序列(命名为TJ1)，该序列位于绿色荧光蛋白(GFP)非选择性标记基因5‘和3’侧翼，位于核基质/支架附着区(MAR/SAR)的两侧。在每个表达盒中，绿色荧光蛋白基因均受花椰菜花叶病毒35 S启动子和诺帕林合成酶多聚糖基化信号的控制。为了研究MAR对转基因整合到寄主基因组中的频率的影响，将这些构建物分别导入烟草细胞。与没有MAR序列的对照相比，盒中MAR序列的存在似乎使转化子的产量增加了2倍以上。由于在本研究中没有获得这些转化子的选择压力，因此可以想象在目前的转化子产量结果中不涉及基于MAR的基因表达增强。因此，观察到的转化子产量应该纯粹反映转基因整合到宿主基因组中的频率。由此得出结论：TJ1MAR使积分频率提高了2倍以上。未观察到MAR序列的取向对转化子产量的显著影响。我们的发现首次提供了从转基因位点克隆的MAR刺激转基因整合到真核基因组中的直接证据

项目成果

K.Shimizu: "Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells : its effect on transformation frequency"Plant Journal. 26・4. 375-384 (2001)

K. Shimizu：“引入的转基因和培养烟草细胞的基因组 DNA 之间的连接区域中存在 SAR 样序列：其对转化频率的影响”《植物杂志》26·4 (2001)。

M.Takahashi: "Phytoremediation of nitrogen dioxide and its metabolism in plants"Acta Biotechnologica. (in press).

M.Takahashi：“二氧化氮的植物修复及其在植物中的代谢”Acta Biotechnologica。

M.Takahashi: "Production and characterization of transgenic plants that were transformed with MnP and LiP from white rot fungi"Acta Biotechnologica. (in press).

M.Takahashi：“用来自白腐真菌的 MnP 和 LiP 转化的转基因植物的生产和表征”Acta Biotechnologica。

M.Takahashi: "Enrichment of nitrite reductase gene improves the ability of Arabidopsis thaliana plants to assimilate nitrogen dioxide"Plant Physiology. 126・2. 731-741 (2001)

M.Takahashi：“亚硝酸还原酶基因的富集提高了拟南芥植物吸收二氧化氮的能力”植物生理学126・2（2001）。

Katsuya Shimizu: "Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells : its effect on transformation frequency."Plant J.. (in press). (2001)

Katsuya Shimizu：“在引入的转基因和培养烟草细胞的基因组 DNA 之间的连接区域中存在类似 SAR 的序列：其对转化频率的影响。”Plant J..（出版中）。