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

用微弹轰击pCaMVNEO质粒，从烟草转基因细胞系中克隆出了一个包含转基因与基因组DNA连接区域的12.5 kb DNA片段。该片段的核苷酸序列分析(DDBJ收录号：。84238)显示其包含一个7.7 kb的核心序列（完整pCaMVNEO和部分pCaMVNEO的串联体）和两个相同的1.3 kb连接序列，分别位于核心序列的5‘和3’端，方向相反。1.3 kb的序列包含拓扑异构酶II （Topo II）一致序列和已知的核支架相关区域（SARs）特异性的AT-rich序列。体外结合实验表明，从1.3 kb序列中提取的507-bp片段（命名为TJ1）能够与培养的烟草细胞核支架制品结合，证实了1.3 kb序列是S/MAR。通过在nptII基因表达盒的5′和3′侧插入507-bp片段，可使nptII基因抗性转化的产量提高5 ~ 10倍。在含有tj1的质粒的转化子中，每拷贝基因表达的uptII酶活性比不含tj1的野生型质粒的转化子高10倍。提示TJ1和TJ2序列在植物转基因技术中具有重要的应用价值。以3个转基因拟南芥株系为探针，分析了具有单个南方杂交带的选择基因。用反聚合酶链反应法克隆了转基因两侧的连接区。除了一个结区外，其余结区都是富含at的序列，包含以S/MAR为特征的基序，计算表明其中7个结区应该具有曲率倾向。与烟草核基质的体外结合实验证明，所有连接区域都具有与核基质结合的能力，而两个输入DNA则没有。少

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