Characterization of DNA sequences of junction regions and development of vectors for high frequency transformation

连接区 DNA 序列的表征和高频转化载体的开发

• 批准号: 08458239
• 负责人: MORIKAWA Hiromichi
• 金额: $ 4.35万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458239/
• 关键词: Trangene; Integration; S; MAR; Position Effect; Particle Bombardment; Site-directed; Topoisomerase; Cultured tobacco cells; 組込み(integration); site-directed; トポイソメラーゼ

A 12.5-kb DNA fragment containing junction regions between transgene and genomic DNA was cloned from a transgenic tobacco cell line that was obtained by microprojectile bombardment of plasmid pCaMVNEO.Nucleotide sequence analysis of the fragement (DDBJ accession no.84238) showed that it contained a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences flanking at both 5' and 3' ends of the core sequence with an inverted orientation. The 1.3-kb sequences contained topoisomerase II (Topo II) consensus sequences and AT-rich sequences that are known to be specific to the nuclear scaffold associated regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated as TJ1) taken from the 1.3-kb sequence had ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirming that the 1.3-kb sequence is an S/MAR.The yield of geneticin-resistant transformant was increased by 5- to 10-fold … More by the insertion of the 507-bp fragment at the 5' and 3' sides of the expresion cassette for the nptII gene in the plasmid. The uptII enzyme activity per copy of the gene expression per copy was 10-fold higher in the transformants that had TJ1-containing plasmid than those that had TJ1-free wild type plasmid. It is suggested that TJ1 and TJ2 sequences were very useful for transgenic technology in plants.Three transgenic Arabidopsis lines indicating a single Southern hybridization band with a selectablegene used as probes were analyzed. The junction regions flanked by transgenes were cloned using inverse polymerase chain reaction methods. All but one of the junction regions were AT-rich sequences containingg motifs characterized by an S/MAR,and calculations showed that seven of them should have a propensity towards curvature. An in vitro binding assay to tobacco nuclear matrices proved that all junction regions had the ability to bind to nuclear matrices and showed that the two input DNA did not. Less

用基因枪法从转基因烟草细胞系中克隆了含有转基因与基因组DNA连接区的12.5kb DNA片段。对该片段进行核苷酸序列分析(DDBJ登录号84238)表明，该片段含有一个7.7kb的核心序列(完整的pCaMVNEO和部分pCaMVNEO的串联)和两个相同的1.3kb的连接序列，分别位于核心序列的5‘和3’端。该1.3kb的序列包含拓扑异构酶II(Topo II)共有序列和已知的核支架相关区域(SARS)特异的AT富含序列。体外结合分析表明，从1.3kb序列中提取的507bp片段(命名为TJ1)能够与培养烟草细胞的核支架材料结合，证实该1.3kb序列为S/MAR。遗传素抗性转化子的产量提高了5-10倍…更多的是通过在表达盒的5‘和3’侧插入507-bp片段，用于在质粒中插入nptII基因。在含有TJ1的转化子中，每个拷贝的基因表达的uptII酶活性是不含TJ1的野生型的10倍。因此，TJ1和TJ2序列在植物转基因技术中具有很大的应用价值。以3个以可选择基因为探针的转基因拟南芥品系为研究对象，对其进行了分析。用反向聚合酶链式反应的方法克隆转基因基因两侧的连接区。除一个外，所有的连接区都是富含AT的序列，含有S/MAR的基序，计算表明其中7个区域应该有曲率的倾向。体外与烟草核基质的结合实验证明，所有的连接区都有与核基质结合的能力，而两个输入的DNA不能。较少

项目成果

M.Takahashi: "Stable transformation of Eustoma grandiflorum by particle bombardment" Plant Cell Reports. (in press). (1998)

M.Takahashi：“通过粒子轰击稳定转化洋桔梗”植物细胞报告。

H.Inada: "Existence of three regulatory regions each containing a highly conserved motif in the promoter of plastid-encoded RNA polymerase gene (rpo B)." Plant Journal. 11・4. 883-890 (1997)

H.Inada：“质体编码的 RNA 聚合酶基因 (rpo B) 启动子中存在三个调控区，每个调控区都包含一个高度保守的基序。”植物杂志 11·4 (1997)。

M.Seki: "In Methods in Molecular Biology,ed.,J.Martinez-Zapater and J.Salinas,Humana Press Inc.," Transient expression of foreign genes in tissues of Arabidopsis thaliana by bombardment-mediated transformation, 300(219-225) (1998)

M.Seki：“分子生物学方法，编辑，J.Martinez-Zapater 和 J.Salinas，Humana Press Inc.”，通过轰击介导的转化在拟南芥组织中瞬时表达外源基因，300（219-

H.Inada, M.Seki, H.Morikawa, M.Nishimura and K.Iba: "Existence of three regulatory regions each containing a highly conserved motif in the promoter of plastid-encoded RNA polymerase gene (rpo B)" Plant Journal. 11 (4). 883-890 (1977)

H.Inada、M.Seki、H.Morikawa、M.Nishimura 和 K.Iba：“质体编码 RNA 聚合酶基因 (rpo B) 启动子中存在三个调控区，每个调控区均包含高度保守的基序”《植物杂志》。

H.Inada: "Existence of three regulatory regions each containing a highly conseved motif in the promoter of plastid-encoded RNA polymerase gene (rpo B)." Plant Journal. 11・4. 883-890 (1997)

H.Inada：“质体编码的 RNA 聚合酶基因 (rpo B) 启动子中存在三个调控区，每个调控区都包含一个高度保守的基序。”植物杂志 11·4 (1997)。